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1. Kaplan  JB, Ragunath  C, Ramasubbu  N, Fine  DH,     ( 2003 )

Detachment of Actinobacillus actinomycetemcomitans biofilm cells by an endogenous beta-hexosaminidase activity.

Journal of bacteriology 185 (16)
PMID : 12896987  :   DOI  :   10.1128/jb.185.16.4693-4698.2003     PMC  :   PMC166467    
Abstract >>
When cultured in broth, fresh clinical isolates of the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans form tenaciously adherent biofilm colonies on surfaces such as plastic and glass. These biofilm colonies release adherent cells into the medium, and the released cells can attach to the surface of the culture vessel and form new colonies, enabling the biofilm to spread. We mutagenized A. actinomycetemcomitans clinical strain CU1000 with transposon IS903phikan and isolated a transposon insertion mutant that formed biofilm colonies which were tightly adherent to surfaces but which lacked the ability to release cells into the medium and disperse. The transposon insertion in the mutant strain mapped to a gene, designated dspB, that was predicted to encode a secreted protein homologous to the catalytic domain of the family 20 glycosyl hydrolases. A plasmid carrying a wild-type dspB gene restored the ability of biofilm colonies of the mutant strain to disperse. We expressed A. actinomycetemcomitans DspB protein engineered to contain a hexahistidine metal-binding site at its C terminus in Escherichia coli and purified the protein by using Ni affinity chromatography. Substrate specificity studies performed with monosaccharides labeled with 4-nitrophenyl groups showed that DspB hydrolyzed the 1-->4 glycosidic bond of beta-substituted N-acetylglucosamine, which is consistent with the known functions of other family 20 glycosyl hydrolases. When added to culture medium, purified DspB protein, but not heat-inactivated DspB, restored the ability of the mutant strain to release cells and disperse. DspB protein also caused the detachment of cells from preformed biofilm colonies of strain CU1000 grown attached to plastic and the disaggregation of highly autoaggregated clumps of CU1000 cells in solution. We concluded that dspB encodes a soluble beta-N-acetylglucosaminidase that causes detachment and dispersion of A. actinomycetemcomitans biofilm cells.
KeywordMeSH Terms
Bacterial Adhesion
2. Wang  Y, Shi  W, Chen  W, Chen  C,     ( 2003 )

Type IV pilus gene homologs pilABCD are required for natural transformation in Actinobacillus actinomycetemcomitans.

Gene 312 (N/A)
PMID : 12909361  :   DOI  :   10.1016/s0378-1119(03)00620-6    
Abstract >>
Some clinical isolates of the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans are naturally competent for DNA uptake. In this study, we examined the sequence and the function of a type IV pilus-like pilABCD gene cluster and its downstream region in a naturally transformable A. actinomycetemcomitans strain D7S. Specific knockout mutants of pilABCD of strain D7S were constructed by replacing individual genes with an antibiotic resistance cassette. The transformation frequency of chromosome markers in the wildtype strain D7S was approximately 10(-3) per CFU. In contrast, the delta pilA, delta pilB, delta pilC, delta pilBC or delta pilD mutants were non-transformable (transformation frequency <10(-8)). Disruption of an ORF downstream of pilD had no apparent effect on the transformability of this bacterium. The pilA or pilBC deletion did not seem to affect fimbria expression or cell surface structure in either rough or smooth strains as determined by scanning and transmission electron microscopy examinations. RT-PCR analysis showed that pilA was expressed in strain D7S under a competence-inducing growth condition. The expression of pilA was barely detectable in strain D7S cultured under a non-competence-inducing condition or in the non-transformable strain JP-2. The results indicate that pilABCD are required for competence but are apparently not involved in fimbria expression of A. actinomycetemcomitans.
KeywordMeSH Terms
Endopeptidases
3. Teng  YT, Hu  W,     ( 2003 )

Expression cloning of a periodontitis-associated apoptotic effector, cagE homologue, in Actinobacillus actinomycetemcomitans.

Biochemical and biophysical research communications 303 (4)
PMID : 12684047  :   DOI  :   10.1016/s0006-291x(03)00471-6    
Abstract >>
To study anti-bacterial immunity and to identify critical bacterial antigens associated with specific periodontal infection, we screened the genomic library of Actinobacillus actinomycetemcomitans, a major Gram(-) anaerobe causing human periodontitis, by expression cloning using disease-associated periodontal CD4(+)T cells derived from HuPBL-engrafted NOD/SCID mice. Here, we report one of the novel genes identified and designated, cagE homologue (in short: cagE) of A. actinomycetemcomitans, which encodes a putative bacterial type IV secretion system with significant homology to Helicobacter pylori CagE and Agrobacterium tumefaciens VirB4. All serum samples from A. actinomycetemcomitans-infected periodontitis patients, but not from the healthy controls, readily recognized CagE by ELISA and Western blot, suggesting its biological and clinical significance. The CagE protein, upon secretion, elicited significant apoptosis on primary human epithelia, endothelia, osteoblasts, and T cells by 4-12h in vitro. Importantly, both cagE(-) mutant strain and N-terminus truncated CagE protein drastically reduced (p<0.001) the induction of apoptosis on human epithelia in vitro. These data strongly suggest that a novel effector protein, CagE in A. actinomycetemcomitans, induces apoptosis of human cells and destructive immunity, thereby it may play an important role in the pathogenesis of A. actinomycetemcomitans-mediated infections.
KeywordMeSH Terms
Apoptosis
4. Planet  PJ, Kachlany  SC, Fine  DH, DeSalle  R, Figurski  DH,     ( 2003 )

The Widespread Colonization Island of Actinobacillus actinomycetemcomitans.

Nature genetics 34 (2)
PMID : 12717435  :   DOI  :   10.1038/ng1154    
Abstract >>
Genomic islands, such as pathogenicity islands, contribute to the evolution and diversification of microbial life. Here we report on the Widespread Colonization Island, which encompasses the tad (tight adherence) locus for colonization of surfaces and biofilm formation by the human pathogen Actinobacillus actinomycetemcomitans. At least 12 of the 14 genes at the tad locus are required for tenacious biofilm formation and synthesis of bundled Flp pili (fibrils) that mediate adherence. The pilin subunit, Flp1, remains inside the cell in tad-locus mutants, indicating that these genes encode a secretion system for export and assembly of fibrils. We found tad-related regions in a wide variety of Bacterial and Archaeal species, and their sequence characteristics indicate possible horizontal transfer. To test the hypothesis of horizontal transfer, we compared the phylogeny of the tad locus to a robust organismal phylogeny using statistical tests of congruence and tree reconciliation techniques. Our analysis strongly supports a complex history of gene shuffling by recombination and multiple horizontal transfers, duplications and losses. We present evidence for a specific horizontal transfer event leading to the establishment of this region as a determinant of disease.
KeywordMeSH Terms
5. Hayashida  H, Poulsen  K, Kilian  M,     ( 2002 )

Differences in iron acquisition from human haemoglobin among strains of Actinobacillus actinomycetemcomitans.

Microbiology (Reading, England) 148 (Pt 12)
PMID : 12480903  :   DOI  :   10.1099/00221287-148-12-3993    
Abstract >>
To get a better insight into the physiology of the high-toxic JP2 clone of Actinobacillus actinomycetemcomitans serotype b, which is strongly associated with juvenile periodontitis in adolescents of African descent, the modes of iron acquisition in this clone were examined and compared to those of other strains of the species. None of the strains examined could utilize human transferrin as a source of iron. This was in accordance with the presence of a non-functional tbpA gene, which normally encodes the A subunit of the transferrin-binding-protein complex. Southern blot analysis indicated that functional duplications of tbpA were not present in the genome. Thus, A. actinomycetemcomitans seems to be in a process of evolution, in which iron acquisition from host transferrin is not essential as in many other members of the pasteurellaceae. All strains could utilize haem as a source of iron. All 11 A. actinomycetemcomitans strains examined harboured a single genomic sequence with homology to the hgpA gene encoding haemoglobin-binding protein A in Haemophilus influenzae. However, in all three strains belonging to the JP2 clone and in one serotype e strain hgpA was a pseudogene. Seven other strains possessed a functional hgpA gene which, according to insertion mutagenesis experiments, was responsible for the ability of these strains to utilize haemoglobin as a source of iron. Thus, the presence of an hgpA pseudogene and the inability to use human haemoglobin as an iron source discriminate the high-toxic JP2 clone from low-toxic serotype b strains and most other strains of A. actinomycetemcomitans.
KeywordMeSH Terms
Bacterial Proteins
6. Suzuki  N, Nakano  Y, Yoshida  Y, Nezu  T, Terada  Y, Yamashita  Y, Koga  T,     ( 2002 )

Guanosine diphosphate-4-keto-6-deoxy-d-mannose reductase in the pathway for the synthesis of GDP-6-deoxy-d-talose in Actinobacillus actinomycetemcomitans.

European journal of biochemistry 269 (23)
PMID : 12444986  :   DOI  :   10.1046/j.1432-1033.2002.03331.x    
Abstract >>
The serotype a-specific polysaccharide antigen of Actinobacillus actinomycetemcomitans is an unusual sugar, 6-deoxy-d-talose. Guanosine diphosphate (GDP)-6-deoxy-d-talose is the activated sugar nucleotide form of 6-deoxy-d-talose, which has been identified as a constituent of only a few microbial polysaccharides. In this paper, we identify two genes encoding GDP-6-deoxy-d-talose synthetic enzymes, GDP-alpha-d-mannose 4,6-dehydratase and GDP-4-keto-6-deoxy-d-mannose reductase, in the gene cluster required for the biosynthesis of serotype a-specific polysaccharide antigen from A. actinomycetemcomitans SUNYaB 75. Both gene products were produced and purified from Escherichia coli transformed with plasmids containing these genes. Their enzymatic reactants were analysed by reversed-phase HPLC (RP-HPLC). The sugar nucleotide produced from GDP-alpha-d-mannose by these enzymes was purified by RP-HPLC and identified by electrospray ionization-MS, 1H nuclear magnetic resonance, and GC/MS. The results indicated that GDP-6-deoxy-d-talose is produced from GDP-alpha-d-mannose. This paper is the first report on the GDP-6-deoxy-d-talose biosynthetic pathway and the role of GDP-4-keto-6-deoxy-d-mannose reductase in the synthesis of GDP-6-deoxy-d-talose.
KeywordMeSH Terms
7. Wang  Y, Goodman  SD, Redfield  RJ, Chen  C,     ( 2002 )

Natural transformation and DNA uptake signal sequences in Actinobacillus actinomycetemcomitans.

Journal of bacteriology 184 (13)
PMID : 12057937  :   DOI  :   10.1128/jb.184.13.3442-3449.2002     PMC  :   PMC135135    
Abstract >>
Actinobacillus actinomycetemcomitans is a member of the family Pasteurellaceae and a major causative agent of periodontitis. While several genera from this family are known to be competent for transformation, A. actinomycetemcomitans has yet to be fully characterized. Here we show that the competence of A. actinomycetemcomitans is remarkably similar to that of Haemophilus influenzae. In addition to having a similar frequency of transformation as H. influenzae, A. actinomycetemcomitans competence could also be induced at least 100-fold by cyclic AMP, suggesting that, as in H. influenzae, at least some competence genes are regulated by catabolite repression. Even more intriguing was the discovery of a putative A. actinomycetemcomitans DNA uptake signal sequence (USS) virtually identical to the USS of H. influenzae. Moreover, we provide evidence that this sequence functions in the same capacity as that from H. influenzae; the sequence appears to be required and sufficient for DNA uptake in a variety of assays. Finally, we have taken advantage of this system to develop a simple, highly efficient competence-based method for generating site-directed mutations in the wild-type fimbriated A. actinomycetemcomitans.
KeywordMeSH Terms
Transformation, Bacterial
8. Haraszthy  VI, Lally  ET, Haraszthy  GG, Zambon  JJ,     ( 2002 )

Molecular cloning of the fur gene from Actinobacillus actinomycetemcomitans.

Infection and immunity 70 (6)
PMID : 12011012  :   DOI  :   10.1128/iai.70.6.3170-3179.2002     PMC  :   PMC127990    
Abstract >>
In several bacterial species, iron availability in host tissues is coordinated with the expression of virulence determinants through the fur gene product. Initial experiments showed that a cloned Escherichia coli fur gene probe hybridized to Southern blots of Actinobacillus actinomycetemcomitans strain JP2 (serotype b) chromosomal DNA. The A. actinomycetemcomitans fur gene was then cloned utilizing partial functional complementation of the fur mutant in E. coli strain H1780. Analysis of the cloned DNA sequence revealed a 438-bp open reading frame with a deduced 146-amino-acid sequence exhibiting 80% identity to Haemophilus influenzae Fur and 62% identity to E. coli Fur. The pUC Aafur gene probe (generated from JP2 serotype b) hybridized to representatives from all five A. actinomycetemcomitans serotypes as well as to two strains derived from monkeys, suggesting that fur is widely distributed in A. actinomycetemcomitans. Open reading frames having >70% identity with the E. coli and H. influenzae flavodoxin and gyrase A genes, respectively, were found. Expression of the A. actinomycetemcomitans fur gene product repressed fiu expression and siderophore production in E. coli. A gel shift assay demonstrated that the expressed A. actinomycetemcomitans Fur protein bound the bacterial fur consensus sequence. Further characterization of the fur gene product in A. actinomycetemcomitans may improve our understanding of its role in the pathogenesis of periodontal disease and may lead to specific therapeutic modalities.
KeywordMeSH Terms
9. Komatsuzawa  H, Asakawa  R, Kawai  T, Ochiai  K, Fujiwara  T, Taubman  MA, Ohara  M, Kurihara  H, Sugai  M,     ( 2002 )

Identification of six major outer membrane proteins from Actinobacillus actinomycetemcomitans.

Gene 288 (1��2��)
PMID : 12034509  :   DOI  :   10.1016/s0378-1119(02)00500-0    
Abstract >>
We have identified six major sarcosyl-insoluble outer membrane proteins (Omp) of Actinobacillus actinomycetemcomitans Y4, and designated them as Omp100, Omp64, Omp39, Omp29, Omp18 and Omp16 according to the molecular mass. A similar N-terminal sequence was found in the first 15 amino acid residues of Omp16 and Omp18. The N-terminal sequence of Omp29 matched perfectly with the sequence previously identified. We cloned and determined the DNA sequences of three complete genes encoding Omp100, Omp64 and Omp18/16, and one incomplete gene encoding Omp39. Each Omp revealed homologies with some bacterial virulence factors responsible for adhesion, invasion, serum resistance, or protein antigenicity. Serum from patients with periodontitis suspected to be related to A. actinomycetemcomintans infection strongly reacted with Omp100, Omp29 and Omp16 as did serum from mice immunized with A. actinomycetemcomitans Y4 whole bacteria. These findings suggest that Omps of A. actinomycetemcomitans can be associated with periodontal disease.
KeywordMeSH Terms
10. Hirosue  M, Kokeguchi  S, Maeda  H, Nishimura  F, Takashiba  S, Murayama  Y,     ( 2001 )

Characterization of two genes encoding ferritin-like protein in Actinobacillus actinomycetemcomitans.

Microbiology and immunology 45 (10)
PMID : 11762755  :   DOI  :   10.1111/j.1348-0421.2001.tb01307.x    
Abstract >>
Two genes encoding ferritin-like protein, designated afnA and afnB, were identified in the upstream region of actX on the Actinobacillus actinomycetemcomitans chromosomal DNA. The actX has been reported to be a regulatory gene homologous to the Escherichia coli fnr, which controls the growth and virulence of A. actinomycetemcomitans under anaerobic conditions. The afnB located 340 bp-upstream from the actX, and the afnA located just 15 bp-upstream from afnB. The afnA and afnB encoded 161 and 165 amino acid residues, respectively, which were similar to ferritin-like proteins of other microorganisms. Western immunoblotting using rabbit antiserum against E. coli ferritin showed these two proteins, which are reactive with the serum with 19-kDa molecular masses, are produced from A. actinomycetemcomitans. The N-terminal amino acid sequences of the two proteins were consequent with those deduced from afnA and afnB. Northern hybridization revealed that the afnA and afnB constituted a bicistronic operon and the accumulation of afnA and afnB mRNA was upregulated under aerobic conditions. These findings suggested that the operon was regulated by the presence of oxygen. The two ferritin-like proteins may have important roles in the adaptation of A. actinomycetemcomitans to oxidative environmental changes.
KeywordMeSH Terms
11. Novak  KF, Dougherty  B, Peláez  M,     ( 2001 )

Actinobacillus actinomycetemcomitans harbours type IV secretion system genes on a plasmid and in the chromosome.

Microbiology (Reading, England) 147 (Pt 11)
PMID : 11700353  :   DOI  :   10.1099/00221287-147-11-3027    
Abstract >>
Nine contiguous genes encoding a potential type IV secretion system have been identified in the chromosome of Actinobacillus actinomycetemcomitans strain VT747 and on a plasmid (pVT745) in strain VT745. Seven of these genes encode predicted proteins that share significant homology with type IV secretion proteins in Bordetella pertussis (ptl operon), Brucella melitensis biovar suis and Agrobacterium tumefaciens (virB operons), where they are involved in protein secretion, pathogen intracellular survival and multiplication, and DNA transport, respectively. Results of previous studies have demonstrated that pVT745 is a conjugative plasmid and that a secondary plasmid, pMMB67, can be mobilized from strain VT745. Given these results, it was hypothesized that (1) the type IV secretion genes on pVT745 are responsible for these two functions and (2) the type IV VT747 chromosomal genes also play a role in the transport of DNA. Wild-type and mutant strains of VT745 were evaluated for their conjugative abilities. Wild-type mating efficiency was 10(-6) transconjugants per donor, while the mutant strain yielded no transconjugants. Wild-type VT745 harbouring a co-resident plasmid, pMMB67, mobilized pMMB67 at a frequency of 10(-6), while VT747 was unable to mobilize this plasmid. These results support the hypothesis that the plasmid-encoded type IV secretion system on pVT745 is involved in DNA transport. However, the chromosomally encoded secretion system may not play a role in DNA transport in strain VT747. While the precise function of these chromosomal genes in strain VT747 has not been determined, Northern blot analyses demonstrated that these genes are expressed in both ACT: actinomycetemcomitans strains VT745 and VT747.
KeywordMeSH Terms
Chromosomes, Bacterial
Plasmids
12. Inoue  T, Tanimoto  I, Tada  T, Ohashi  T, Fukui  K, Ohta  H,     ( 2001 )

Fermentable-sugar-level-dependent regulation of leukotoxin synthesis in a variably toxic strain of Actinobacillus actinomycetemcomitans.

Microbiology (Reading, England) 147 (Pt 10)
PMID : 11577154  :   DOI  :   10.1099/00221287-147-10-2749    
Abstract >>
Actinobacillus actinomycetemcomitans, a Gram-negative periodontopathic bacterium, produces a leukotoxin belonging to the RTX family. The production of leukotoxin varies greatly among different strains of this species and under different culture conditions. A toxin-production-variable strain, 301-b, stably produces significant amounts of leukotoxin in anaerobic fructose-limited chemostat cultures, but does not do so in the presence of excess fructose. This communication describes the cloning and sequencing of the leukotoxin promoter region from 301-b, showing that this strain has a promoter region similar to that from strain 652, a moderately toxic strain. Northern blot analysis using a leukotoxin gene probe demonstrated that change in toxin production in response to the level of external fructose was due to alteration in the transcriptional level of the leukotoxin gene. Pulsing of fructose into the fructose-limited chemostat culture remarkably reduced the intracellular cAMP level from 40 pmol (mg dry wt cells)(-1) to 3.1 pmol (mg dry wt cells)(-1), which was restored when the culture was returned to fructose-limited conditions. Further, it was found that addition of external cAMP to the culture with excess fructose resulted in an apparent recovery of leukotoxin production. Taken together, these findings indicate that a cAMP-dependent mechanism, possibly a catabolite-repression-like system, may be involved in the regulation of leukotoxin production in this bacterium.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
13. Kachlany  SC, Planet  PJ, DeSalle  R, Fine  DH, Figurski  DH,     ( 2001 )

Genes for tight adherence of Actinobacillus actinomycetemcomitans: from plaque to plague to pond scum.

Trends in microbiology 9 (9)
PMID : 11553455  :  
Abstract >>
The Gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans forms an extremely tenacious biofilm on solid surfaces such as glass, plastic and hydroxyapatite. This characteristic is likely to be important for colonization of the oral cavity and initiation of a potentially devastating form of periodontal disease. Genetic analysis has revealed a cluster of tad genes responsible for tight adherence to surfaces. Evidence indicates that the tad genes are part of a locus encoding a novel secretion system for the assembly and release of long, bundled Flp pili. Remarkably similar tad loci appear in the genomes of a wide variety of Gram-negative and Gram-positive bacteria, including many significant pathogens, and in Archaea. We propose that the tad loci are important for microbial colonization in a variety of environmental niches.
KeywordMeSH Terms
14. Saiki  K, Konishi  K, Gomi  T, Nishihara  T, Yoshikawa  M,     ( 2001 )

Reconstitution and purification of cytolethal distending toxin of Actinobacillus actinomycetemcomitans.

Microbiology and immunology 45 (6)
PMID : 11497226  :   DOI  :   10.1111/j.1348-0421.2001.tb02650.x    
Abstract >>
Cytolethal distending toxin (CDT) has been found in various pathogenic bacterial species and causes a cell distending and a G2 arrest against eukaryotic cells. All the cdtABC genes, which encode CDT, are known to be required for the CDT activities although the CDT holotoxin structure has not been elucidated. We cloned the cdtABC genes of Actinobacillus actinomycetemcomitans and constructed an Escherichia coli expression system for them. We found that crude extracts from six deletion mutants (delta cdtA, delta cdtB, delta cdtC, delta cdtBC, delta cdtAC, and delta cdtAB) of recombinant E. coli, which showed very weak or no detectable CDT activities, restored the CDT activities when pre-mixing and pre-incubation of them were performed in combinations to contain all the CdtA, CdtB, and CdtC proteins. These results indicate that all the Cdt proteins are required for the CDT activities. We also found that the chimera CdtB protein, CdtB-intein-CBD (chitin binding domain) like CdtB protein itself assembled with CdtA and CdtC. The reconstituted CDT containing the chimera CdtB protein was specifically extracted by chitin beads and the only CDT portion was isolated from the chitin beads by a cleavage reaction of the intein. The purified reconstituted-CDT was found to consist of CdtA, CdtB, and CdtC proteins, and showed appreciable CDT activities, indicating that the CDT holotoxin structure is the CdtABC complex. To our knowledge, this is the first report succeeded in complete purification of an active CDT and may offer useful tools for elucidation of the toxic mechanism of CDT.
KeywordMeSH Terms
15. Kaplan  JB, Perry  MB, MacLean  LL, Furgang  D, Wilson  ME, Fine  DH,     ( 2001 )

Structural and genetic analyses of O polysaccharide from Actinobacillus actinomycetemcomitans serotype f.

Infection and immunity 69 (9)
PMID : 11500407  :   DOI  :   10.1128/iai.69.9.5375-5384.2001     PMC  :   PMC98647    
Abstract >>
The oral bacterium Actinobacillus actinomycetemcomitans is implicated as a causative agent of localized juvenile periodontitis (LJP). A. actinomycetemcomitans is classified into five serotypes (a to e) corresponding to five structurally and antigenically distinct O polysaccharide (O-PS) components of their respective lipopolysaccharide molecules. Serotype b has been reported to be the dominant serotype isolated from LJP patients. We determined the lipopolysaccharide O-PS structure from A. actinomycetemcomitans CU1000, a strain isolated from a 13-year-old African-American female with LJP which had previously been classified as serotype b. The O-PS of strain CU1000 consisted of a trisaccharide repeating unit composed of L-rhamnose and 2-acetamido-2-deoxy-D-galactose (molar ratio, 2:1) with the structure -->2)-alpha-L-Rhap-(1-3)-2-O-(beta-D-GalpNAc)-alpha-L-Rhap-(1-->* O-PS from strain CU1000 was structurally and antigenically distinct from the O-PS molecules of the five known A. actinomycetemcomitans serotypes. Strain CU1000 was mutagenized with transposon IS903phikan, and three mutants that were deficient in O-PS synthesis were isolated. All three transposon insertions mapped to a single 1-kb region on the chromosome. The DNA sequence of a 13.1-kb region surrounding these transposon insertions contained a cluster of 14 open reading frames that was homologous to gene clusters responsible for the synthesis of A. actinomycetemcomitans serotype b, c, and e O-PS antigens. The CU1000 gene cluster contained two genes that were not present in serotype-specific O-PS antigen clusters of the other five known A. actinomycetemcomitans serotypes. These data indicate that strain CU1000 should be assigned to a new A. actinomycetemcomitans serotype, designated serotype f. A PCR assay using serotype-specific PCR primers showed that 3 out of 20 LJP patients surveyed (15%) harbored A. actinomycetemcomitans strains carrying the serotype f gene cluster. The finding of an A. actinomycetemcomitans serotype showing serological cross-reactivity with anti-serotype b-specific antiserum suggests that a reevaluation of strains previously classified as serotype b may be warranted.
KeywordMeSH Terms
16. Hedegaard  J, Okkels  H, Bruun  B, Kilian  M, Mortensen  KK, Nørskov-Lauritsen  N,     ( 2001 )

Phylogeny of the genus Haemophilus as determined by comparison of partial infB sequences.

Microbiology (Reading, England) 147 (Pt 9)
PMID : 11535800  :   DOI  :   10.1099/00221287-147-9-2599    
Abstract >>
A 453 bp fragment of infB, the gene encoding translation initiation factor 2, was sequenced and compared from 66 clinical isolates and type strains of Haemophilus species and related bacteria. Analysis of the partial infB sequences obtained suggested that the human isolates dependent on X and V factor, H. influenzae, H. haemolyticus, H. aegyptius and some cryptic genospecies of H. influenzae, were closely related to each other. H. parainfluenzae constituted a heterogeneous group within the boundaries of the genus, whereas H. aphrophilus/paraphrophilus and Actinobacillus actinomycetemcomitans were only remotely related to the type species of the genus Haemophilus H. parahaemolyticus and H. paraphrohaemolyticus took up an intermediary position and may not belong in the genus Haemophilus sensu stricto. Ambiguous results were obtained with seven isolates tentatively identified as H. segnis, which fell into two discrete clusters. The delineation of 'Haemophilus sensu stricto' as suggested by infB analysis supports previous results obtained by DNA hybridization, in contrast to the delineation inferred from 16S rRNA sequence comparison.
KeywordMeSH Terms
Genes, Bacterial
Phylogeny
17. Kachlany  SC, Planet  PJ, Desalle  R, Fine  DH, Figurski  DH, Kaplan  JB,     ( 2001 )

flp-1, the first representative of a new pilin gene subfamily, is required for non-specific adherence of Actinobacillus actinomycetemcomitans.

Molecular microbiology 40 (3)
PMID : 11359562  :   DOI  :   10.1046/j.1365-2958.2001.02422.x    
Abstract >>
Actinobacillus actinomycetemcomitans, a Gram-negative bacterium responsible for localized juvenile periodontitis and other infections such as endocarditis, produces long fibrils of bundled pili that are believed to mediate non-specific, tenacious adherence to surfaces. Previous investigations have implicated an abundant, small (approximately 6.5 kDa), fibril-associated protein (Flp/Fap) as the primary fibril subunit. Here, we report studies on fibril structure and on the function and evolution of Flp. High-resolution electron microscopy of adherent clinical strain CU1000N revealed long bundles of 5- to 7-nm-diameter pili, whose subunits appear to be arranged in a helical array similar to that observed for type IV pili in other bacteria. Fibrils were found to be associated with the bacterial cell surface and smaller structures thought to be membrane vesicles. A modified version of the CU1000N Flp1 polypeptide with the T7-TAG epitope fused to its C-terminus was expressed in the wild-type strain, and the presence of the modified Flp1 in fibrils was confirmed by immunogold electron microscopy with monoclonal antibody to T7-TAG. To determine the importance of Flp1 in fibril formation and cell adherence, we used transposon IS903phikan to isolate insertion mutations in the flp-1 gene (formerly designated flp). Mutants with insertions early in flp-1 fail to produce fibrils and do not adhere to surfaces. Both fibril production and adherence were restored by cloned flp-1 in trans, thus providing the first evidence that flp-1 is required for fibril formation and tight, non-specific adherence. One mutant was found to have an insertion near the 3' end of flp-1 that results in the expression of a truncated and altered C-terminus of Flp1. This mutant produced short, unbundled pili, and its adherence to surfaces was significantly less than that of wild-type bacteria. These findings and related observations with the Flp1-T7-TAG protein indicate that the C-terminus of Flp1 is important for the bundling and adherence properties of pili. Extensive sequence comparisons and phylogenetic analysis of 61 predicted prepilin genes of bacteria revealed flp-1 to be a member of a novel and widespread subfamily of type IV prepilin genes. Thus, Flp pili are likely to be expressed by diverse bacterial species. Furthermore, we found that it is common for bacterial genomes to contain multiple alleles of flp-like genes, including the open reading frame (flp-2, previously designated orfA) immediately downstream of flp-1 in A. actinomycetemcomitans. The duplication and divergence of flp genes in bacteria may be important to the diversification of the colonization properties of these organisms.
KeywordMeSH Terms
18. Galli  DM, Chen  J, Novak  KF, Leblanc  DJ,     ( 2001 )

Nucleotide sequence and analysis of conjugative plasmid pVT745.

Journal of bacteriology 183 (5)
PMID : 11160089  :   DOI  :   10.1128/JB.183.5.1585-1594.2001     PMC  :   PMC95043    
Abstract >>
The complete nucleotide sequence and genetic map of pVT745 are presented. The 25-kb plasmid was isolated from Actinobacillus actinomycetemcomitans, a periodontal pathogen. Two-thirds of the plasmid encode functions related to conjugation, replication, and replicon stability. Among potential gene products with a high degree of similarity to known proteins are those associated with plasmid conjugation. It was shown that pVT745 derivatives not only mobilized a coresident nontransmissible plasmid, pMMB67, but also mediated their own conjugative transfer to different A. actinomycetemcomitans strains. However, transfer of pVT745 derivatives from A. actinomycetemcomitans to Escherichia coli JM109 by conjugation was successful only when an E. coli origin of replication was present on the pVT745 construct. Surprisingly, 16 open reading frames encode products of unknown function. The plasmid contains a conserved replication region which belongs to the HAP (Haemophilus-Actinobacillus-Pasteurella) theta replicon family. However, its host range appears to be rather narrow compared to other members of this family. Sequences homologous to pVT745 have previously been detected in the chromosomes of numerous A. actinomycetemcomitans strains. The nature and origin of these homologs are discussed based on information derived from the nucleotide sequence.
KeywordMeSH Terms
19. Eberhard  J, Oza  J, Reich  NO,     ( 2001 )

Cloning, sequence analysis and heterologous expression of the DNA adenine-(N(6)) methyltransferase from the human pathogen Actinobacillus actinomycetemcomitans.

FEMS microbiology letters 195 (2)
PMID : 11179656  :   DOI  :   10.1111/j.1574-6968.2001.tb10525.x    
Abstract >>
We cloned and sequenced the DNA adenine-N(6) methyltransferase gene of the human pathogen Actinobacillus actinomycetemcomitans (M.AacDAM). Restriction digestion shows that the enzyme methylates adenine in the sequence GATC. Expression of the enzyme in a DAM(-) background shows in vivo activity. A PSI-BLAST search revealed that M.AacDAM is most related to M.HindIV, M.EcoDAM, M.StyDAM, and M.SmaII. The ClustalW alignment shows highly conserved regions in the enzyme characteristic for type a MTases. Phylogenetic tree analysis shows a cluster of enzymes recognizing the sequence GATC, within a branch of orphan MTases harboring M.AacDAM. The cloning and sequencing of this first methyltransferase gene described for A. actinomycetemcomitans open the path for studies on the potential regulatory impact of DNA methylation on gene regulation and virulence in this organism.
KeywordMeSH Terms
20. Suzuki  N, Nakano  Y, Yoshida  Y, Nakao  H, Yamashita  Y, Koga  T,     ( 2000 )

Genetic analysis of the gene cluster for the synthesis of serotype a-specific polysaccharide antigen in Aactinobacillus actinomycetemcomitans.

Biochimica et biophysica acta 1517 (1)
PMID : 11118626  :   DOI  :   10.1016/s0167-4781(00)00229-3    
Abstract >>
The serotype a-specific polysaccharide antigen (SPA) of Actinobacillus actinomycetemcomitans consists of 6-deoxy-D-talose. A gene cluster associated with the biosynthesis of SPA was cloned and sequenced from the chromosomal DNA of A. actinomycetemcomitans SUNYaB 75 (serotype a). This cluster consisted of 14 open reading frames. Insertional inactivation of eight genes in this cluster resulted in loss of the ability of A. actinomycetemcomitans SUNYaB 75 cells to produce the polysaccharide. A protein database search revealed that the 11 sequential genes containing these eight genes were not found in SPA-associated gene clusters of the other serotypes of A. actinomycetemcomitans. These results suggest that the gene cluster is unique to serotype a and is essential to the synthesis of the SPA.
KeywordMeSH Terms
Hexoses
21. Kokeguchi  S, Hirosue  M, Maeda  H, Miyamoto  M, Takashiba  S, Murayama  Y,     ( 2000 )

Molecular characterization of the hlyX-like gene of Actinobacillus actinomycetemcomitans Y4.

Research in microbiology 151 (9)
PMID : 11130862  :  
Abstract >>
We isolated and characterized a possible regulatory gene, designated actX gene, from Actinobacillus actinomyctemcomitans Y4, which defined the Actinobacillus pleuropneumoniae hlyX-like regulatory gene. DNA sequence analysis for plasmid clone pKM317 containing a 1.6-kb DNA insert indicated an open reading frame encoding a polypeptide of 257 amino acid residues. Analysis of the deduced amino acid sequence showed the presence of five characteristic cysteine residues in the N-terminal region and a putative DNA binding residue in the C-terminal region, indicating that actX might belong to a regulatory gene family. Escherichia coli DH5alpha and a mutant strain JRG1728 transformed by plasmid carrying actX manifested apparent hemolytic activity on sheep blood agar and grew anaerobically, although the original strains did not.
KeywordMeSH Terms
DNA-Binding Proteins
Genes, Regulator
Transcription Factors
22. Kachlany  SC, Fine  DH, Figurski  DH,     ( 2000 )

Secretion of RTX leukotoxin by Actinobacillus actinomycetemcomitans.

Infection and immunity 68 (11)
PMID : 11035711  :   DOI  :   10.1128/iai.68.11.6094-6100.2000     PMC  :   PMC97685    
Abstract >>
Actinobacillus actinomycetemcomitans, the etiologic agent for localized juvenile periodontitis and certain other human infections, such as endocarditis, expresses a leukotoxin that acts on polymorphonuclear leukocytes and macrophages. Leukotoxin is a member of the highly conserved repeat toxin (RTX) family of bacterial toxins expressed by a variety of pathogenic bacteria. While the RTX toxins of other bacterial species are secreted, the leukotoxin of A. actinomycetemcomitans is thought to remain associated with the bacterial cell. We have examined leukotoxin production and localization in rough (adherent) and smooth (nonadherent) strains of A. actinomycetemcomitans. We found that leukotoxin expressed by the rough, adherent, clinical isolate CU1000N is indeed cell associated, as expected. However, we were surprised to find that smooth, nonadherent strains of A. actinomycetemcomitans, including Y4, JP2 (a strain expressing a high level of toxin), and CU1060N (an isogenic smooth variant of CU1000N), secrete an abundance of leukotoxin into the culture supernatants during early stages of growth. After longer times of incubation, leukotoxin disappears from the supernatants, and its loss is accompanied by the appearance of a number of low-molecular-weight polypeptides. The secreted leukotoxin is active, as evidenced by its ability to kill HL-60 cells in vitro. We found that the growth phase and initial pH of the growth medium significantly affect the abundance of secreted leukotoxin, and we have developed a rapid (<2 h) method to partially purify large amounts of leukotoxin. Remarkably, mutations in the tad genes, which are required for tight nonspecific adherence of A. actinomycetemcomitans to surfaces, cause leukotoxin to be released from the bacterial cell. These studies show that A. actinomycetemcomitans has the potential to secrete abundant leukotoxin. It is therefore appropriate to consider a possible role for leukotoxin secretion in the pathogenesis of A. actinomycetemcomitans.
KeywordMeSH Terms
23. Kachlany  SC, Planet  PJ, Bhattacharjee  MK, Kollia  E, DeSalle  R, Fine  DH, Figurski  DH,     ( 2000 )

Nonspecific adherence by Actinobacillus actinomycetemcomitans requires genes widespread in bacteria and archaea.

Journal of bacteriology 182 (21)
PMID : 11029439  :   DOI  :   10.1128/jb.182.21.6169-6176.2000     PMC  :   PMC94753    
Abstract >>
The gram-negative coccobacillus, Actinobacillus actinomycetemcomitans, is the putative agent for localized juvenile periodontitis, a particularly destructive form of periodontal disease in adolescents. This bacterium has also been isolated from a variety of other infections, notably endocarditis. Fresh clinical isolates of A. actinomycetemcomitans form tenacious biofilms, a property likely to be critical for colonization of teeth and other surfaces. Here we report the identification of a locus of seven genes required for nonspecific adherence of A. actinomycetemcomitans to surfaces. The recently developed transposon IS903phikan was used to isolate mutants of the rough clinical isolate CU1000 that are defective in tight adherence to surfaces (Tad(-)). Unlike wild-type cells, Tad(-) mutant cells adhere poorly to surfaces, fail to form large autoaggregates, and lack long, bundled fibrils. Nucleotide sequencing and genetic complementation analysis revealed a 6.7-kb region of the genome with seven adjacent genes (tadABCDEFG) required for tight adherence. The predicted TadA polypeptide is similar to VirB11, an ATPase involved in macromolecular transport. The predicted amino acid sequences of the other Tad polypeptides indicate membrane localization but no obvious functions. We suggest that the tad genes are involved in secretion of factors required for tight adherence of A. actinomycetemcomitans. Remarkably, complete and highly conserved tad gene clusters are present in the genomes of the bubonic plague bacillus Yersinia pestis and the human and animal pathogen Pasteurella multocida. Partial tad loci also occur in strikingly diverse Bacteria and Archaea. Our results show that the tad genes are required for tight adherence of A. actinomycetemcomitans to surfaces and are therefore likely to be essential for colonization and pathogenesis. The occurrence of similar genes in a wide array of microorganisms indicates that they have important functions. We propose that tad-like genes have a significant role in microbial colonization.
KeywordMeSH Terms
Genes, Archaeal
Genes, Bacterial
Virulence Factors
24. Nakano  Y, Yoshida  Y, Suzuki  N, Yamashita  Y, Koga  T,     ( 2000 )

A gene cluster for the synthesis of serotype d-specific polysaccharide antigen in Actinobacillus actinomycetemcomitans.

Biochimica et biophysica acta 1493 (1��2��)
PMID : 10978535  :   DOI  :   10.1016/s0167-4781(00)00179-2    
Abstract >>
The serotype d antigen of Actinobacillus actinomycetemcomitans consists of D-glucose, D-mannose, and L-rhamnose in a molar ratio of 1:2:1. A gene cluster involved in the synthesis of serotype-specific polysaccharide antigen was cloned from the chromosomal DNA of A. actinomycetemcomitans IDH 781 (serotype d). This cluster consisted of 12 open reading frames. Insertional inactivation of six genes in this cluster resulted in loss of ability of A. actinomycetemcomitans IDH 781 cells to produce the polysaccharide. Comparing the structure of the gene cluster with similar clusters from other serotypes of A. actinomycetemcomitans, showed that eight genes are unique to serotype d; the other four genes are involved in the biosynthesis of dTDP-L-rhamnose. These results suggest that the synthesis and structure of serotype d-specific polysaccharide of A. actinomycetemcomitans is quite different from those of other serotype strains.
KeywordMeSH Terms
Genes, Bacterial
25. Shenker  BJ, Hoffmaster  RH, McKay  TL, Demuth  DR,     ( 2000 )

Expression of the cytolethal distending toxin (Cdt) operon in Actinobacillus actinomycetemcomitans: evidence that the CdtB protein is responsible for G2 arrest of the cell cycle in human T cells.

Journal of immunology (Baltimore, Md. : 1950) 165 (5)
PMID : 10946289  :   DOI  :   10.4049/jimmunol.165.5.2612    
Abstract >>
We have previously shown that Actinobacillus actinomycetemcomitans produces an immunosuppressive factor that is encoded by the cdtB gene, which is homologous to a family of cytolethal distending toxins (Cdt) expressed by several gram-negative bacteria. In this study, we report that the cdt locus in A. actinomycetemcomitans is composed of five open reading frames, designated orf1, orf2, cdtA, cdtB, and cdtC. The deduced amino acid sequences of the five open reading frames are highly conserved among A. actinomycetemcomitans strains 652, Y4, 29522, and HK1651. There is also strong homology with the Cdt proteins of Haemophilus ducreyi (87-91%), but only partial homology with that of Campylobacter jejuni and Escherichia coli (29-48%). Analysis of A. actinomycetemcomitans mRNA by RT-PCR suggests that the two small open reading frames upstream of cdtA are coexpressed with cdtA, cdtB, and cdtC. We next utilized a series of plasmids that express various combinations of the cdt genes to determine their requirement for expression of immunoinhibitory activity. Cell extracts of E. coli transformed with each of the plasmids were tested for their capacity to induce G2 arrest in the cell cycle of PHA-activated human T cells. These experiments suggest that expression of cdtB alone is sufficient to induce G2 arrest in human T cells, but do not exclude the possibility that cdtC also contributes to cell cycle arrest. The implications of our results with respect to the function of the individual Cdt proteins are discussed.
KeywordMeSH Terms
26. Suzuki  N, Yoshida  Y, Nakano  Y,     ( 2000 )

Thymidine diphosphate-6-deoxy-L-lyxo-4-hexulose reductase synthesizing dTDP-6-deoxy-L-talose from Actinobacillus actinomycetemcomitans.

The Journal of biological chemistry 275 (10)
PMID : 10702238  :   DOI  :   10.1074/jbc.275.10.6806    
Abstract >>
The serotype c-specific polysaccharide antigen of Actinobacillus actinomycetemcomitans NCTC 9710 contains an unusual sugar, 6-deoxy-L-talose, which has been identified as a constituent of cell wall components in some bacteria. Two genes coding for thymidine diphosphate (dTDP)-6-deoxy-L-lyxo-4-hexulose reductases were identified in the gene cluster required for biosynthesis of serotype c-specific polysaccharide. Both dTDP-6-deoxy-L-lyxo-4-hexulose reductases were overproduced and purified from Escherichia coli transformed with the plasmids containing these genes. The sugar nucleotides converted by both reductases were purified by reversed-phase high performance liquid chromatography and identified by (1)H nuclear magnetic resonance and gas-liquid chromatography. The results indicated that one of two reductases produced dTDP-6-deoxy-L-talose and the other produced dTDP-L-rhamnose (dTDP-6-deoxy-L-mannose). The amino acid sequence of the dTDP-6-deoxy-L-lyxo-4-hexulose reductase forming dTDP-6-deoxy-L-talose shared only weak homology with that forming dTDP-L-rhamnose, despite the fact that these two enzymes catalyze the reduction of the same substrate and the products are determined by the stereospecificity of the reductase activity. Neither the gene for dTDP-6-deoxy-L-talose biosynthesis nor its corresponding protein product has been found in other bacteria; this biosynthetic pathway is identified here for the first time.
KeywordMeSH Terms
Hexoses
27. Suzuki  N, Nakao  H, Nakano  Y, Yoshida  Y,     ( 1999 )

Genetic analysis of the gene cluster responsible for synthesis of serotype e-specific polysaccharide antigen in Actinobacillus actinomycetemcomitans.

Biochimica et biophysica acta 1489 (2��3��)
PMID : 10673051  :   DOI  :   10.1016/s0167-4781(99)00192-x    
Abstract >>
A gene cluster associated with the biosynthesis of the serotype e-specific polysaccharide antigen (SPA) of Actinobacillus actinomycetemcomitans IDH1705 belonging to serotype e was cloned and sequenced. This cluster consisted of 18 open reading frames. Escherichia coli produced the polysaccharide that reacts with the serotype e-specific antiserum when transformed with a plasmid containing the cluster. Comparing the structure of the gene cluster with similar clusters from A. actinomycetemcomitans strains Y4 (serotype b) and NCTC9710 (serotype c) revealed that a 5.3-kb region containing the distal half of one gene and two entire genes in the cluster from strain IDH1705 replaced a 6.2-kb region containing eight genes in the cluster from strain Y4, and a 4.7-kb region containing four genes in the cluster from strain NCTC9710. These results suggest that this region is essential to the antigenic specificity of serotype e A. actinomycetemcomitans.
KeywordMeSH Terms
Multigene Family
28. Bhattacharjee  MK, Fine  DH, Thomson  VJ,     ( 1999 )

Direct selection of IS903 transposon insertions by use of a broad-host-range vector: isolation of catalase-deficient mutants of Actinobacillus actinomycetemcomitans.

Journal of bacteriology 181 (23)
PMID : 10572134  :   PMC  :   PMC103693    
Abstract >>
Transposon mutagenesis in bacteria generally requires efficient delivery of a transposon suicide vector to allow the selection of relatively infrequent transposition events. We have developed an IS903-based transposon mutagenesis system for diverse gram-negative bacteria that is not limited by transfer efficiency. The transposon, IS903phikan, carries a cryptic kan gene, which can be expressed only after successful transposition. This allows the stable introduction of the transposon delivery vector into the host. Generation of insertion mutants is then limited only by the frequency of transposition. IS903phikan was placed on an IncQ plasmid vector with the transposase gene located outside the transposon and expressed from isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoters. After transposase induction, IS903phikan insertion mutants were readily selected in Escherichia coli by their resistance to kanamycin. We used IS903phikan to isolate three catalase-deficient mutants of the periodontal pathogen Actinobacillus actinomycetemcomitans from a library of random insertions. The mutants display increased sensitivity to hydrogen peroxide, and all have IS903phikan insertions within an open reading frame whose predicted product is closely related to other bacterial catalases. Nucleotide sequence analysis of the catalase gene (designated katA) and flanking intergenic regions also revealed several occurrences of an 11-bp sequence that is closely related to the core DNA uptake signal sequence for natural transformation of Haemophilus influenzae. Our results demonstrate the utility of the IS903phikan mutagenesis system for the study of A. actinomycetemcomitans. Because IS903phikan is carried on a mobilizable, broad-host-range IncQ plasmid, this system is potentially useful in a variety of bacterial species.
KeywordMeSH Terms
Arabidopsis Proteins
DNA Transposable Elements
Genetic Vectors
29. Koga  T, Nakano  Y, Nezu  T, Yamashita  Y,     ( 1999 )

A novel NDP-6-deoxyhexosyl-4-ulose reductase in the pathway for the synthesis of thymidine diphosphate-D-fucose.

The Journal of biological chemistry 274 (24)
PMID : 10358040  :   DOI  :   10.1074/jbc.274.24.16933    
Abstract >>
The serotype-specific polysaccharide antigen of Actinobacillus actinomycetemcomitans Y4 (serotype b) consists of D-fucose and L-rhamnose. Thymidine diphosphate (dTDP)-D-fucose is the activated nucleotide sugar form of D-fucose, which has been identified as a constituent of structural polysaccharides in only a few bacteria. In this paper, we show that three dTDP-D-fucose synthetic enzymes are encoded by genes in the gene cluster responsible for the synthesis of serotype b-specific polysaccharide in A. actinomycetemcomitans. The first and second steps of the dTDP-D-fucose synthetic pathway are catalyzed by D-glucose-1-phosphate thymidylyltransferase and dTDP-D-glucose 4,6-dehydratase, which are encoded by rmlA and rmlB in the gene cluster, respectively. These two reactions are common to the well studied dTDP-L-rhamnose synthetic pathway. However, the enzyme catalyzing the last step of the dTDP-D-fucose synthetic pathway has never been reported. We identified the fcd gene encoding a dTDP-4-keto-6-deoxy-D-glucose reductase. After purifying the three enzymes, their enzymatic activities were analyzed by reversed-phase high performance liquid chromatography. In addition, nuclear magnetic resonance analysis and gas-liquid chromatography analysis proved that the fcd gene product converts dTDP-4-keto-6-deoxy-D-glucose to dTDP-D-fucose. Moreover, kinetic analysis of the enzyme indicated that the Km values for dTDP-4-keto-6-deoxy-D-glucose and NADPH are 97.3 and 28.7 microM, respectively, and that the enzyme follows the sequential mechanism. This paper is the first report on the dTDP-D-fucose synthetic pathway and dTDP-4-keto-6-deoxy-D-glucose reductase.
KeywordMeSH Terms
Genes, Bacterial
30. Shenker  BJ, Demuth  D, Chowhan  R, Miller  M,     ( 1999 )

Actinobacillus actinomycetemcomitans immunosuppressive protein is a member of the family of cytolethal distending toxins capable of causing a G2 arrest in human T cells.

Journal of immunology (Baltimore, Md. : 1950) 162 (8)
PMID : 10202019  :  
Abstract >>
We have previously shown that Actinobacillus actinomycetecomitans produces an immunosuppressive factor (ISF) capable of impairing human lymphocyte function by perturbing cell cycle progression. We now report that ISF is the product of the cdtB gene, one of three genes encoding the family of cytolethal distending toxins (Cdt). The ISF polypeptide exhibits >/=95% identity with Hemophilus ducreyi CdtB protein and
KeywordMeSH Terms
31. Zmuda  JL, Scannapieco  FA,     ( 1999 )

Identification and molecular analysis of rough-colony-specific outer membrane proteins of Actinobacillus actinomycetemcomitans.

Infection and immunity 67 (6)
PMID : 10338497  :   PMC  :   PMC96598    
Abstract >>
Actinobacillus actinomycetemcomitans, a gram-negative bacterium isolated from the human mouth, has been implicated in the pathogenesis of early-onset periodontitis. Primary isolates cultured from subgingival plaque exhibit an adherent, rough colony phenotype which spontaneously converts to a nonadherent, smooth phenotype upon in vitro subculture. The rough colony variant produces abundant fimbriae and autoaggregates, while the smooth colony variant is planktonic and produces scant fimbriae. To begin to understand the significance of colony variation in biofilm formation by A. actinomycetemcomitans, outer membrane protein profiles of four isogenic rough and smooth colony variants were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two proteins with relative molecular masses of 43 and 20 kDa were expressed by the rough colony variants exclusively. Expression of these proteins was not found to be dependent on growth phase, oxygen tension, or type of complex medium. N-terminal amino acid sequences of these proteins obtained by Edman degradation were compared with sequences from the University of Oklahoma A. actinomycetemcomitans genome database. Two contiguous open reading frames (ORFs) encoding proteins having sequence homology with these proteins were identified. The 43-kDa protein (RcpA [rough colony protein A]) was similar to precursor protein D of the general secretion pathway of gram-negative bacilli, while the 20-kDa protein (RcpB [rough colony protein B]) appeared to be unique. The genes encoding these proteins have been cloned from A. actinomycetemcomitans 283 and sequenced. A BLASTX (gapped BLAST) search of the surrounding ORFs revealed homology with other fimbria-related proteins. These data suggest that the genes encoding the 43-kDa (rcpA) and 20-kDa (rcpB) proteins may be functionally related to each other and to genes that may encode fimbria-associated proteins.
KeywordMeSH Terms
32. Izano  EA, Sadovskaya  I, Wang  H, Vinogradov  E, Ragunath  C, Ramasubbu  N, Jabbouri  S, Perry  MB, Kaplan  JB,     ( 2008 )

Poly-N-acetylglucosamine mediates biofilm formation and detergent resistance in Aggregatibacter actinomycetemcomitans.

Microbial pathogenesis 44 (1)
PMID : 17851029  :   DOI  :   10.1016/j.micpath.2007.08.004     PMC  :   PMC2253675    
Abstract >>
Clinical isolates of the periodontopathogen Aggregatibacter actinomycetemcomitans form matrix-encased biofilms on abiotic surfaces in vitro. A major component of the A. actinomycetemcomitans biofilm matrix is poly-beta-1,6-N-acetyl-d-glucosamine (PGA), a hexosamine-containing polysaccharide that mediates intercellular adhesion. In this report, we describe studies on the purification, structure, genetics and function of A. actinomycetemcomitans PGA. We found that PGA was very tightly attached to A. actinomycetemcomitans biofilm cells and could be efficiently separated from the cells only by phenol extraction. A. actinomycetemcomitans PGA copurified with LPS on a gel filtration column. (1)H NMR spectra of purified A. actinomycetemcomitans PGA were consistent with a structure containing a linear chain of N-acetyl-d-glucosamine residues in beta(1,6) linkage. Genetic analyses indicated that all four genes of the pgaABCD locus were required for PGA production in A. actinomycetemcomitans. PGA mutant strains still formed biofilms in vitro. Unlike wild-type biofilms, however, PGA mutant biofilms were sensitive to detachment by DNase I and proteinase K. Treatment of A. actinomycetemcomitans biofilms with the PGA-hydrolyzing enzyme dispersin B made them 3 log units more sensitive to killing by the cationic detergent cetylpyridinium chloride. Our findings suggest that PGA, extracellular DNA and proteinaceous adhesins all contribute to the structural integrity of the A. actinomycetemcomitans biofilm matrix.
KeywordMeSH Terms
33. Mayer  MP, Bueno  LC, Hansen  EJ,     ( 1999 )

Identification of a cytolethal distending toxin gene locus and features of a virulence-associated region in Actinobacillus actinomycetemcomitans.

Infection and immunity 67 (3)
PMID : 10024565  :   PMC  :   PMC96451    
Abstract >>
A genetic locus for a cytolethal distending toxin (CDT) was identified in a polymorphic region of the chromosome of Actinobacillus actinomycetemcomitans, a predominant oral pathogen. The locus was comprised of three open reading frames (ORFs) that had significant amino acid sequence similarity and more than 90% sequence identity to the cdtABC genes of some pathogenic Escherichia coli strains and Haemophilus ducreyi, respectively. Sonic extracts from recombinant E. coli, containing the A. actinomycetemcomitans ORFs, caused the distension and killing of Chinese hamster ovary cells characteristic of a CDT. Monoclonal antibodies made reactive with the CdtA, CdtB, and CdtC proteins of H. ducreyi recognized the corresponding gene products from the recombinant strain. CDT-like activities were no longer expressed by the recombinant strain when an OmegaKan-2 interposon was inserted into the cdtA and cdtB genes. Expression of the CDT-like activities in A. actinomycetemcomitans was strain specific. Naturally occurring expression-negative strains had large deletions within the region of the cdt locus. The cdtABC genes were flanked by an ORF (virulence plasmid protein), a partial ORF (integrase), and DNA sequences (bacteriophage integration site) characteristic of virulence-associated regions. These results provide evidence for a functional CDT in a human oral pathogen.
KeywordMeSH Terms
34. Tang  G, Ruiz  T, Barrantes-Reynolds  R, Mintz  KP,     ( 2007 )

Molecular heterogeneity of EmaA, an oligomeric autotransporter adhesin of Aggregatibacter (Actinobacillus) actinomycetemcomitans.

Microbiology (Reading, England) 153 (Pt 8)
PMID : 17660409  :   DOI  :   10.1099/mic.0.2007/005892-0    
Abstract >>
Adhesion of Aggregatibacter actinomycetemcomitans to extracellular matrix proteins is mediated by antennae-like surface structures composed of EmaA oligomers. EmaA is an outer-membrane protein orthologous to the autotransporter YadA, a virulence determinant of Yersinia. emaA was present in the 27 strains examined, covering the six serotypes of A. actinomycetemcomitans. Ten individual genotypes and three different forms of the protein (full-length, intermediate and truncated) were predicted. The prototypic, full-length EmaA (202 kDa) was only associated with serotypes b and c, which displayed antennae-like surface structures. These strains bound to collagen embedded in a 3D matrix. The intermediate form of EmaA (173 kDa) was exclusively associated with serotypes d and a, which contained a 279 aa in-frame deletion, as well as a different N-terminal head domain sequence. These differences modified the appearance of the EmaA structures on the cell surface but maintained collagen-binding activity. Strains containing the truncated form of EmaA had single or multiple substitutions, deletions or insertions in the sequences, which resulted in the absence of EmaA molecules on the outer membrane and loss of collagen-binding activity. Population structure analyses of this organism, based on emaA, indicated that serotypes b and c belonged to one subpopulation, which was independent of the other serotypes. The main divergence was found in the functional head domain. The conserved emaA genotype within serotypes suggests a stable clonal linkage between this autotransporter protein and other virulence determinants.
KeywordMeSH Terms
Genetic Variation
35. Haubek  D, Poulsen  K, Kilian  M,     ( 2007 )

Microevolution and patterns of dissemination of the JP2 clone of Aggregatibacter (Actinobacillus) actinomycetemcomitans.

Infection and immunity 75 (6)
PMID : 17353281  :   DOI  :   10.1128/IAI.01734-06     PMC  :   PMC1932881    
Abstract >>
The natural history, microevolution, and patterns of interindividual transmission and global dissemination of the JP2 clone of Aggregatibacter (Actinobacillus) actinomycetemcomitans were studied by population genetic analysis. The JP2 clone is strongly associated with aggressive periodontitis in adolescents of African descent and differs from other clones of the species by several genetic peculiarities, including a 530-bp deletion in the promoter region of the leukotoxin gene operon, which results in increased leukotoxic activity. Multilocus sequence analysis of 82 A. actinomycetemcomitans strains, 66 of which were JP2 clone strains collected over a period of more than 20 years, confirmed that there is a clonal population structure with evolutionary lineages corresponding to serotypes. Although genetically highly conserved, as shown by alignment of sequences of eight housekeeping genes, strains belonging to the JP2 clone had a number of point mutations, particularly in the pseudogenes hbpA and tbpA. Characteristic mutations allowed isolates from individuals from the Mediterranean area and from West Africa, including the Cape Verde Islands, to be distinguished. The patterns of mutations indicate that the JP2 clone initially emerged as a distinct genotype in the Mediterranean part of Africa approximately 2,400 years ago and subsequently spread to West Africa, from which it was transferred to the American continents during the transatlantic slave trade. The sustained exclusive colonization of individuals of African descent despite geographical separation for centuries suggests that the JP2 clone has a distinct host tropism. The colonization of family members by JP2 clone strains with unique point mutations provides strong evidence that there is intrafamilial transmission and suggests that dissemination of the JP2 clone is restricted to close contacts.
KeywordMeSH Terms
Evolution, Molecular
36. Yamada  H, Takashima  E, Konishi  K,     ( 2007 )

Molecular characterization of the membrane-bound quinol peroxidase functionally connected to the respiratory chain.

The FEBS journal 274 (3)
PMID : 17288564  :   DOI  :   10.1111/j.1742-4658.2006.05637.x    
Abstract >>
Here, we report for the first time quinol peroxidase (QPO), an enzyme that uses ubiquinol-1 as an electron donor for the reduction of H(2)O(2) to water. We purified QPO to > 90% purity from the membrane fraction of Actinobacillus actinomycetemcomitans. QPO is a 53.6-kDa protein that contains three heme c molecules. The qpo gene was predicted to encode a putative bacterial cytochrome c peroxidase with N-terminal extensions containing an additional potential heme c-binding motif. Although qpo has high sequence homology to bacterial cytochrome c peroxidases, QPO did not catalyze peroxidation in the presence of horse heart cytochrome c. In addition, the cytoplasmic membrane of A. actinomycetemcomitans had apparent QPO-dependent peroxidase activity in the presence of NADH or succinate, which are substrates for the respiratory chain. Based on these findings, we present a new mechanism for the scavenging of reactive oxygen species in which quinol in the respiratory chain is consumed.
KeywordMeSH Terms
Electron Transport
37. Crosby  JA, Kachlany  SC,     ( 2007 )

TdeA, a TolC-like protein required for toxin and drug export in Aggregatibacter (Actinobacillus) actinomycetemcomitans.

Gene 388 (1��2��)
PMID : 17116373  :   DOI  :   10.1016/j.gene.2006.10.004     PMC  :   PMC1831674    
Abstract >>
Aggregatibacter actinomycetemcomitans is an oral bacterium that causes localized aggressive periodontitis (LAP) and extra-oral infections such as sub-acute infective endocarditis. As part of its array of virulence factors, A. actinomycetemcomitans produces leukotoxin (LtxA), a member of the RTX family of toxins. LtxA kills human leukocytes and we have recently shown that the toxin is required for beta-hemolysis by A. actinomycetemcomitans on solid medium. In other RTX toxin-producing bacteria, an outer membrane channel-forming protein, TolC, is required for toxin secretion and drug export. We have identified an ORF in A. actinomycetemcomitans that encodes a putative protein having predicted structural properties similar to TolC. Inactivation of this ORF resulted in a mutant that was no longer beta-hemolytic and did not secrete LtxA. This mutant was significantly more sensitive to antimicrobial agents compared to the wild type strain and was unable to export the antimicrobial agent berberine. Thus, this ORF was named tdeA for "toxin and drug export". Examination of the DNA sequence surrounding tdeA revealed two upstream ORFs that encode proteins similar to the drug efflux proteins, MacA and MacB. Inactivation of macB in A. actinomycetemcomitans did not alter the drug sensitivity profile or the hemolytic activity of the mutant. The genes macA, macB and tdeA are organized as an operon and are constitutively expressed as a single transcript. These results show that A. actinomycetemcomitans indeed requires a TolC-like protein for LtxA secretion and that this protein, TdeA, also functions as part of a drug efflux system.
KeywordMeSH Terms
38. Cattoir  V, Lemenand  O, Avril  JL, Gaillot  O,     ( 2006 )

The sodA gene as a target for phylogenetic dissection of the genus Haemophilus and accurate identification of human clinical isolates.

International journal of medical microbiology : IJMM 296 (8)
PMID : 17049306  :   DOI  :   10.1016/j.ijmm.2006.06.005    
Abstract >>
The genus Haemophilus constitutes a heterogeneous group of Pasteurellaceae species, and conventional identification of isolates other than Haemophilus influenzae and Haemophilus parainfluenzae is often challenging. Here, simple colony-PCR and sequencing assays with the same pair of degenerate primers were used to characterize a 449- to 458-bp fragment (sodA(int)) internal to the sodA gene encoding the manganese-dependent superoxide dismutase in type strains of all 15 Haemophilus species and Actinobacillus actinomycetemcomitans. The topology of a sodA(int)-based phylogenetic tree was in general agreement with that inferred from the analysis of 16S rRNA and other housekeeping gene sequences, but allowed more confident delineation of the main clusters of species. The sodA(int) sequences showed a markedly higher divergence than those of the corresponding 16S rRNA genes, and 38 independent human clinical isolates were identified by comparing their sodA(int) sequence to those of the type species. Except for one Haemophilus aphrophilus strain, all isolates were unambiguously characterized in spite of a high intraspecific sodA(int) sequence diversity. This study provides a comprehensive sequence-based phylogenetic analysis of the entire genus Haemophilus, and confirms that sodA is a potent target for the identification of clinical isolates of Pasteurellaceae. This approach might contribute to the taxonomic reappraisal of this family, and to the development of diagnostic tools.
KeywordMeSH Terms
Phylogeny
39. Balashova  NV, Diaz  R, Balashov  SV, Crosby  JA, Kachlany  SC,     ( 2006 )

Regulation of Aggregatibacter (Actinobacillus) actinomycetemcomitans leukotoxin secretion by iron.

Journal of bacteriology 188 (24)
PMID : 17041062  :   DOI  :   10.1128/JB.01253-06     PMC  :   PMC1698250    
Abstract >>
The gram-negative oral and systemic pathogen Aggregatibacter (Actinobacillus) actinomycetemcomitans produces a leukotoxin (LtxA) that is a member of the RTX (repeats in toxin) family of secreted bacterial toxins. We have recently shown that LtxA has the ability to lyse erythrocytes, which results in a beta-hemolytic phenotype on Columbia blood agar. To determine if LtxA is regulated by iron, we examined beta-hemolysis under iron-rich and iron-limiting conditions. Beta-hemolysis was suppressed in the presence of FeCl3. In contrast, strong beta-hemolysis occurred in the presence of the iron chelator deferoxamine. We found that secretion of LtxA was completely inhibited by free iron, but expression of ltxA was not regulated by iron. Free chromium, cobalt, and magnesium did not affect LtxA secretion. Other LtxA-associated genes were not regulated by iron. Thus, iron appears to play an important role in the regulation of LtxA secretion in A. actinomycetemcomitans in a manner independent of gene regulation.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
40. Nørskov-Lauritsen  N, Kilian  M,     ( 2006 )

Reclassification of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Haemophilus paraphrophilus and Haemophilus segnis as Aggregatibacter actinomycetemcomitans gen. nov., comb. nov., Aggregatibacter aphrophilus comb. nov. and Aggregatibacter segnis comb. nov., and emended description of Aggregatibacter aphrophilus to include V factor-dependent and V factor-independent isolates.

International journal of systematic and evolutionary microbiology 56 (Pt 9)
PMID : 16957111  :   DOI  :   10.1099/ijs.0.64207-0     DOI  :   10.1099/ijs.0.64207-0    
Abstract >>
The aim of this study was to reinvestigate the relationships and the generic affiliations of the species Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Haemophilus paraphrophilus and Haemophilus segnis. The nicotinamide phosphoribosyltransferase gene (nadV) conferring V factor-independent growth was identified in Haemophilus aphrophilus. The gene encodes a polypeptide of 462 amino acids that shows 74.5 % amino acid sequence identity to the corresponding enzyme from Actinobacillus actinomycetemcomitans. Ten isolates of Haemophilus paraphrophilus all carried a nadV pseudogene. DNA from Haemophilus aphrophilus was able to transform Haemophilus paraphrophilus into the NAD-independent phenotype. The transformants carried a full-length nadV inserted in the former locus of the pseudogene. The DNA-DNA relatedness between the type strains of Haemophilus aphrophilus and Haemophilus paraphrophilus was 77 %. We conclude that the division into two species Haemophilus aphrophilus and Haemophilus paraphrophilus is not justified and that Haemophilus paraphrophilus should be considered a later heterotypic synonym of Haemophilus aphrophilus. Forty strains of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus and Haemophilus segnis were investigated by multilocus sequence analysis. The 40 strains form a monophyletic group clearly separate from other evolutionary lineages of the family Pasteurellaceae. We propose the transfer of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus and Haemophilus segnis to a new genus Aggregatibacter gen. nov. as Aggregatibacter actinomycetemcomitans comb. nov. (the type species; type strain ATCC 33384(T)=CCUG 13227(T)=CIP 52.106(T)=DSM 8324(T)=NCTC 9710(T)), Aggregatibacter aphrophilus comb. nov. (type strain ATCC 33389(T)=CCUG 3715(T)=CIP 70.73(T)=NCTC 5906(T)) and Aggregatibacter segnis comb. nov. (type strain HK316(T)=ATCC 33393(T)=CCUG 10787(T)=CCUG 12838(T)=CIP 103292(T)=NCTC 10977(T)). The species of the genus Aggregatibacter are independent of X factor and variably dependent on V factor for growth in vitro.
KeywordMeSH Terms
41. Perez  BA, Planet  PJ, Kachlany  SC, Tomich  M, Fine  DH, Figurski  DH,     ( 2006 )

Genetic analysis of the requirement for flp-2, tadV, and rcpB in Actinobacillus actinomycetemcomitans biofilm formation.

Journal of bacteriology 188 (17)
PMID : 16923904  :   DOI  :   10.1128/JB.00496-06     PMC  :   PMC1595400    
Abstract >>
The tad locus of Actinobacillus actinomycetemcomitans encodes a molecular transport system required for tenacious, nonspecific adherence to surfaces and formation of extremely strong biofilms. This locus is dedicated to the biogenesis of Flp pili, which are required for colonization and virulence. We have previously shown that 11 of the 14 tad locus genes are required for adherence and Flp pilus production. Here, we present genetic and phylogenetic analyses of flp-2, tadV, and rcpB genes in biofilm formation. We show that tadV, predicted to encode prepilin peptidase, is required for adherence. In contrast, targeted insertional inactivation of flp-2, a gene closely related to the prepillin gene flp-1, did not abrogate biofilm formation. Expression studies did not detect Flp2-T7 protein under standard laboratory conditions. We present phylogenetic data showing that there is no significant evidence for natural selection in the available flp-2 sequences from A. actinomycetemcomitans, suggesting that flp-2 does not play a significant role in the biology of this organism. Mutants with insertions at the 3' end of rcpB formed biofilms equivalent to wild-type A. actinomycetemcomitans. Surprisingly, 5' end chromosomal insertion mutants in rcpB were obtained only when a wild-type copy of the rcpB gene was provided in trans or when the Tad secretion system was inactivated. Together, our results strongly suggest that A. actinomycetemcomitans rcpB is essential in the context of a functional tad locus. These data show three different phenotypes for the three genes.
KeywordMeSH Terms
42. Balashova  NV, Crosby  JA, Al Ghofaily  L, Kachlany  SC,     ( 2006 )

Leukotoxin confers beta-hemolytic activity to Actinobacillus actinomycetemcomitans.

Infection and immunity 74 (4)
PMID : 16552030  :   DOI  :   10.1128/IAI.74.4.2015-2021.2006     PMC  :   PMC1418943    
Abstract >>
Actinobacillus actinomycetemcomitans is the etiologic agent of localized aggressive periodontitis, a rapidly progressing oral disease that occurs in adolescents. A. actinomycetemcomitans can also cause systemic disease, including infective endocarditis. In early work on A. actinomycetemcomitans workers concluded that this bacterium is not beta-hemolytic. More recent reports have suggested that A. actinomycetemcomitans does have the potential to be beta-hemolytic. While growing A. actinomycetemcomitans on several types of growth media, we noticed a beta-hemolytic reaction on media from one manufacturer. Beta-hemolysis occurred on Columbia agar from Accumedia with either sheep or horse blood, but not on similar media from other manufacturers. A surprising result was that mutants of A. actinomycetemcomitans defective for production of leukotoxin, a toxin that is reportedly highly specific for only human and primate white blood cells, are not beta-hemolytic. Purified leukotoxin was able to lyse sheep and human erythrocytes in vitro. This work showed that in contrast to the accepted view, A. actinomycetemcomitans leukotoxin can indeed destroy erythrocytes and that the production of this toxin results in beta-hemolytic colonies on solid medium. In light of these results, the diagnostic criteria for clinical identification of A. actinomycetemcomitans and potentially related bacteria should be reevaluated. Furthermore, in studies on A. actinomycetemcomitans leukotoxin workers should now consider this toxin's ability to destroy red blood cells.
KeywordMeSH Terms
43. Yamada  T, Komoto  J, Saiki  K, Konishi  K, Takusagawa  F,     ( 2006 )

Variation of loop sequence alters stability of cytolethal distending toxin (CDT): crystal structure of CDT from Actinobacillus actinomycetemcomitans.

Protein science : a publication of the Protein Society 15 (2)
PMID : 16434747  :   DOI  :   10.1110/ps.051790506     PMC  :   PMC2242449    
Abstract >>
Cytolethal distending toxin (CDT) secreted by Actinobacillus actinomycetemcomitans induces cell cycle arrest of cultured cells in the G2 phase. The crystal structure of the natural form of A. actinomycetemcomitans DCT (aCDT) has been determined at 2.4 A resolution. aCDT is a heterotrimer consisting of the three subunits, aCdtA, aCdtB, and aCdtC. Two crystallographically independent aCDTs form a dimer through interactions of the aCdtB subunits. The primary structure of aCDT has 94.3% identity with that of Haemophilus ducreyi CDT (hCDT), and the structure of aCDT is quite similar to that of hCDT reconstituted from the three subunits determined recently. However, the molecular packings in the crystal structures of aCDT and hCDT are quite different. A careful analysis of molecular packing suggests that variation of the amino acid residues in a nonconserved loop (181TSSPSSPERRGY192 of aCdtB and 181NSSSSPPERRVY192 of hCdtB) is responsible for the different oligomerization of very similar CDTs. The loop of aCdtB has a conformation to form a dimer, while the loop conformation of hCdtB prevents hCDT from forming a dimer. Although dimerization of aCDT does not affect toxic activity, it changes the stability of protein. aCDT rapidly aggregates and loses toxic activity in the absence of sucrose in a buffered solution, while hCDT is stable and retains toxic activity. Another analysis of crystal structures of aCDT and hCDT suggests that the receptor contact area is in the deep groove between CdtA and CdtC, and the characteristic "aromatic patch" on CdtA.
KeywordMeSH Terms
44. Rhodes  ER, Tomaras  AP, McGillivary  G, Connerly  PL, Actis  LA,     ( 2005 )

Genetic and functional analyses of the Actinobacillus actinomycetemcomitans AfeABCD siderophore-independent iron acquisition system.

Infection and immunity 73 (6)
PMID : 15908408  :   DOI  :   10.1128/IAI.73.6.3758-3763.2005     PMC  :   PMC1111845    
Abstract >>
The Actinobacillus actinomycetemcomitans afeABCD iron transport system, the expression of which is controlled by iron and Fur, was identified in three different isolates. The protein products of this locus are related to bacterial ABC transporters involved in metal transport. Transformation of the Escherichia coli 1017 iron acquisition mutant with a plasmid harboring afeABCD promoted cell growth under iron-chelated conditions. However, insertion disruption of each of the afeABCD coding regions abolished this growth-relieving effect. The replacement of the parental afeA allele with the derivative afeA::EZ::TN drastically reduced the ability of A. actinomycetemcomitans cells to grow under iron-chelated conditions.
KeywordMeSH Terms
45. Wang  Y, Liu  A, Chen  C,     ( 2005 )

Genetic basis for conversion of rough-to-smooth colony morphology in Actinobacillus actinomycetemcomitans.

Infection and immunity 73 (6)
PMID : 15908406  :   DOI  :   10.1128/IAI.73.6.3749-3753.2005     PMC  :   PMC1111812    
Abstract >>
The basis of the rough-to-smooth conversion of Actinobacillus actinomycetemcomitans was examined. Smooth variants often contained mutations at the flp promoter region. Replacing the mutated flp promoter with the wild-type promoter restored the rough phenotype. The expression level of the flp promoter was approximately 100-fold lower in smooth than in rough strains. Mutations of the flp promoter are a cause of the rough-to-smooth conversion.
KeywordMeSH Terms
46. Ramasubbu  N, Thomas  LM, Ragunath  C, Kaplan  JB,     ( 2005 )

Structural analysis of dispersin B, a biofilm-releasing glycoside hydrolase from the periodontopathogen Actinobacillus actinomycetemcomitans.

Journal of molecular biology 349 (3)
PMID : 15878175  :   DOI  :   10.1016/j.jmb.2005.03.082    
Abstract >>
Bacteria in a biofilm are enmeshed in a self-synthesized extracellular polysaccharide matrix that holds the bacteria together in a mass and firmly attaches the bacterial mass to the underlying surface. A major component of the extracellular polysaccharide matrix in several phylogenetically diverse bacteria is PGA, a linear polymer of N-acetylglucosamine residues in beta(1,6)-linkage. PGA is produced by the Gram-negative periodontopathogen Actinobacillus actinomycetemcomitans as well as by the Gram-positive device-associated pathogen Staphylococcus epidermidis. We recently reported that A.actinomycetemcomitans produces a soluble glycoside hydrolase named dispersin B, which degrades PGA. Here, we present the crystal structure of dispersin B at 2.0A in complex with a glycerol and an acetate ion at the active site. The enzyme crystallizes in the orthorhombic space group C222(1) with cell dimensions a=41.02A, b=86.13A, c=185.77A. The core of the enzyme consists a (beta/alpha)(8) barrel topology similar to other beta-hexosaminidases but significant differences exist in the arrangement of loops hovering in the vicinity of the active site. The location and interactions of the glycerol and acetate moieties in conjunction with the sequence analysis suggest that dispersin B cleaves beta(1,6)-linked N-acetylglucosamine polymer using a catalytic machinery similar to other family 20 hexosaminidases which cleave beta(1,4)-linked N-acetylglucosamine residues.
KeywordMeSH Terms
47. Wang  Y, Chen  C,     ( 2005 )

Mutation analysis of the flp operon in Actinobacillus actinomycetemcomitans.

Gene 351 (N/A)
PMID : 15837433  :   DOI  :   10.1016/j.gene.2005.02.010    
Abstract >>
Fresh clinical isolates of the periodontal pathogen Actinobacillus actinomycetemcomitans live as autoaggregates, in which cells are densely packed and embedded in an extracellular matrix composed of bundled fimbriae, exopolymers, and vesicles. The expression of fimbriae is known to be determined by the flp operon of 14 genes, flp-1-flp-2-tadV-rcpCAB-tadZABCDEFG. We generated mutations of each gene of this operon in A. actinomycetemcomitans strain D7S. All mutants expressed some changes in the production of extracellular matrix materials that include vesicles, exopolymers, and fimbriae. The expression of fimbriae required the function of flp-1, rcpA, rcpB, tadB, tadD, tadE, and tadF. Mutants of flp-2, tadZ, tadA, tadC, and tadG expressed reduced levels of fimbriae, or fimbriae that had different gross appearance. Importantly, the expression of the non-fimbrial matrix materials was affected by all mutations, suggesting that the flp operon was involved in production of these materials. The flp locus apparently plays a central role in autoaggregation of A. actinomycetemcomitans, which may be the primary survival strategy of this bacterium in vivo.
KeywordMeSH Terms
Mutation
48. Fine  DH, Velliyagounder  K, Furgang  D, Kaplan  JB,     ( 2005 )

The Actinobacillus actinomycetemcomitans autotransporter adhesin Aae exhibits specificity for buccal epithelial cells from humans and old world primates.

Infection and immunity 73 (4)
PMID : 15784534  :   DOI  :   10.1128/IAI.73.4.1947-1953.2005     PMC  :   PMC1087452    
Abstract >>
Cells of the gram-negative periodontopathogen Actinobacillus actinomycetemcomitans express a surface-exposed, outer membrane autotransporter protein, designated Aae, which has been implicated in epithelial cell binding. We constructed a mutant strain of A. actinomycetemcomitans that contained a transposon insertion in the Aae structural gene (aae) and tested the mutant to determine its ability to bind to buccal epithelial cells (BECs) isolated from healthy volunteers. Significantly fewer mutant cells than wild-type cells bound to BECs. A broad-host-range plasmid that contained an intact aae gene driven by a heterologous tac promoter restored the ability of the mutant strain to bind to BECs at wild-type levels. This plasmid also conferred upon Escherichia coli the ability to express the Aae protein on its surface and to bind to human BECs. Aae-expressing E. coli also bound to BECs isolated from six Old World primates but not to BECs isolated from four New World primates or nine other nonprimate mammals, as well as to human gingival epithelial cells but not to human pharyngeal, palatal, tongue, bronchial, or cervical epithelial cells. Our findings indicate that Aae mediates binding of A. actinomycetemcomitans to BECs from humans and Old World primates and that this process may contribute to the host range specificity and tissue tropism exhibited by this bacterium.
KeywordMeSH Terms
49. Nørskov-Lauritsen  N, Bruun  B, Kilian  M,     ( 2005 )

Multilocus sequence phylogenetic study of the genus Haemophilus with description of Haemophilus pittmaniae sp. nov.

International journal of systematic and evolutionary microbiology 55 (Pt 1)
PMID : 15653917  :   DOI  :   10.1099/ijs.0.63325-0    
Abstract >>
The phylogeny of human isolates of Haemophilus species was estimated based on partial sequences of four separate housekeeping genes. The clustering of each set of sequences was in accordance with speciation of the strains with few exceptions: of 108 gene fragments examined, only three appeared to have been subject to recombination events across the species barrier. Housekeeping gene similarity supported previous DNA-DNA hybridization data for the genus rather than the phylogeny inferred from 16S rRNA gene sequence comparison. The similarity of sequences of Haemophilus parainfluenzae with those of Haemophilus influenzae suggested preservation of the former species in the genus Haemophilus. Three strains representing a novel taxon were unique with respect to the four investigated gene loci. 16S rRNA gene sequence analysis suggested that this taxon belonged to the Parainfluenzae cluster. DNA-DNA hybridization data supported this generic placement. Nine strains of the novel taxon were available for analysis. They were distinct from representatives of all current species of the genus Haemophilus by conventional phenotypic characterization. Genotypic and phenotypic data show that the strains merit recognition as a novel species of Haemophilus. The name Haemophilus pittmaniae sp. nov. is proposed, with HK 85T (=CCUG 48703T=NCTC 13334T) as the type strain.
KeywordMeSH Terms
Phylogeny
Sequence Analysis, DNA
50. Kaplan  JB, Velliyagounder  K, Ragunath  C, Rohde  H, Mack  D, Knobloch  JK, Ramasubbu  N,     ( 2004 )

Genes involved in the synthesis and degradation of matrix polysaccharide in Actinobacillus actinomycetemcomitans and Actinobacillus pleuropneumoniae biofilms.

Journal of bacteriology 186 (24)
PMID : 15576769  :   DOI  :   10.1128/JB.186.24.8213-8220.2004     PMC  :   PMC532409     DOI  :   10.1128/JB.186.24.8213-8220.2004     PMC  :   PMC532409    
Abstract >>
Biofilms are composed of bacterial cells embedded in an extracellular polysaccharide matrix. A major component of the Escherichia coli biofilm matrix is PGA, a linear polymer of N-acetyl-D-glucosamine residues in beta(1,6) linkage. PGA mediates intercellular adhesion and attachment of cells to abiotic surfaces. In this report, we present genetic and biochemical evidence that PGA is also a major matrix component of biofilms produced by the human periodontopathogen Actinobacillus actinomycetemcomitans and the porcine respiratory pathogen Actinobacillus pleuropneumoniae. We also show that PGA is a substrate for dispersin B, a biofilm-releasing glycosyl hydrolase produced by A. actinomycetemcomitans, and that an orthologous dispersin B enzyme is produced by A. pleuropneumoniae. We further show that A. actinomycetemcomitans PGA cross-reacts with antiserum raised against polysaccharide intercellular adhesin, a staphylococcal biofilm matrix polysaccharide that is genetically and structurally related to PGA. Our findings confirm that PGA functions as a biofilm matrix polysaccharide in phylogenetically diverse bacterial species and suggest that PGA may play a role in intercellular adhesion and cellular detachment and dispersal in A. actinomycetemcomitans and A. pleuropneumoniae biofilms.
KeywordMeSH Terms
Genes, Bacterial
Genes, Bacterial
51. Mintz  KP,     ( 2004 )

Identification of an extracellular matrix protein adhesin, EmaA, which mediates the adhesion of Actinobacillus actinomycetemcomitans to collagen.

Microbiology (Reading, England) 150 (Pt 8)
PMID : 15289564  :   DOI  :   10.1099/mic.0.27110-0    
Abstract >>
Actinobacillus actinomycetemcomitans is an aetiologic agent in the development of periodontal and some systemic diseases in humans. This pathogen localizes to the underlying connective tissue of the oral cavity in individuals with periodontal disease. The adhesion of A. actinomycetemcomitans to extracellular matrix components of the connective tissue prompted this study to identify gene products mediating the interaction of A. actinomycetemcomitans to these molecules. A transposon mutagenesis system was optimized for use in A. actinomycetemcomitans and used to generate an insertional mutant library. A total of 2300 individual insertion transposon mutants were screened for changes in the adhesion to collagen and fibronectin. Mutants were identified which exhibited the following phenotypes: a decrease in collagen binding; a decrease in fibronectin binding; a decrease in binding to both proteins; and an increase in binding to both collagen and fibronectin. The identification of mutants defective in adhesion to the individual proteins indicates that distinct adhesins are expressed by this organism. Molecular analysis of these mutants implicated 11 independent loci in protein adhesion. One gene, emaA, is likely to encode a direct mediator of collagen adhesion, based on predicted protein features homologous to the collagen-binding protein YadA of Yersinia enterocolitica. EmaA was localized to the outer membrane, as expected for an adhesin. Reduction in fibronectin adhesion appeared to be influenced by abrogation of proteins involved in molybdenum-cofactor biosynthesis. Several other loci identified as reducing or increasing adhesion to both collagen and fibronectin are suggested to be involved in regulatory cascades that promote or repress expression of collagen and fibronectin adhesins. Collectively, the results support the hypothesis that A. actinomycetemcomitans host colonization involves afimbrial adhesins for extracellular matrix proteins, and that the expression of adhesion is modulated by global regulatory mechanisms.
KeywordMeSH Terms
52. Korczak  B, Christensen  H, Emler  S, Frey  J, Kuhnert  P,     ( 2004 )

Phylogeny of the family Pasteurellaceae based on rpoB sequences.

International journal of systematic and evolutionary microbiology 54 (Pt 4)
PMID : 15280320  :   DOI  :   10.1099/ijs.0.03043-0    
Abstract >>
Sequences of the gene encoding the beta-subunit of the RNA polymerase (rpoB) were used to delineate the phylogeny of the family Pasteurellaceae. A total of 72 strains, including the type strains of the major described species as well as selected field isolates, were included in the study. Selection of universal rpoB-derived primers for the family allowed straightforward amplification and sequencing of a 560 bp fragment of the rpoB gene. In parallel, 16S rDNA was sequenced from all strains. The phylogenetic tree obtained with the rpoB sequences reflected the major branches of the tree obtained with the 16S rDNA, especially at the genus level. Only a few discrepancies between the trees were observed. In certain cases the rpoB phylogeny was in better agreement with DNA-DNA hybridization studies than the phylogeny derived from 16S rDNA. The rpoB gene is strongly conserved within the various species of the family of Pasteurellaceae. Hence, rpoB gene sequence analysis in conjunction with 16S rDNA sequencing is a valuable tool for phylogenetic studies of the Pasteurellaceae and may also prove useful for reorganizing the current taxonomy of this bacterial family.
KeywordMeSH Terms
53. Hill  JE, Penny  SL, Crowell  KG, Goh  SH, Hemmingsen  SM,     ( 2004 )

cpnDB: a chaperonin sequence database.

Genome research 14 (8)
PMID : 15289485  :   DOI  :   10.1101/gr.2649204     PMC  :   PMC509277    
Abstract >>
Type I chaperonins are molecular chaperones present in virtually all bacteria, some archaea and the plastids and mitochondria of eukaryotes. Sequences of cpn60 genes, encoding 60-kDa chaperonin protein subunits (CPN60, also known as GroEL or HSP60), are useful for phylogenetic studies and as targets for detection and identification of organisms. Conveniently, a 549-567-bp segment of the cpn60 coding region can be amplified with universal PCR primers. Here, we introduce cpnDB, a curated collection of cpn60 sequence data collected from public databases or generated by a network of collaborators exploiting the cpn60 target in clinical, phylogenetic, and microbial ecology studies. The growing database currently contains approximately 2000 records covering over 240 genera of bacteria, eukaryotes, and archaea. The database also contains over 60 sequences for the archaeal Type II chaperonin (thermosome, a homolog of eukaryotic cytoplasmic chaperonin) from 19 archaeal genera. As the largest curated collection of sequences available for a protein-encoding gene, cpnDB provides a resource for researchers interested in exploiting the power of cpn60 as a diagnostic or as a target for phylogenetic or microbial ecology studies, as well as those interested in broader subjects such as lateral gene transfer and codon usage. We built cpnDB from open source tools and it is available at http://cpndb.cbr.nrc.ca.
KeywordMeSH Terms
54. Li  L, Matevski  D, Aspiras  M, Ellen  RP, Lépine  G,     ( 2004 )

Two epithelial cell invasion-related loci of the oral pathogen Actinobacillus actinomycetemcomitans.

Oral microbiology and immunology 19 (1)
PMID : 14678470  :  
Abstract >>
Two invasion-related loci, apiA and the two-gene operon apiBC, were isolated from the oral pathogen Actinobacillus actinomycetemcomitans UT32. apiA encodes a 32.5 kDa protein that migrates on SDS-PAGE as a 101 kDa protein as detected by Western blot analysis or silver staining of an outer membrane-enriched fraction of Escherichia coli transformants. E. coli expressing ApiA have a different phenotype than the host vector, in broth and on solid media, and a colony morphology that resembles that of fresh A. actinomycetemcomitans isolates. These E. coli transformants bound to chicken collagen type II, human collagen type II, III, V and fibronectin. apiB and apiC encode proteins of 130.1 and 70.6 kDa, respectively. ApiBC conferred on E. coli a slightly enhanced ability to bind to collagen type III. ApiA- and ApiB-deficient mutants were constructed in A. actinomycetemcomitans. The ApiB-mutant had 4-fold diminished invasion of KB cells; the ApiA-mutant had increased invasion. Both loci were found in all A. actinomycetemcomitans strains, although polymorphism was detected only for apiBC. The deduced sequences of these invasion-related proteins are homologous to members of the YadA adhesin/invasin family.
KeywordMeSH Terms
55. Cobbe  N, Heck  MM,     ( 2004 )

The evolution of SMC proteins: phylogenetic analysis and structural implications.

Molecular biology and evolution 21 (2)
PMID : 14660695  :   DOI  :   10.1093/molbev/msh023    
Abstract >>
The SMC proteins are found in nearly all living organisms examined, where they play crucial roles in mitotic chromosome dynamics, regulation of gene expression, and DNA repair. We have explored the phylogenetic relationships of SMC proteins from prokaryotes and eukaryotes, as well as their relationship to similar ABC ATPases, using maximum-likelihood analyses. We have also investigated the coevolution of different domains of eukaryotic SMC proteins and attempted to account for the evolutionary patterns we have observed in terms of available structural data. Based on our analyses, we propose that each of the six eukaryotic SMC subfamilies originated through a series of ancient gene duplication events, with the condensins evolving more rapidly than the cohesins. In addition, we show that the SMC5 and SMC6 subfamily members have evolved comparatively rapidly and suggest that these proteins may perform redundant functions in higher eukaryotes. Finally, we propose a possible structure for the SMC5/SMC6 heterodimer based on patterns of coevolution.
KeywordMeSH Terms
Evolution, Molecular
Phylogeny
56. Fong  KP, Tang  HY, Brown  AC, Kieba  IR, Speicher  DW, Boesze-Battaglia  K, Lally  ET,     ( 2011 )

Aggregatibacter actinomycetemcomitans leukotoxin is post-translationally modified by addition of either saturated or hydroxylated fatty acyl chains.

Molecular oral microbiology 26 (4)
PMID : 21729247  :   DOI  :   10.1111/j.2041-1014.2011.00617.x     PMC  :   PMC3404814    
Abstract >>
Aggregatibacter actinomycetemcomitans, a common inhabitant of the human upper aerodigestive tract, produces a repeat in toxin (RTX), leukotoxin (LtxA). The LtxA is transcribed as a 114-kDa inactive protoxin with activation being achieved by attachment of short chain fatty acyl groups to internal lysine residues. Methyl esters of LtxA that were isolated from A. actinomycetemcomitans strains JP2 and HK1651 and subjected to gas chromatography/mass spectrometry contained palmitoyl (C16:0, 27-29%) and palmitolyl (C16:1 cis �G9, 43-44%) fatty acyl groups with smaller quantities of myristic (C14:0, 14%) and stearic (C18:0, 12-14%) fatty acids. Liquid chromatography/mass spectrometry of tryptic peptides from acylated and unacylated recombinant LtxA confirmed that Lys(562) and Lys(687) are the sites of acyl group attachment. During analysis of recombinant LtxA peptides, we observed peptide spectra that were not observed as part of the RTX acylation schemes of either Escherichia coli�\-hemolysin or Bordetella pertussis cyclolysin. Mass calculations of these spectra suggested that LtxA was also modified by the addition of monohydroxylated forms of C14 and C16 acyl groups. Multiple reaction monitoring mass spectrometry identified hydroxymyristic and hydroxypalmitic acids in wild-type LtxA methyl esters. Single or tandem replacement of Lys(562) and Lys(687) with Arg blocks acylation, resulting in a >75% decrease in cytotoxicity when compared with wild-type toxin, suggesting that these post-translational modifications are playing a critical role in LtxA-mediated target cell cytotoxicity.
KeywordMeSH Terms
Protein Processing, Post-Translational
57. Potron  A, Mainardi  JL, Podglajen  I, Meunier  F, Sanson-le Pors  MJ, Berçot  B,     ( 2010 )

Recurrent infective endocarditis due to Aggregatibacter actinomycetemcomitans: reinfection or relapse?

Journal of medical microbiology 59 (Pt 12)
PMID : 20724510  :   DOI  :   10.1099/jmm.0.024380-0    
Abstract >>
Aggregatibacter actinomycetemcomitans is commonly part of the normal microflora of the human upper respiratory tract. It has been implicated in periodontal disease and various infections, particularly endocarditis. We report here what we believe to be the first case of recurrent infective endocarditis due to A. actinomycetemcomitans in a 44-year-old woman occurring 5 years after the initial episode. Genomic analysis proved that the strains were closely related. Despite efficient antibiotic treatment, surgery was necessary for recovery.
KeywordMeSH Terms
58. Fine  DH, Kaplan  JB, Furgang  D, Karched  M, Velliyagounder  K, Yue  G,     ( 2010 )

Mapping the epithelial-cell-binding domain of the Aggregatibacter actinomycetemcomitans autotransporter adhesin Aae.

Microbiology (Reading, England) 156 (Pt 11)
PMID : 20688817  :   DOI  :   10.1099/mic.0.037606-0     PMC  :   PMC3090143    
Abstract >>
The Gram-negative periodontopathogen Aggregatibacter actinomycetemcomitans (Aa) binds selectively to buccal epithelial cells (BECs) of human and Old World primates by means of the outer-membrane autotransporter protein Aae. We speculated that the exposed N-terminal portion of the passenger domain of Aae would mediate binding to BECs. By using a series of plasmids that express full-length or truncated Aae proteins in Escherichia coli, we found that the BEC-binding domain of Aae was located in the N-terminal surface-exposed region of the protein, specifically in the region spanning amino acids 201-284 just upstream of the repeat region within the passenger domain. Peptides corresponding to amino acids 201-221, 222-238 and 201-240 were synthesized and tested for their ability to reduce Aae-mediated binding to BECs based on results obtained with truncated Aae proteins expressed in E. coli. BEC-binding of E. coli expressing Aae was reduced by as much as 50 % by pre-treatment of BECs with a 40-mer peptide (201-240; P40). Aae was also shown to mediate binding to cultured human epithelial keratinocytes (TW2.6), OBA9 and TERT, and endothelial (HUVEC) cells. Pre-treatment of epithelial cells with P40 resulted in a dose-dependent reduction in binding and reduced the binding of both full-length and truncated Aae proteins expressed in E. coli, as well as Aae expressed in Aa. Fluorescently labelled P40 peptides reacted in a dose-dependent manner with BEC receptors. We propose that these proof-of-principle experiments demonstrate that peptides can be designed to interfere with Aa binding mediated by host-cell receptors specific for Aae adhesins.
KeywordMeSH Terms
Bacterial Adhesion
59. Lally  ET, Golub  EE, Kieba  IR, Taichman  NS, Decker  S, Berthold  P, Gibson  CW, Demuth  DR, Rosenbloom  J,     ( 1991 )

Structure and function of the B and D genes of the Actinobacillus actinomycetemcomitans leukotoxin complex.

Microbial pathogenesis 11 (2)
PMID : 1961107  :  
Abstract >>
The Actinobacillus actinomycetemcomitans leukotoxin gene complex, consisting of four genes, has been cloned and the sequence of the AaLtC and AaLtA genes reported. The present paper details the sequences of the AaLtB and AaLtD genes which, like AaLtC and AaLTA, are also homologues of genes found in other cytolytic toxin complexes of several other Gram-negative bacterial pathogens. When tested in a recombinant expression system, the AaLtB and/or AaLtD genes are required for the translocation and insertion of the A. actinomycetemcomitans leukotoxin (AaLtA) into the cell membrane of Escherichia coli.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
60. Kokeguchi  S, Kato  K, Nishimura  F, Kurihara  H, Murayama  Y,     ( 1991 )

Isolation and partial characterization of a 39 kDa major outer membrane protein of Actinobacillus actinomycetemcomitans Y4.

FEMS microbiology letters 61 (1)
PMID : 2004699  :   DOI  :   10.1016/0378-1097(91)90018-6    
Abstract >>
The outer membrane fractions of Actinobacillus actinomycetemcomitans, which were extracted from whole cells with cetyl trimethyl ammonium bromide and CaCl2, contained four major outer membrane proteins (MOMP) of 39, 37, 36 and 30 kDa. The 39 kDa MOMP of A. actinomycetemcomitans was sequentially purified by extraction with Zwittergent 3-14 detergent, anion-exchange chromatography and gel filtration chromatography. Analysis of amino acid composition and N-terminal amino acid sequence of 20 residues of purified 39 kDa MOMP was performed. Although some of the periodontitis patient sera reacted strongly with 39 kDa and 30 kDa MOMP in crude outer membrane fractions, purified 39 kDa MOMP showed decreased immunoreactivity with the human sera.
KeywordMeSH Terms
61. Balashova  NV, Shah  C, Patel  JK, Megalla  S, Kachlany  SC,     ( 2009 )

Aggregatibacter actinomycetemcomitans LtxC is required for leukotoxin activity and initial interaction between toxin and host cells.

Gene 443 (1��2��)
PMID : 19450669  :   DOI  :   10.1016/j.gene.2009.05.002    
Abstract >>
Aggregatibacter actinomycetemcomitans is a human pathogen that produces the RTX toxin (repeats in toxin), leukotoxin (LtxA). Based on other RTX toxin systems, the product of ltxC, the first gene of the ltx operon, is predicted to be involved in fatty acid modification of LtxA. To determine the function of ltxC in A. actinomycetemcomitans, we generated an ltxC mutation in the highly leukotoxic strain JP2N using random mutagenesis. The toxin from the ltxC mutant (LtxA(ltxC)) was expressed and secreted into the cell culture supernatant but could not lyse human leukocytes or erythrocytes. Mass spectrometric analysis of LtxA(ltxC) and LtxA from strain JP2N (LtxA(wt)) revealed two peptides that differed and this data suggests that two internal lysine residues of LtxA from the wild-type strain are modified. In blocking experiments, pre-treatment of cells with LtxA(ltxC) was unable to prevent LtxA(wt) from killing cells. Furthermore, in contrast to LtxA(wt), LtxA(ltxC) did not cause an increase in intracellular calcium levels in human leukocytes. Taken together, our data show that ltxC is required for full activity and modification of LtxA in A. actinomycetemcomitans and that modification is important for initial binding of toxin to host cells, as defined by an increase in intracellular calcium levels.
KeywordMeSH Terms
Host-Pathogen Interactions
62. Xu  Y, Sim  SH, Nam  KH, Jin  XL, Kim  HM, Hwang  KY, Lee  K, Ha  NC,     ( 2009 )

Crystal structure of the periplasmic region of MacB, a noncanonic ABC transporter.

Biochemistry 48 (23)
PMID : 19432486  :   DOI  :   10.1021/bi900415t    
Abstract >>
MacB is a noncanonic ABC-type transporter within Gram-negative bacteria, which is responsible both for the efflux of macrolide antibiotics and for the secretion of heat-stable enterotoxin II. In Escherichia coli, MacB requires the membrane fusion protein MacA and the multifunctional outer membrane channel TolC to pump substrates to the external medium. Sequence analysis of MacB suggested that MacB has a relatively large periplasmic region. To gain insight into how MacB assembles with MacA and TolC, we determined the crystal structure of the periplasmic region of Actinobacillus actinomycetemcomitans MacB. Fold matching program reveals that parts of the MacB periplasmic region have structural motifs in common with the RND-type transporter AcrB. Since it behaved as a monomer in solution, our finding is consistent with the dimeric nature of full-length MacB, providing an insight into the assembly in the tripartite efflux pump.
KeywordMeSH Terms
63. Ohta  H, Kato  K, Kokeguchi  S, Hara  H, Fukui  K, Murayama  Y,     ( 1991 )

Nuclease-sensitive binding of an Actinobacillus actinomycetemcomitans leukotoxin to the bacterial cell surface.

Infection and immunity 59 (12)
PMID : 1937819  :   PMC  :   PMC259083    
Abstract >>
A leukotoxin of Actinobacillus actinomycetemcomitans 301-b was solubilized from cell-associated membrane vesicles by treatment with externally added DNase and RNase and was further purified by a procedure which included ammonium sulfate fractionation, gel filtration chromatography, and ion-exchange chromatography. The purified toxin had a molecular mass of 113,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a high isoelectric point (approximately 8.8). From these characteristics, it was to be expected that the membrane vesicle toxin was almost identical to the leukotoxin extracted with polymyxin B in an earlier study (C.-C. Tsai, B. J. Shenker, J. M. DiRienzo, D. Malamud, and N. S. Taichman, Infect, Immun. 43:700-705, 1984). The treatment with DNase and RNase was also highly effective for solubilizing the leukotoxin directly from whole cells, suggesting that the toxin is secreted extracellularly but retained in nucleic acids on the outermost surface of bacterial cells.
KeywordMeSH Terms
64. Kim  S, Yum  S, Jo  WS, Lee  BL, Jeong  MH, Ha  NC,     ( 2008 )

Expression and biochemical characterization of the periplasmic domain of bacterial outer membrane porin TdeA.

Journal of microbiology and biotechnology 18 (5)
PMID : 18633280  :  
Abstract >>
TolC is an outer membrane porin protein and an essential component of drug efflux and type-I secretion systems in Gram-negative bacteria. TolC comprises a periplasmic alpha- helical barrel domain and a membrane-embedded beta-barrel domain. TdeA, a functional and structural homolog of TolC, is required for toxin and drug export in the pathogenic oral bacterium Actinobacillus actinomycetemcomitans. Here, we report the expression of the periplasmic domain of TdeA as a soluble protein by substitution of the membraneembedded domain with short linkers, which enabled us to purify the protein in the absence of detergent. We confirmed the structural integrity of the TdeA periplasmic domain by size-exclusion chromatography, circular dichroism spectroscopy, and electron microscopy, which together showed that the periplasmic domain of the TolC protein family can fold correctly on its own. We further demonstrated that the periplasmic domain of TdeA interacts with peptidoglycans of the bacterial cell wall, which supports the idea that completely folded TolC family proteins traverse the peptidoglycan layer to interact with inner membrane transporters.
KeywordMeSH Terms
Gene Expression
65. Dileepan  T, Kachlany  SC, Balashova  NV, Patel  J, Maheswaran  SK,     ( 2007 )

Human CD18 is the functional receptor for Aggregatibacter actinomycetemcomitans leukotoxin.

Infection and immunity 75 (10)
PMID : 17635865  :   DOI  :   10.1128/IAI.00314-07     PMC  :   PMC2044523    
Abstract >>
Aggregatibacter (Actinobacillus) actinomycetemcomitans is the causative organism of localized aggressive periodontitis, a rapidly progressing degenerative disease of the gingival and periodontal ligaments, and is also implicated in causing subacute infective endocarditis in humans. The bacterium produces a variety of virulence factors, including an exotoxic leukotoxin (LtxA) that is a member of the repeats-in-toxin (RTX) family of bacterial cytolysins. LtxA exhibits a unique specificity to macrophages and polymorphonuclear cells of humans and other primates. Human lymphocyte function-associated antigen 1 (LFA-1) has been implicated as the putative receptor for LtxA. Human LFA-1 comprises the CD11a and CD18 subunits. It is not clear, however, which of its subunits serves as the functional receptor that confers species-specific susceptibility to LtxA. Here we demonstrate that the human CD18 is the receptor for LtxA based on experiments performed with chimeric beta2-integrins recombinantly expressed in a cell line that is resistant to LtxA effects. In addition, we show that the cysteine-rich tandem repeats encompassing integrin-epidermal growth factor-like domains 2, 3, and 4 of the extracellular region of human CD18 are critical for conferring susceptibility to LtxA-induced biological effects.
KeywordMeSH Terms
66. Balashova  NV, Park  DH, Patel  JK, Figurski  DH, Kachlany  SC,     ( 2007 )

Interaction between leukotoxin and Cu,Zn superoxide dismutase in Aggregatibacter actinomycetemcomitans.

Infection and immunity 75 (9)
PMID : 17635874  :   DOI  :   10.1128/IAI.00288-07     PMC  :   PMC1951164    
Abstract >>
Aggregatibacter (Actinobacillus) actinomycetemcomitans is a gram-negative oral pathogen that is the etiologic agent of localized aggressive periodontitis and systemic infections. A. actinomycetemcomitans produces leukotoxin (LtxA), which is a member of the RTX (repeats in toxin) family of secreted bacterial toxins and is known to target human leukocytes and erythrocytes. To better understand how LtxA functions as a virulence factor, we sought to detect and study potential A. actinomycetemcomitans proteins that interact with LtxA. We found that Cu,Zn superoxide dismutase (SOD) interacts specifically with LtxA. Cu,Zn SOD was purified from A. actinomycetemcomitans to homogeneity and remained enzymatically active. Purified Cu,Zn SOD allowed us to isolate highly specific anti-Cu,Zn SOD antibody and this antibody was used to further confirm protein interaction. Cu,Zn SOD-deficient mutants displayed decreased survival in the presence of reactive oxygen and nitrogen species and could be complemented with wild-type Cu,Zn SOD in trans. We suggest that A. actinomycetemcomitans Cu,Zn SOD may protect both bacteria and LtxA from reactive species produced by host inflammatory cells during disease. This is the first example of a protein-protein interaction involving a bacterial Cu,Zn SOD.
KeywordMeSH Terms
67. Doungudomdacha  S, Volgina  A, DiRienzo  JM,     ( 2007 )

Evidence that the cytolethal distending toxin locus was once part of a genomic island in the periodontal pathogen Aggregatibacter (Actinobacillus) actinomycetemcomitans strain Y4.

Journal of medical microbiology 56 (Pt 11)
PMID : 17965355  :   DOI  :   10.1099/jmm.0.47273-0     PMC  :   PMC2717017    
Abstract >>
The authors have previously shown that the periodontal pathogen Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans Y4 contains an operon for a genotoxin known as the cytolethal distending toxin (Cdt). The cdt locus in strain Y4 is flanked by remnants of heterologous plasmid and integrase sequences. In this study, the DNA sequence immediately downstream from the cdt locus on the Y4 chromosome was examined. The extended sequence contained a region that had all the characteristics of a typical bacterial pathogenicity or genomic island. The genomic island (GIY4-1) was approximately 22 kb long, was flanked by a bacteriophage attachment (att) sequence and contained a full-length integrase/resolvase gene (xerD). A total of 22 complete and partial ORFs represented putative DNA replication/DNA binding/conjugation proteins as well as hypothetical proteins. GIY4-1 was most closely related to putative genomic islands in Haemophilus ducreyi 35000HP and Haemophilus influenzae 86-028NP and to a chromosomal region in Haemophilus somnus 129PT. GIY4-1 was not present in HK1651, which was used as the prototype strain for genomic sequencing of A. actinomycetemcomitans. Several sequences in GIY4-1 were homologous to ORFs found on the A. actinomycetemcomitans plasmid pVT745. None of the identified ORFs in GIY4-1 appeared to encode potential virulence genes. However, several unique observations supported the possibility that the cdt locus of A. actinomycetemcomitans Y4 was originally contained within the genomic island.
KeywordMeSH Terms
68. Obradovi?  D, Gašperši?  R, Caserman  S, Leonardi  A, Jamnik  M, Podlesek  Z, Seme  K, Anderluh  G, Križaj  I, Ma?ek  P, Butala  M,     ( 2016 )

A Cytolethal Distending Toxin Variant from Aggregatibacter actinomycetemcomitans with an Aberrant CdtB That Lacks the Conserved Catalytic Histidine 160.

PloS one 11 (7)
PMID : 27414641  :   DOI  :   10.1371/journal.pone.0159231     PMC  :   PMC4945079    
Abstract >>
The periodontopathogen Aggregatibacter actinomycetemcomitans synthesizes several virulence factors, including cytolethal distending toxin (CDT). The active CDT holoenzyme is an AB-type tripartite genotoxin that affects eukaryotic cells. Subunits CdtA and CdtC (B-components) allow binding and intracellular translocation of the active CdtB (A-component), which elicits nuclear DNA damage. Different strains of A. actinomycetemcomitans have diverse virulence genotypes, which results in varied pathogenic potential and disease progression. Here, we identified an A. actinomycetemcomitans strain isolated from two patients with advance chronic periodontitis that has a regular cdtABC operon, which, however, codes for a unique, shorter, variant of the CdtB subunit. We describe the characteristics of this CdtB�G116-188, which lacks the intact nuclear localisation signal and the catalytic histidine 160. We show that the A. actinomycetemcomitans DO15 isolate secretes CdtB�G116-188, and that this subunit cannot form a holotoxin and is also not genotoxic if expressed ectopically in HeLa cells. Furthermore, the A. actinomycetemcomitans DO15 isolate is not toxic, nor does it induce cellular distention upon infection of co-cultivated HeLa cells. Biological significance of this deletion in the cdtB remains to be explained.
KeywordMeSH Terms
69. Lally  ET, Golub  EE, Kieba  IR, Taichman  NS, Rosenbloom  J, Rosenbloom  JC, Gibson  CW, Demuth  DR,     ( 1989 )

Analysis of the Actinobacillus actinomycetemcomitans leukotoxin gene. Delineation of unique features and comparison to homologous toxins.

The Journal of biological chemistry 264 (26)
PMID : 2670940  :  
Abstract >>
Actinobacillus actinomycetemcomitans leukotoxin has been implicated as a virulence factor in human infections. To initiate delineation of leukotoxin structure/function relationships, molecular cloning of the leukotoxin gene was carried out. When an A. actinomycetemcomitans genomic DNA library in lambda EMBL3 was screened using a 1.3-kilobase pair restriction fragment containing a portion of the leukotoxin gene, 13 positive recombinants were identified. One recombinant, designated lambda OP8, containing a 16-kilobase pair insert was selected for detailed study. Lysates from lambda OP8, but not control lysates, exhibited leukotoxic activity with target cell specificity identical to the native toxin. Western blots identified the recombinant-produced toxin as a 125-kDa protein doublet identical in mobility to the native toxin. Restriction enzyme and extensive DNA analyses demonstrated that the leukotoxin gene showed strong homology to two other toxins produced by Escherichia coli and Pasteurella haemolytica. As in the other two species, the A. actinomycetemcomitans toxin is contained in a cluster of four genes in which the A gene encodes the toxin and the products of the B, C, and D genes are involved in posttranslational modification of the toxin and its membrane insertion and secretion. The target cell specificity of the A. actinomycetemcomitans toxin differs from the other two toxins and is restricted to human and some non-human primate cells of the monomyelocytic lineage. The A. actinomycetemcomitans leukotoxin is not secreted but remains associated with the bacterial membrane, possibly through a hydrophobic domain at the carboxyl terminus which distinguishes it from the E. coli and P. haemolytica toxins.
KeywordMeSH Terms
Genes
Genes, Bacterial
70. Tsuzukibashi  O, Saito  M, Kobayashi  T, Umezawa  K, Nagahama  F, Hiroi  T, Hirasawa  M, Takada  K,     ( 2014 )

A gene cluster for the synthesis of serotype g-specific polysaccharide antigen in Aggregatibacter actinomycetemcomitans.

Archives of microbiology 196 (4)
PMID : 24562973  :   DOI  :   10.1007/s00203-014-0965-3    
Abstract >>
Aggregatibacter actinomycetemcomitans is an important pathogen related to aggressively progressive periodontal breakdown in adolescents and adults. The species can be divided into six serotypes (a-f) according to their surface carbohydrate antigens. Recently, a new serotype g of A. actinomycetemcomitans was proposed. The aim of the present study was to sequence the gene cluster associated with the biosynthesis of the serotype g-specific polysaccharide antigen and develop serotype-specific primers for PCR assay to identify serotype g strains of A. actinomycetemcomitans. The serotype-specific polysaccharide (SSPS) gene cluster of the NUM-Aa 4039 strain contained 21 genes in 21,842-bp nucleotides. The similarity of the SSPS gene cluster sequence was 96.7 % compared with that of the serotype e strain. Seventeen serotype g genes showed more than 90 % homology both in nucleotide and amino acids to the serotype e strain. Three additional genes with 1,579 bp in NUM-Aa 4039 were inserted into the corresponding ORF13 of the serotype e strain. The serotype g-specific primers were designed from the insertion region of NUM-Aa 4039. Serotypes of the a-f strains were not amplified by serotype-specific g primers; only NUM-Aa 4039 showed an amplicon band. The NUM-Aa 4039 strain was three genes in the SSPS gene cluster different from those of serotype e strain. The specific primers derived from these different regions are useful for identification and distribution of serotype g strain among A. actinomycetemcomitans from clinical samples.
KeywordMeSH Terms
Multigene Family
71. Sun  R, Kittichotirat  W, Wang  J, Jan  M, Chen  W, Asikainen  S, Bumgarner  R, Chen  C,     ( 2013 )

Genomic Stability of Aggregatibacter actinomycetemcomitans during Persistent Oral Infection in Human.

PloS one 8 (6)
PMID : 23824402  :   DOI  :   10.1371/journal.pone.0066472     PMC  :   PMC3688926    
Abstract >>
The genome of periodontal pathogen Aggregatibacter actinomycetemcomitans exhibits substantial variations in gene content among unrelated strains primarily due to the presence or absence of genomic islands. This study examined the genomic stability of A. actinomycetemcomitans during its persistent infection in the same host. Four pairs of A. actinomycetemcomitans strains, each pair isolated from an individual over time (0-10 years), were examined for their gains/losses of genes by whole genome sequencing, comparative genomic hybridization by microarray and PCR analysis. Possible effects due to genomic changes were further assessed by comparative transcriptome analysis using microarrays. The results showed that each pair of strains was clonally identical based on phylogenetic analysis of 150 core genes. A novel 24.1-Kb plasmid found in strain S23A was apparently lost in the sibling strain I23C. A 353-bp inversion affecting two essential genes of the serotype-specific gene cluster was found in the serotype antigen-nonexpressing strain I23C, while the same gene cluster was intact in the serotype-expressing sibling strain S23A. A 2,293-bp deletion affecting a gene encoding oxaloacetate decarboxylase and its neighbor region was found in strain SCC2302 but not in the sibling strain AAS4a. However, no evidence of gains or losses of genomic islands was found in the paired strains. Transcriptome profiles showed little or no difference in the paired strains. In conclusion, the genome of A. actinomycetemcomitans appears to be relatively stable during short-term infection. Several types of genomic changes were observed in the paired strains of A. actinomycetemcomitans recovered from the same subjects, including a mutation in serotype-specific gene cluster that may allow the bacteria to evade host immune response.
KeywordMeSH Terms
Genomic Instability
72. Guthmiller  JM, Kraig  E, Cagle  MP, Kolodrubetz  D,     ( 1990 )

Sequence of the lktD gene from Actinobacillus actinomycetemcomitans.

Nucleic acids research 18 (17)
PMID : 2402458  :   DOI  :   10.1093/nar/18.17.5292     PMC  :   PMC332165    
Abstract >>
N/A
KeywordMeSH Terms
73. Guthmiller  JM, Kolodrubetz  D, Cagle  MP, Kraig  E,     ( 1990 )

Sequence of the lktB gene from Actinobacillus actinomycetemcomitans.

Nucleic acids research 18 (17)
PMID : 2402457  :   DOI  :   10.1093/nar/18.17.5291     PMC  :   PMC332164    
Abstract >>
N/A
KeywordMeSH Terms
74. Brown  AC, Balashova  NV, Epand  RM, Epand  RF, Bragin  A, Kachlany  SC, Walters  MJ, Du  Y, Boesze-Battaglia  K, Lally  ET,     ( 2013 )

Aggregatibacter actinomycetemcomitans leukotoxin utilizes a cholesterol recognition/amino acid consensus site for membrane association.

The Journal of biological chemistry 288 (32)
PMID : 23792963  :   DOI  :   10.1074/jbc.M113.486654     PMC  :   PMC3949334    
Abstract >>
Aggregatibacter actinomycetemcomitans produces a repeats-in-toxin (RTX) leukotoxin (LtxA) that selectively kills human immune cells. Binding of LtxA to its �]2 integrin receptor (lymphocyte function-associated antigen-1 (LFA-1)) results in the clustering of the toxin�Preceptor complex in lipid rafts. Clustering occurs only in the presence of LFA-1 and cholesterol, and LtxA is unable to kill cells lacking either LFA-1 or cholesterol. Here, the interaction of LtxA with cholesterol was measured using surface plasmon resonance and differential scanning calorimetry. The binding of LtxA to phospholipid bilayers increased by 4 orders of magnitude in the presence of 40% cholesterol relative to the absence of cholesterol. The affinity was specific to cholesterol and required an intact secondary structure. LtxA contains two cholesterol recognition/amino acid consensus (CRAC) sites; CRAC(336) ((333)LEEYSKR(339)) is highly conserved among RTX toxins, whereas CRAC(503) ((501)VDYLK(505)) is unique to LtxA. A peptide corresponding to CRAC(336) inhibited the ability of LtxA to kill Jurkat (Jn.9) cells. Although peptides corresponding to both CRAC(336) and CRAC(503) bind cholesterol, only CRAC(336) competitively inhibited LtxA binding to this sterol. A panel of full-length LtxA CRAC mutants demonstrated that an intact CRAC(336) site was essential for LtxA cytotoxicity. The conservation of CRAC(336) among RTX toxins suggests that this mechanism may be conserved among RTX toxins.
KeywordMeSH Terms
Bacterial Toxins
Cholesterol
Integrins
Lipid Raft
Lipid-Protein Interaction
Microbial Pathogenesis
75. Xu  Y, Moeller  A, Jun  SY, Le  M, Yoon  BY, Kim  JS, Lee  K, Ha  NC,     ( 2012 )

Assembly and channel opening of outer membrane protein in tripartite drug efflux pumps of Gram-negative bacteria.

The Journal of biological chemistry 287 (15)
PMID : 22308040  :   DOI  :   10.1074/jbc.M111.329375     PMC  :   PMC3320922    
Abstract >>
Gram-negative bacteria are capable of expelling diverse xenobiotic substances from within the cell by use of three-component efflux pumps in which the energy-activated inner membrane transporter is connected to the outer membrane channel protein via the membrane fusion protein. In this work, we describe the crystal structure of the membrane fusion protein MexA from the Pseudomonas aeruginosa MexAB-OprM pump in the hexameric ring arrangement. Electron microscopy study on the chimeric complex of MexA and the outer membrane protein OprM reveals that MexA makes a tip-to-tip interaction with OprM, which suggests a docking model for MexA and OprM. This docking model agrees well with genetic results and depicts detailed interactions. Opening of the OprM channel is accompanied by the simultaneous exposure of a protein structure resembling a six-bladed cogwheel, which intermeshes with the complementary cogwheel structure in the MexA hexamer. Taken together, we suggest an assembly and channel opening model for the MexAB-OprM pump. This study provides a better understanding of multidrug resistance in Gram-negative bacteria.
KeywordMeSH Terms
Drug Resistance, Multiple, Bacterial
Protein Multimerization
Pseudomonas aeruginosa
76. Velliyagounder  K, Ganeshnarayan  K, Velusamy  SK, Fine  DH,     ( 2012 )

In vitro efficacy of diallyl sulfides against the periodontopathogen Aggregatibacter actinomycetemcomitans.

Antimicrobial agents and chemotherapy 56 (5)
PMID : 22330917  :   DOI  :   10.1128/AAC.00020-12     PMC  :   PMC3346624    
Abstract >>
The in vitro antibacterial effects of diallyl sulfide (DAS) against the Gram-negative periodontopathogen Aggregatibacter actinomycetemcomitans, the key etiologic agent of the severe form of localized aggressive periodontitis and other nonoral infections, were studied. A. actinomycetemcomitans was treated with garlic extract, allicin, or DAS, and the anti-A. actinomycetemcomitans effects of the treatment were evaluated. Garlic extract, allicin, and DAS significantly inhibited the growth of A. actinomycetemcomitans (greater than 3 log; P < 0.01) compared to control cells. Heat inactivation of the garlic extracts significantly reduced the protein concentration; however, the antimicrobial effect was retained. Purified proteins from garlic extract did not exhibit antimicrobial activity. Allicin lost all its antimicrobial effect when it was subjected to heat treatment, whereas DAS demonstrated an antimicrobial effect similar to that of the garlic extract, suggesting that the antimicrobial activity of garlic extract is mainly due to DAS. An A. actinomycetemcomitans biofilm-killing assay performed with DAS showed a significant reduction in biofilm cell numbers, as evidenced by both confocal microscopy and culture. Scanning electron microscopy (SEM) analysis of DAS-treated A. actinomycetemcomitans biofilms showed alterations of colony architecture indicating severe stress. Flow cytometry analysis of OBA9 cells did not demonstrate apoptosis or cell cycle arrest at therapeutic concentrations of DAS (0.01 and 0.1 �gg/ml). DAS-treated A. actinomycetemcomitans cells demonstrated complete inhibition of glutathione (GSH) S-transferase (GST) activity. However, OBA9 cells, when exposed to DAS at similar concentrations, showed no significant differences in GST activity, suggesting that DAS-induced GST inhibition might be involved in A. actinomycetemcomitans cell death. These findings demonstrate that DAS exhibits significant antibacterial activity against A. actinomycetemcomitans and that this property might be utilized for exploring its therapeutic potential in treatment of A. actinomycetemcomitans-associated oral and nonoral infections.
KeywordMeSH Terms
77.     ( 1997 )

Characterization of a periplasmic protein involved in iron utilization of Actinobacillus actinomycetemcomitans.

Journal of bacteriology 179 (15)
PMID : 9244288  :   DOI  :   10.1128/jb.179.15.4949-4952.1997     PMC  :   PMC179347    
Abstract >>
The periodontopathic bacterium Actinobacillus actinomycetemcomitans possesses a 35-kDa periplasmic iron-repressible protein. Its regulation is mediated by the Fur protein, as was inferred from the Fur-binding consensus sequence at the -35 position of the gene for the 35-kDa protein and from the relaxed expression of the gene in a mutant with an altered Fur-binding sequence. The 35-kDa protein, designated AfuA, has strong homology to HitA and FbpA of Haemophilus influenzae and Neisseria meningitidis, respectively, which serve as periplasmic iron transport proteins.
KeywordMeSH Terms
78.     ( 1996 )

Differential regulation of the leukotoxin operon in highly leukotoxic and minimally leukotoxic strains of Actinobacillus actinomycetemcomitans.

Infection and immunity 64 (7)
PMID : 8698501  :   PMC  :   PMC174132    
Abstract >>
The expression of the leukotoxin (ltx) operon varies significantly among Actinobacillus actinomycetemcomitans strains. The dual promoters driving ltx expression in the highly toxic strain JP2 have been previously characterized (J. M. Brogan, E. T. Lally, K. Poulsen, M. Kilian, and D. R. Demuth, Infect. Immun. 62:501-508, 1994), and genetic analyses of A. actinomycetemcomitans suggest that highly toxic strains like JP2 arose from minimally toxic strains, presumably by deletion of a 530-bp domain within the ltx promoter region (K. Poulsen, E. Theilade, E.T. Lally, D. R. Demuth, and M. Kilian, Microbiology 140:2049-2060, 1994). However, the ltx promoter of minimally toxic A. actinomycetemcomitans strains has not been well characterized. In this study, deletion and primer extension analyses showed that the ltx promoter of A. actinomycetemcomitans 652 is situated approximately 150 bp upstream of the ltxC gene and initiates transcription 138 nucleotides upstream of ltxC. In contrast to strain JP2, only a single promoter appears to drive ltx expression in 652. The 652 promoter resides within the 530-bp region that is absent from the JP2 promoter sequence, suggesting that the specific sequences controlling ltx expression differ in highly toxic and minimally toxic A. actinomycetemcomitans strains. In addition, ltx expression in strain 652 was shown to be induced three- to fourfold when cells were grown under anaerobic conditions. The induction of whole cell leukotoxicity, was accompanied by increases in the levels of Ltx polypeptide and the steady-state levels of ltx mRNA, suggesting that regulation occurred at the level of transcription. In contrast, the levels of leukotoxicity, Ltx polypeptide, and fix mRNA in strain JP2 were unaffected by anaerobic growth. These results suggest that the ltx operon is differentially regulated in highly toxic and minimally toxic A. actinomycetemcomitans strains and that the sequences controlling the oxygen-dependent regulation of ltx expression may reside within the 530-bp domain that is not present in highly toxic A. actinomycetemcomitans.
KeywordMeSH Terms
Operon
79.     ( 1996 )

Novel FNR homologues identified in four representative oral facultative anaerobes: Capnocytophaga ochracea, Capnocytophaga sputigena, Haemophilus aphrophilus, and Actinobacillus actinomycetemcomitans.

FEMS microbiology letters 137 (2��3��)
PMID : 8998988  :   DOI  :   10.1111/j.1574-6968.1996.tb08108.x    
Abstract >>
Based upon DNA sequence data and positive immunochemical reactivity of expressed protein, novel homologues of the FNR family were identified in four representative oral facultative anaerobes: Capnocytophaga ochracea, Capnocytophaga sputigena, Haemophilus aphrophilus, and Actinobacillus actinomycetemcomitans. The similarity to E. coli FNR and to HlyX (itself 71% similar to E. coli FNR, while regulating expression of hemolysin operon in Actinobacillus pleuropneumoniae) was estimated from the deduced partial amino acid sequence to be, in the above order of tested species, 98, 98, 86, and 85%, and 75, 75, 88, and 88%, respectively. The phylogenetic relatedness indicates a rather closer link of HlyX to the FNR homologues from both pathogens, H. aphrophilus and A. actinomycetemcomitans. The possibility that the A. actinomycetemcomitans FNR homologue functions as a redox-sensing transcriptional factor to regulate, in addition to anaerobic respiration, microaerobic expression of the leukotoxin operon (ltx gene) is suggested.
KeywordMeSH Terms
DNA-Binding Proteins
Escherichia coli Proteins
Transcription Factors
80.     ( 1996 )

Molecular analysis of a new insertion sequence from Actinobacillus (Haemophilus) actinomycetemcomitans FDC Y4.

Microbiology (Reading, England) 142 (Pt 9) (N/A)
PMID : 8828211  :   DOI  :   10.1099/00221287-142-9-2449    
Abstract >>
We have found a new insertion sequence (IS), designated ISAa1, downstream of the S10 operon in Actinobacillus (Haemophilus) actinomycetemcomitans FDC Y4. ISAa1, the first IS element characterized in this organism, is 705 bp long and lacks terminal inverted repeats. This element displayed significant homology with IS200. Hybridization patterns of genomic DNA of seven A. actinomycetemcomitans strains with an internal ISAa1 probe varied depending on the serotypes, suggesting that ISAa1 might be a useful tool for epidemiological studies.
KeywordMeSH Terms
81.     ( 1996 )

Cloning and characterization of the Actinobacillus actinomycetemcomitans gene encoding a heat-shock protein 90 homologue.

Gene 179 (2)
PMID : 8972900  :   DOI  :   10.1016/s0378-1119(96)00317-4    
Abstract >>
In a search for clones from a lambda gt11 expression library of Actinobacillus actinomycetemcomitans (Aa) genomic DNA that expressed epitopes from a 70-kDa iron-repressible membrane protein, we inadvertently identified clones that encoded a member of the 90-kDa heat-shock protein (HSP 90) family. The gene appears to encode a homologue of HtpG, as the nucleotide sequence has approximately 70% identity with the Escherichia coli (Ec) and Vibrio fischeri htpG. Growth of an Aa htpG insertion mutant at 42 degrees C was reduced to 50% of the parent strain, similar to an Ec htpG deletion mutant. These data suggest that Aa HtpG performs a function similar to Ec HtpG.
KeywordMeSH Terms
Escherichia coli Proteins
82.     ( 1997 )

The gnd gene encoding a novel 6-phosphogluconate dehydrogenase and its adjacent region of Actinobacillus actinomycetemcomitans chromosomal DNA.

Biochemical and biophysical research communications 230 (1)
PMID : 9020051  :   DOI  :   10.1006/bbrc.1996.5917    
Abstract >>
A 10-kb DNA fragment containing the gnd gene from Actinobacillus actinomy-cetemcomitans Y4 was isolated and sequenced. The structural gnd gene codes for 6-phosphogluconate dehydrogenase that consists of 484 amino acids. In contrast to the gnd gene in Escherichia coli, Salmonella typhimurium, or Klebsiella pneumoniae, the gnd gene of A. actinomycetemcomitans was not located in the rfb or cps operon. The zwf gene encoding glucose 6-phosphate dehydrogenase, which is another enzyme consisting of pentose-phosphate pathway, sided at 3.8-kb upstream from the gnd gene. A phylogenetic tree based on sequence analyses showed higher homology of 6-phospho-gluconate dehydrogenase of A. actinomycetemcomitans with the eucaryotic enzymes rather than with bacterial enzymes.
KeywordMeSH Terms
Chromosomes, Bacterial
Genes, Bacterial
Regulatory Sequences, Nucleic Acid
83.     ( 1996 )

cis Elements and trans factors are both important in strain-specific regulation of the leukotoxin gene in Actinobacillus actinomycetemcomitans.

Infection and immunity 64 (9)
PMID : 8751884  :   PMC  :   PMC174248    
Abstract >>
Actinobacillus actinomycetemcomitans, the etiologic agent of localized juvenile periodontitis, produces a potent leukotoxin that kills human neutrophils. The production of leukotoxin RNA can vary more than 50-fold among isolates of A. actinomycetemcomitans, and strains expressing high levels of leukotoxin RNA are most often found at sites of periodontal disease. To assess the relative contributions of transcription factors and promoter sequences in setting the disparate levels of leukotoxin RNA found, we have undertaken classical cis/trans analyses. First, the leukotoxin promoter regions from moderately leukotoxic (Y4) and minimally leukotoxic (ATCC 33384) strains of A. actinomycetemcomitans were cloned, sequenced, and compared with the previously sequences leukotoxin promoter region of the high-producer strain JP2. The Y4 and ATCC 33384 promoter regions each contain a 528-bp segment that is absent from JP2. Interestingly, the analysis of various deletion constructs in A. actinomycetemcomitans indicated that Y4, despite the large insertion, initiates leukotoxin RNA synthesis at the same promoter as JP2 does. To perform cis/trans analyses, these three leukotoxin promoter regions were cloned into a plasmid upstream of the reporter gene beta-galactosidase. Each plasmid was transformed into JP2, Y4, and ATCC 33384, and the beta-galactosidase levels were determined. The results indicated that the sequences responsible for down-regulating leukotoxin RNA levels in Y4 relative to JP2 are found within the transcribed region of the Y4 leukotoxin operon. Importantly, in ATCC 33384, strain-specific trans factors and promoter sequence differences are equally significant in determining the lower levels of leukotoxin RNA. We hypothesize that either strain ATCC 33384 has a negative regulatory protein (which is missing or mutated in JP2/Y4) or that JP2 and Y4 carry an activator that is missing or mutated in ATCC 33384.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
84.     ( 1994 )

Regulation of Actinobacillus actinomycetemcomitans leukotoxin expression: analysis of the promoter regions of leukotoxic and minimally leukotoxic strains.

Infection and immunity 62 (2)
PMID : 8300209  :   PMC  :   PMC186135    
Abstract >>
The leukotoxin of Actinobacillus actinomycetemcomitans has been implicated as a virulence determinant in various human infections and is encoded by a multigene operon consisting of four known genes, designated ltxC, ltxA, ltxB, and ltxD. The ltx operon appears to be present in all A. actinomycetemcomitans strains, but levels of toxin expression vary greatly among strains. Thus, to gain a better understanding of the expression and regulation of the ltx operon, we have analyzed the ltx promoters of a highly toxic (JP2) and a minimally toxic (652) strain of A. actinomycetemcomitans. The nucleotide sequence of the JP2 ltx promoter contains -10 and -35 elements situated 350 bases upstream of ltxC, and primer extension of JP2 RNA confirmed that they are functional in vivo. However, a second primer extension product of 40 bases was present, and analysis of a series of truncated JP2 promoters fused to lacZ suggested that the region immediately upstream of ltxC also promotes transcription in Escherichia coli. These results suggest that two promoters may direct ltx expression in JP2. In addition, a small open reading frame capable of encoding a peptide of 78 amino acids was identified upstream of ltxC. Northern blots showed that this open reading frame is transcribed as part of a 4.2-kb mRNA, a transcript not previously identified as being derived from the ltx operon. In contrast, strain 652 expresses low steady-state levels of ltx mRNA, and its intact ltx promoter was inefficient in transcribing lacZ in E. coli. The nucleotide sequence of the 652 promoter is similar to that of the JP2 promoter but contains a region of 530 bp that is not present in JP2. Of 15 additional strains of A. actinomycetemcomitans that were analyzed, 13 contained promoters resembling the 652 sequence and 2 possessed JP2-like promoters. Both strains possessing the JP2-like promoter expressed 10- to 20-fold-higher levels of leukotoxin than did the strains possessing promoters resembling the 652 promoter. These results suggest that high levels of leukotoxin expression may correlate with the presence of the JP2-like promoter.
KeywordMeSH Terms
Genes, Bacterial
85. Galli  DM, Leblanc  DJ,     ( 1995 )

Transcriptional analysis of rolling circle replicating plasmid pVT736-1: evidence for replication control by antisense RNA.

Journal of bacteriology 177 (15)
PMID : 7543479  :   DOI  :   10.1128/jb.177.15.4474-4480.1995     PMC  :   PMC177199    
Abstract >>
Several plasmids have been described in Actinobacillus actinomycetemcomitans, a gram-negative coccobacillus. Recently, the nucleotide sequence of pVT736-1, a cryptic plasmid of A. actinomycetemcomitans VT736, was determined. This plasmid possesses all the features necessary for rolling circle replication. The present study involved a transcriptional analysis of pVT736-1. Results of Northern (RNA) blot analyses and primer extension studies indicated that the two open reading frames identified in pVT736-1 are each preceded by at least one promoter. Expression of these promoters varied with growth phase. In addition, an antisense RNA (Cop RNA) appeared to control the synthesis of the putative replication protein. To our knowledge, this is the first rolling circle replicating plasmid isolated from a gram-negative organism that has been subjected to such detailed analysis.
KeywordMeSH Terms
DNA Helicases
DNA Replication
Gene Expression Regulation, Bacterial
Transcription, Genetic
86. Nakano  Y, Inai  Y, Yamashita  Y, Nagaoka  S, Kusuzaki-Nagira  T, Nishihara  T, Okahashi  N, Koga  T,     ( 1995 )

Molecular and immunological characterization of a 64-kDa protein of Actinobacillus actinomycetemcomitans.

Oral microbiology and immunology 10 (3)
PMID : 7567064  :  
Abstract >>
The 64-kDa protein to which about half the sera from patients with localized juvenile periodontitis and rapidly progressive periodontitis reacted strongly was purified from Actinobacillus actinomycetemcomitans Y4. Determination of the N-terminal sequence of the protein revealed that it was a GroEL-like protein. The DNA fragment containing the groEL gene of A. actinomycetemcomitans was amplified by polymerase chain reaction, and the groESL operon was cloned by using colony hybridization with the amplified fragment from A. actinomycetemcomitans chromosomal DNA. Sequence analysis revealed that structures of the operon and its products were typical in gram-negative bacteria. Rabbit polyclonal antibodies to the 64-kDa protein cross-reacted with approximately 65-kDa proteins of Haemophilus aphrophilus, Haemophilus influenzae, Haemophilus paraphrophilus, Escherichia coli and Eikenella corrodens but not with any cellular proteins of Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum. It is possible that antibodies reactive to the 64-kDa protein in periodontitis patients are induced by the cross-reactivity with the hsp60 proteins of other bacteria.
KeywordMeSH Terms
87. Guthmiller  JM, Kolodrubetz  D, Kraig  E,     ( 1995 )

Mutational analysis of the putative leukotoxin transport genes in Actinobacillus actinomycetemcomitans.

Microbial pathogenesis 18 (5)
PMID : 7476096  :   DOI  :   10.1006/mpat.1995.0028    
Abstract >>
The periodontal pathogen, Actinobacillus actinomycetemcomitans, produces leukotoxin, a protein that specifically lyses host defense cells. The leukotoxin is similar in sequence and operon organization to the Escherichia coli alpha-hemolysin and other members of the RTX family of toxins. However, unlike the other RTX toxins, the A. actinomycetemcomitans leukotoxin is not secreted from the cell and instead remains associated with the outer membrane. Nonetheless, the A. actinomycetemcomitans Ikt operon contains two genes, IktB and IktD, that appear analagous to the toxin localization genes found in the other Gram-negative bacteria. Thus, to determine the roles of these putative transport genes in A. actinomycetemcomitans, we have used insertional mutagenesis to generate mutant strains lacking functional LktB and/or LktD. When either IktD or both IktB and IktD were inactivated, the level of detectable leukotoxin protein in the cell decreased significantly. However, the IktB and IktD mutations had no effect on the levels of leukotoxin RNA. Thus, the lack of LktB and LktD proteins must affect LktA synthesis post-transcriptionally. It is proposed that this is an indirect effect of leukotoxin mislocalization in IktB- and IktD- mutants. Finally, analysis of the mutants revealed that LktB and LktD are not essential for the formation of extracellular membrane vesicles in A. actinomycetemcomitans.
KeywordMeSH Terms
Carrier Proteins
Membrane Transport Proteins
88. Kroll  JS, Langford  PR, Wilks  KE, Keil  AD,     ( 1995 )

Bacterial [Cu,Zn]-superoxide dismutase: phylogenetically distinct from the eukaryotic enzyme, and not so rare after all!

Microbiology (Reading, England) 141 (Pt 9) (N/A)
PMID : 7496539  :   DOI  :   10.1099/13500872-141-9-2271    
Abstract >>
Copper- and zinc-containing superoxide dismutases ([Cu,Zn]-SODs) are generally considered almost exclusively eukaryotic enzymes, protecting the cytosol and extracellular compartments of higher organisms from damage by oxygen free-radicals. The recent description of a few examples of bacterial forms of the enzyme, located in the periplasm of different Gram-negative micro-organisms, prompted a re-evaluation of this general perception. A PCR-based approach has been developed and used successfully to identify bacterial genes encoding [Cu,Zn]-SOD in a wide range of important human and animal pathogens-members of the Haemophilus, Actinobacillus and Pasteurella (HAP) group, and Neisseria meningitidis. Comparison of [Cu,Zn]-SOD peptide sequences found in Haemophilus ducreyi, Actinobacillus pleuropneumoniae, Actinobacillus actinomycetemcomitans, Pasteurella multocida, and N. meningitidis with previously described bacterial proteins and examples of eukaryotic [Cu,Zn]-SOD has shown that the bacterial proteins constitute a distinct family apparently widely separated in evolutionary terms from the eukaryotic examples. The widespread occurrence of [Cu,Zn]-SOD in the periplasm of bacterial pathogens, appropriately located to dismute exogenously derived superoxide radical anions, suggests that this enzyme may play a role in the interactive biology of organisms with their hosts and so contribute to their capacity to cause disease.
KeywordMeSH Terms
Evolution, Molecular
89. Simpson  DL, Berthold  P, Taichman  NS,     ( 1988 )

Killing of human myelomonocytic leukemia and lymphocytic cell lines by Actinobacillus actinomycetemcomitans leukotoxin.

Infection and immunity 56 (5)
PMID : 3258584  :   PMC  :   PMC259778    
Abstract >>
The purified leukotoxin of Actinobacillus actinomycetemcomitans kills human leukemic cell lines (e.g., HL-60, U937, and KG-1) and human T- and B-cell lines (e.g., JURKAT, MOLT-4, Daudi, and Raji) in a dose- and time-dependent manner. The 50% effective doses for these cell lines are similar to those established for human polymorphonuclear leukocytes and monocytes. In contrast, other human and nonhuman tumor cell lines are not susceptible to the leukotoxin. These human leukemia and lymphoid cell lines will serve as useful model systems with which to study the molecular specificity and mechanism(s) of action of the actinobacillus leukotoxin.
KeywordMeSH Terms
90.     ( 1999 )

Cloning of the gene encoding the Actinobacillus actinomycetemcomitans serotype b OmpA-like outer membrane protein.

Infection and immunity 67 (2)
PMID : 9916112  :   PMC  :   PMC96408    
Abstract >>
The gene encoding an outer membrane protein A (OmpA)-like, heat-modifiable Omp of Actinobacillus actinomycetemcomitans ATCC 43718 (strain Y4, serotype b) was cloned by a PCR cloning procedure. DNA sequence analysis revealed that the gene encodes a protein of 346 amino acid residues with a molecular mass of 36.9 kDa. The protein expressed by the cloned gene reacted with a monoclonal antibody to the previously described 29-kDa Omp (Omp29) of strain Y4. This monoclonal antibody reacted specifically with Omp29 of A. actinomycetemcomitans (serotype b), but not with any Omp of Escherichia coli, including OmpA. This protein exhibited characteristic heat modifiability on sodium dodecyl sulfate-polyacrylamide gels, showing an apparent molecular mass of 29 kDa when unheated and a mass of 34 kDa when heated. The N-terminal amino acid sequence of the protein expressed in E. coli perfectly matched those deduced from the purified Omp29 of strain Y4. The deduced amino acid sequence of the gene coding for Omp29 from serotype b matched completely (except for valine at position 321) that of a recently reported omp34 gene described for A. actinomycetemcomitans serotype c (NCTC 9710). Because of the conserved nature of the gene within these serotypes, we designated the gene described herein from serotype b as omp34.
KeywordMeSH Terms
91.     ( 1990 )

Nucleotide sequence of the leukotoxin gene from Actinobacillus actinomycetemcomitans: homology to the alpha-hemolysin/leukotoxin gene family.

Infection and immunity 58 (4)
PMID : 2318535  :   PMC  :   PMC258561    
Abstract >>
The leukotoxin produced by Actinobacillus actinomycetemcomitans has been implicated in the etiology of localized juvenile periodontitis. To initiate a genetic analysis into the role of this protein in disease, we have cloned its gene, lktA. We now present the complete nucleotide sequence of the lktA gene from A. actinomycetemcomitans. When the deduced amino acid sequence of the leukotoxin protein was compared with those of other proteins, it was found to be homologous to the leukotoxin from Pasteurella haemolytica and to the alpha-hemolysins from Escherichia coli and Actinobacillus pleuropneumoniae. Each alignment showed at least 42% identity. As in the other organisms, the lktA gene of A. actinomycetemcomitans was linked to another gene, lktC, which is thought to be involved in the activation of the leukotoxin. The predicted LktC protein was related to the leukotoxin/hemolysin C proteins from the other bacteria, since they shared a minimum of 49% amino acid identity. Surprisingly, although actinobacillus species are more closely related to pasteurellae than to members of the family Enterobacteriaciae, LktA and LktC from A. actinomycetemcomitans shared significantly greater sequence identity with the E. coli alpha-hemolysin proteins than with the P. haemolytica leukotoxin proteins. Despite the overall homology to the other leukotoxin/hemolysin proteins, the LktA protein from A. actinomycetemcomitans has several unique properties. Most strikingly, it is a very basic protein with a calculated pI of 9.7; the other toxins have estimated pIs around 6.2. The unusual features of the A. actinomycetemcomitans protein are discussed in light of the different species and target-cell specificities of the hemolysins and the leukotoxins.
KeywordMeSH Terms
Base Sequence
Escherichia coli Proteins
Genes, Bacterial
Hemolysin Proteins
Sequence Homology, Nucleic Acid
92.     ( 1998 )

A gene cluster for 6-deoxy-L-talan synthesis in Actinobacillus actinomycetemcomitans.

Biochimica et biophysica acta 1442 (2��3��)
PMID : 9805002  :   DOI  :   10.1016/s0167-4781(98)00174-2    
Abstract >>
The serotype c antigen of Actinobacillus actinomycetemcomitans consists of 6-deoxy-l-talose. A gene cluster involved in the synthesis of serotype-specific polysaccharide antigen was cloned from the chromosomal DNA of A. actinomycetemcomitans NCTC 9710 (serotype c). This cluster consisted of 17 open reading frames. Escherichia coli produced the polysaccharide that reacts with the serotype c-specific antibody when transformed with a plasmid containing the cluster. Comparing the structure of the gene cluster with a similar cluster from A. actinomycetemcomitans Y4 (serotype b), which produces a polysaccharide consisting of l-rhamnose and d-fucose, revealed that a 5.7 kb region containing seven genes in the cluster from strain Y4 was replaced by a 3.8 kb region containing three genes in strain NCTC 9710. The results suggest that these region, as well as dTDP-6-deoxy-l-talose-forming dTDP-4-keto-l-rhamnose reductase, is essential to the production of extracellular polysaccharide specific to serotype c.
KeywordMeSH Terms
Genes, Bacterial
Multigene Family
93.     ( 1998 )

The cell cycle-specific growth-inhibitory factor produced by Actinobacillus actinomycetemcomitans is a cytolethal distending toxin.

Infection and immunity 66 (10)
PMID : 9746611  :   PMC  :   PMC108622    
Abstract >>
Actinobacillus actinomycetemcomitans has been shown to produce a soluble cytotoxic factor(s) distinct from leukotoxin. We have identified in A. actinomycetemcomitans Y4 a cluster of genes encoding a cytolethal distending toxin (CDT). This new member of the CDT family is similar to the CDT produced by Haemophilus ducreyi. The CDT from A. actinomycetemcomitans was produced in Escherichia coli and was able to induce cell distension, growth arrest in G2/M phase, nucleus swelling, and chromatin fragmentation in HeLa cells. The three proteins, CDTA, -B and -C, encoded by the cdt locus were all required for toxin activity. Antiserum raised against recombinant CDTC completely inhibited the cytotoxic activity of culture supernatant and cell homogenate fractions of A. actinomycetemcomitans Y4. These results strongly suggest that the CDT is responsible for the cytotoxic activity present in the culture supernatant and cell homogenate fractions of A. actinomycetemcomitans Y4. This CDT is a new putative virulence factor of A. actinomycetemcomitans and may play a role in the pathogenesis of periodontal diseases.
KeywordMeSH Terms
94.     ( 1998 )

Codon usage in Actinobacillus actinomycetemcomitans.

FEMS microbiology letters 163 (1)
PMID : 9631542  :   DOI  :   10.1111/j.1574-6968.1998.tb13022.x    
Abstract >>
The codon usage patterns of 21 genes encompassing 5800 codons from Actinobacillus actinomycetemcomitans were analyzed. A. actinomycetemcomitans genes could be divided into two groups based on their function and G + C content. One group included those genes encoding basic cellular functions. This group displayed an average G + C content of 48%. A second group comprised genes encoding the leukotoxin determinant, an insertion sequence and a plasmid. This group displayed an average G + C content of 36%. These findings suggest that portions of the A. actinomycetemcomitans genome may have been acquired by horizontal gene transfer from one or more distantly related species. We present a table of A. actinomycetemcomitans codon usage. These data may be used to establish standards for computer programs that predict A. actinomycetemcomitans protein coding regions and may be useful in designing degenerate oligonucleotide probes.
KeywordMeSH Terms
95.     ( 1997 )

Cloning and sequence analysis of the fimbriae associated protein (fap) gene from Actinobacillus actinomycetemcomitans.

Microbial pathogenesis 23 (2)
PMID : 9245617  :   DOI  :   10.1006/mpat.1997.0137    
Abstract >>
Fimbrial-associated protein, an attachment factor of Actinobacillus actinomycetemcomitans, was genetically analysed by cloning. The plasmid obtained was found to harbor a 1.7kb fragment which contained a 228bp open reading frame encoding 76 amino acids (7.970kDa). The fimbriae associated protein gene (fap) was strongly expressed in fimbriated A. actinomycetemcomitans 310a but not in non-fimbriated strains.
KeywordMeSH Terms
Fimbriae Proteins
Fimbriae, Bacterial
96.     ( 1997 )

Isolation and characterization of the dnaKJ operon from Actinobacillus actinomycetemcomitans.

DNA sequence : the journal of DNA sequencing and mapping 8 (1��2��)
PMID : 9522128  :  
Abstract >>
The dnaKJ operon of Actinobacillus actinomycetemcomitans Y4 was cloned by the DNA-probing method using synthetic oligonucleotides designed on the basis of two conserved regions in DnaK/hsp70 proteins and sequenced by inverse PCR. The sequenced region was shown to contain two open reading frames coding for proteins analogous to DnaK and DnaJ.
KeywordMeSH Terms
Escherichia coli Proteins
97.     ( 1998 )

Molecular characterization of low-molecular-weight component protein, Flp, in Actinobacillus actinomycetemcomitans fimbriae.

Microbiology and immunology 42 (4)
PMID : 9623911  :   DOI  :   10.1111/j.1348-0421.1998.tb02280.x    
Abstract >>
Fimbriae preparation from Actinobacillus actinomycetemcomitans was found to contain an abundant low-molecular-weight protein (termed Flp) with an apparent molecular mass of approximately 6.5 kDa, in addition to a small amount of 54-kDa protein. Immunogold electron microscopy localized the Flp protein at the bacterial fimbriae but not at the cell surface. The DNA fragment including the flp gene was cloned from A. actinomycetemcomitans 304-a and its nucleotide sequence was determined. An open reading frame of the flp gene was composed of 225 bp encoding a protein of 75 amino acids. Comparison of the translated amino acid sequence with the sequence of native Flp determined by Edman degradation indicated that the N-terminal part of 26 amino acids is leader peptide. The N-terminal sequence of mature Flp exhibited some similarity to type-IV pilin. Furthermore, the processing site of premature Flp is also similar to that of type-IV prepilin, and a gene encoding a protein homologous to type-IV prepilin-like protein leader peptidase was found downstream of the flp gene. These findings indicate that Flp is the major component protein of A. actinomycetemcomitans fimbriae.
KeywordMeSH Terms
Genes, Bacterial
98.     ( 1998 )

Identification of a genetic locus essential for serotype b-specific antigen synthesis in Actinobacillus actinomycetemcomitans.

Infection and immunity 66 (1)
PMID : 9423846  :   PMC  :   PMC107865    
Abstract >>
A large gene cluster associated with the biosynthesis of the serotype-specific polysaccharide antigen (SPA) of Actinobacillus actinomycetemcomitans Y4 (serotype b) was cloned and characterized. Western blot analysis showed that Escherichia coli DH5alpha, containing a plasmid carrying this cluster, produced a polysaccharide which reacted with a monoclonal antibody directed against the SPA of A. actinomycetemcomitans Y4. High-performance liquid chromatography analysis indicated that the polysaccharide produced by an E. coli transformant, as well as A. actinomycetemcomitans Y4 SPA, was composed of rhamnose and fucose. Furthermore, using various derivatives of the plasmid, we demonstrated that the cloned 13-kb BssHII-BspHI fragment was indispensable for SPA synthesis in E. coli DH5alpha. The 24,909-bp nucleotide sequence, which included this fragment and its flanking regions, was determined. In the sequenced area, 24 open reading frames (ORFs) with the same orientation were found. Most of these were located sequentially within a short distance of each other. Many of the deduced amino acid sequences were similar to the gene products of the polysaccharide synthetic genes of other bacteria. The average G+C content (37.7%) of all 24 ORFs in the sequenced area was lower than that (45.6%) of the whole chromosome of A. actinomycetemcomitans Y4. It is noteworthy the average G+C content of the nine ORFs in the 8.5-kb central region of the 13-kb BssHII-BspHI fragment indispensable for SPA synthesis in E. coli was found to be especially low (27.0%).
KeywordMeSH Terms
99.     ( 1998 )

Molecular characterization of an outer membrane protein of Actinobacillus actinomycetemcomitans belonging to the OmpA family.

Infection and immunity 66 (1)
PMID : 9423883  :   PMC  :   PMC107906    
Abstract >>
The major outer membrane protein (OMP) of Actinobacillus actinomycetemcomitans is an OmpA homolog that demonstrates electrophoretic heat modifiability. The gene encoding this protein was isolated from a genomic library of A. actinomycetemcomitans NCTC 9710 by immunoscreening with serum from a patient with localized juvenile periodontitis. Expression of the cloned gene in Escherichia coli and subsequent Western blot analysis revealed a protein with an approximate molecular mass of 34 kDa. The amino acid sequence predicted from the cloned gene demonstrated that the mature protein had a molecular mass of 34,911 Da and significant identity to members of the OmpA family of proteins. We have named the major OMP of A. actinomycetemcomitans Omp34, and its corresponding gene has been named omp34.
KeywordMeSH Terms
Escherichia coli Proteins

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