| 1. |
Wu H,
( 1999 ) Identification of dipeptide repeats and a cell wall sorting signal in the fimbriae-associated adhesin, Fap1, of Streptococcus parasanguis. PMID : 10594831 : DOI : 10.1046/j.1365-2958.1999.01670.x Abstract >>
Fap1, a fimbriae-associated protein, is involved in fimbriae assembly and adhesion of Streptococcus parasanguis FW213 (Wu et al., 1998). In this study, the sequence of the fap1 gene was resolved using a primer island transposition system. Sequence analysis indicated that fap1 was composed of 7659 nucleotides. The predicted Fap1 protein contains an unusually long signal sequence (50 amino acid residues), a cell wall sorting signal and two repeat regions. Repeat regions I and II have a similar dipeptide composition (E/V/I)S, composed of 28 and 1000 repeats respectively. The two regions combined accounted for 80% of the Fap1 coding region. The experimental amino acid composition and isoelectric point (pI) of Fap1 were similar to that predicted from the deduced Fap1 protein. Results of Northern analyses revealed that the fap1 open reading frame (ORF) was transcribed as a 7.8 kb monocistronic message. Insertional inactivation at the 3' end, downstream of the fap1 ORF, did not affect Fap1, fimbrial expression or bacterial adhesion. Insertional inactivation of fap1 immediately upstream of the repeat region II abolished expression of Fap1 and fimbriae, and was concurrent with a diminution in adhesion of FW213. Inactivation of the cell wall sorting signal of fap1 also eliminated long fimbrial formation and reduced the ability of FW213 to bind to SHA. Fap1 was no longer anchored on the cell surface. Large quantities of truncated Fap1 were found in the growth medium instead. These results suggest that the fap1 ORF alone is sufficient to support Fap1 expression and adhesion, and demonstrate that anchorage of Fap1 on the cell surface is required for long fimbriae formation. These data further document the role of long fimbriae in adhesion of S. parasanguis FW213 to SHA.
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2. |
Whiley RA,
Shu SE,
Kawamura Y,
( 1999 ) Genetic approaches to the identification of the mitis group within the genus Streptococcus. PMID : 10517614 : DOI : 10.1099/00221287-145-9-2605 Abstract >>
The usefulness and reliability of partial sequence analysis of the manganese-dependent superoxide dismutase gene (sodA), autolysin (lytA) gene amplification and species-specific PCR based on the D-alanine:D-alanine ligase (ddl) gene for differentiating each member of the mitis group of the genus Streptococcus was investigated. On the phylogenetic tree based on sodA partial sequences (366 bp) from 96 strains, including all species currently within the mitis group isolated in different geographic areas (mainly Japan and the UK), eight well separated clusters were generated corresponding to recognized species, and all strains fell into those clusters to which they had also been assigned by DNA-DNA hybridization. The Streptococcus pneumoniae sub-cluster was located within the Streptococcus mitis cluster, but the sodA gene of S. pneumoniae was very conserved and therefore could be separated from all other species examined. Furthermore, the lytA gene amplification approach could also be used to differentiate S. pneumoniae from other species. The species-specific amplification product of the ddl gene was successfully detected in Streptococcus sanguinis and Streptococcus gordonii, but failed to be detected in some strains of Streptococcus oralis including the type strain and S. mitis. We conclude that the partial sequence analysis of the sodA gene could be applied globally as a reliable and easy method for the accurate identification of all species currently within the mitis group.
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3. |
Wu H,
Zeng M,
Fives-Taylor P,
( 2007 ) The glycan moieties and the N-terminal polypeptide backbone of a fimbria-associated adhesin, Fap1, play distinct roles in the biofilm development of Streptococcus parasanguinis. PMID : 17296746 : DOI : 10.1128/IAI.01544-06 PMC : PMC1865748 Abstract >>
Fap1, a fimbria-associated glycoprotein, is essential for biofilm formation of Streptococcus parasanguinis and mediates bacterial attachment to saliva-coated hydroxylapatite, an in vitro tooth model (E. H. Froeliger and P. M. Fives-Taylor, Infect. Immun. 69:2512-2519, 2001; H. Wu and P. M. Fives-Taylor, Mol. Microbiol. 34:1070-1081, 1999; H. Wu et al., Mol. Microbiol. 28:487-500, 1998). Fap1 belongs to a growing family of high-molecular-weight serine-rich proteins found in streptococcal and staphylococcal species and possesses two serine-rich repeat regions. The glycan moiety of Fap1 appears to be O linked within the repeat regions (A. E. Stephenson et al., Mol. Microbiol. 43:147-157, 2002). In the present study, we identified a gene cluster immediately upstream of fap1 that encodes three putative glycosyltransferases and one nucleotide-sugar synthetase-like protein. Inactivation of one glycosyltransferase gene galT2 abolished the expression of two glycan epitopes; however, it did not alter bacterial ability to adhere to both SHA and saliva-conditioned biofilm surfaces. In contrast, the biofilms formed by the galT2 mutant were shallow and had a 70% decrease in biomass accumulation, suggesting that these glycan moieties mediated by GalT2 are not required for the initial adhesion but are important for biofilm formation. A recombinant N-terminal Fap1 polypeptide was shown to interact with a 53-kDa salivary protein and block and displace bacterial attachment, further demonstrating the role of the Fap1 polypeptide in bacterial adhesion. Taken together, these results suggest that Fap1 glycosylation plays an important role in bacterial biofilm formation, whereas the nonglycosylated Fap1 peptide mediates bacterial initial attachment during the process of biofilm formation.
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4. |
Wu H,
Bu S,
Newell P,
Chen Q,
Fives-Taylor P,
( 2007 ) Two gene determinants are differentially involved in the biogenesis of Fap1 precursors in Streptococcus parasanguis. PMID : 16997950 : DOI : 10.1128/JB.00836-06 PMC : PMC1797361 Abstract >>
Mature Fap1, a 200-kDa fimbria-associated adhesin, is required for fimbrial biogenesis and biofilm formation in Streptococcus parasanguis. Fap1-like proteins are found in the genomes of many streptococcal and staphylococcal species. Fap1 is a serine-rich glycoprotein modified by O-linked glycan moieties. In this study, we identified a seven-gene cluster including secY2, orf1, orf2, orf3, secA2, gtf1, and gtf2 that is localized immediately downstream of fap1. The lower G+C contents and the presence of a putative transposase element suggest that this gene cluster was horizontally transferred from other bacteria and represents a genomic island. At least two genes in this island mediated Fap1 biogenesis. Mutation of a glucosyltransferase (Gtf1) gene led to accumulation of a Fap1 precursor, which had no detectable glycan moieties. Inactivation of a gene coding for an accessory Sec protein (SecY2) resulted in expression of a distinct Fap1 precursor, which reacted with one glycan-specific Fap1 antibody but not with another glycan-specific antibody. Furthermore, partially glycosylated Fap1 was detected on the cell surface and in the culture supernatant. These data suggest that SecY2 has a role in complete glycosylation of Fap1 and imply that SecY2 is not the only translocation channel for the Fap1 precursor and that alternative secretion machinery exists. Together, Gtf1 and SecY2 are involved in biogenesis of two distinct Fap1 precursors in S. parasanguis. Discovery of the effect of an accessory Sec protein on Fap1 glycosylation suggests that Fap1 secretion and glycosylation are coupled during Fap1 biogenesis.
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5. |
Ganeshkumar N,
Hannam PM,
Kolenbrander PE,
McBride BC,
( 1991 ) Nucleotide sequence of a gene coding for a saliva-binding protein (SsaB) from Streptococcus sanguis 12 and possible role of the protein in coaggregation with actinomyces. PMID : 1671775 : PMC : PMC258372 Abstract >>
The nucleotide sequence of a 2.9-kb streptococcal DNA fragment which codes for two proteins with MrS of 36,000 (Streptococcus sanguis adhesin B [SsaB]) and 20,000 has been determined. The ssaB gene is 927 bp and codes for a 34,684-Da protein. The open reading frame coding for the 20-kDa protein is 489 bp and codes for a protein of 17,885 Da. The SsaB protein has a putative hydrophobic 19-amino-acid signal sequence resulting in a 32,620-Mr secreted protein, whereas the 20-kDa protein has no signal sequence. Both proteins are hydrophilic, and neither appears to have a hydrophobic membrane anchor sequence in the carboxy-terminal region. A DNA sequence homology of 73% exists between the cloned fragment containing the ssaB gene from S. sanguis 12 and the cloned fragment containing the type 1 fimbrial gene of S. sanguis FW213 (J.C. Fenno, D.J. LeBlanc, and P. Fives-Taylor, Infect. Immun. 57:3527-3533, 1989). Amino acid comparisons of the SsaB and type 1 fimbrial proteins show 87% homology, indicating a close similarity of the two proteins. Antiserum raised against the cloned SsaB protein cross-reacts with a 38-kDa protein identified from Streptococcus gordonii (S. sanguis) PK488 which was proposed to mediate coaggregation with Actinomyces naeslundii PK606 (P.E. Kolenbrander and R.N. Andersen, Infect. Immun. 58:3064-3072, 1990). The SsaB adhesion may play a role in oral colonization by binding either to a receptor on saliva or to a receptor on actinomyces.
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6. |
Glazunova OO,
Raoult D,
Roux V,
( 2006 ) Streptococcus massiliensis sp. nov., isolated from a patient blood culture. PMID : 16627666 : DOI : 10.1099/ijs.0.64009-0 Abstract >>
An unidentified strain of the viridans group of streptococci was isolated from a human blood sample. It was distinguished from all other recognized species of the Streptococcus sanguinis group by several biochemical characteristics. Phylogenetic analysis based on 16S rRNA gene sequence comparisons clustered this strain with Streptococcus ferus (mutans group) but phylogenetic analysis based on rpoB and sodA gene sequence comparisons included it in the S. sanguinis group. The isolate showed 95.4 and 95.2 % 16S rRNA gene sequence similarity to S. ferus and S. sanguinis, respectively, confirming it as belonging to a novel taxon, for which the name Streptococcus massiliensis sp. nov. is proposed. The type strain is 4401825T (=CIP 108498T=CCUG 49690T).
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7. |
Itoh Y,
Kawamura Y,
Kasai H,
Shah MM,
Nhung PH,
Yamada M,
Sun X,
Koyana T,
Hayashi M,
Ohkusu K,
Ezaki T,
( 2006 ) dnaJ and gyrB gene sequence relationship among species and strains of genus Streptococcus. PMID : 16487673 : DOI : 10.1016/j.syapm.2005.12.003 Abstract >>
The dnaJ and gyrB nucleotide sequences were determined for members of the genus Streptococcus. The average similarity between the species tested was 76.4% (69.7-100%) for dnaJ and 75.9 (70.1-98.7%) for gyrB. These data indicated that the dnaJ and gyrB genes are more divergent and more discriminatory than the 16S rDNA gene. Furthermore, the variation in the dnaJ nucleotide sequences among the mitis group was greater than that of the gyrB nucleotide sequences, especially between Streptococcus pneumoniae and Streptococcus mitis. Subsequently, the high discrimination power of dnaJ within the mitis group was confirmed. Thus, we conclude that the dnaJ and gyrB genes are efficient alternative targets for the classification of the genus Streptococcus, and that dnaJ is suitable for phylogenetic analysis of closely related Streptococcus strains.
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8. |
Hoshino T,
Fujiwara T,
Kilian M,
( 2005 ) Use of phylogenetic and phenotypic analyses to identify nonhemolytic streptococci isolated from bacteremic patients. PMID : 16333101 : DOI : 10.1128/JCM.43.12.6073-6085.2005 PMC : PMC1317212 Abstract >>
The aim of this study was to evaluate molecular and phenotypic methods for the identification of nonhemolytic streptococci. A collection of 148 strains consisting of 115 clinical isolates from cases of infective endocarditis, septicemia, and meningitis and 33 reference strains, including type strains of all relevant Streptococcus species, were examined. Identification was performed by phylogenetic analysis of nucleotide sequences of four housekeeping genes, ddl, gdh, rpoB, and sodA; by PCR analysis of the glucosyltransferase (gtf) gene; and by conventional phenotypic characterization and identification using two commercial kits, Rapid ID 32 STREP and STREPTOGRAM and the associated databases. A phylogenetic tree based on concatenated sequences of the four housekeeping genes allowed unequivocal differentiation of recognized species and was used as the reference. Analysis of single gene sequences revealed deviation clustering in eight strains (5.4%) due to homologous recombination with other species. This was particularly evident in S. sanguinis and in members of the anginosus group of streptococci. The rate of correct identification of the strains by both commercial identification kits was below 50% but varied significantly between species. The most significant problems were observed with S. mitis and S. oralis and 11 Streptococcus species described since 1991. Our data indicate that identification based on multilocus sequence analysis is optimal. As a more practical alternative we recommend identification based on sodA sequences with reference to a comprehensive set of sequences that is available for downloading from our server. An analysis of the species distribution of 107 nonhemolytic streptococci from bacteremic patients showed a predominance of S. oralis and S. anginosus with various underlying infections.
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9. |
Maeda K,
Nagata H,
Nonaka A,
Kataoka K,
Tanaka M,
Shizukuishi S,
( 2004 ) Oral streptococcal glyceraldehyde-3-phosphate dehydrogenase mediates interaction with Porphyromonas gingivalis fimbriae. PMID : 15488735 : DOI : 10.1016/j.micinf.2004.06.005 Abstract >>
Interaction of Porphyromonas gingivalis with plaque-forming bacteria is necessary for its colonization in periodontal pockets. Participation of Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and P. gingivalis fimbriae in this interaction has been reported. In this investigation, the contribution of various oral streptococcal GAPDHs to interaction with P. gingivalis fimbriae was examined. Streptococcal cell surface GAPDH activity was measured by incubation of a constant number of streptococci with glyceraldehyde-3-phosphate and analysis for the conversion of NAD+ to NADH based on the absorbance at 340 nm. Coaggregation activity was measured by a turbidimetric assay. Cell surface GAPDH activity was correlated with coaggregation activity (r = 0.854, P < 0.01) with Spearman's rank correlation coefficient. S. oralis ATCC 9811 and ATCC 10557, Streptococcus gordonii G9B, Streptococcus sanguinis ATCC 10556, and Streptococcus parasanguinis ATCC 15909 exhibited high cell surface GAPDH activity and coaggregation activity; consequently, their cell surface GAPDHs were extracted with mutanolysin and purified on a Cibacron Blue Sepharose column. Subsequently, their DNA sequences were elucidated. Purified GAPDHs bound P. gingivalis recombinant fimbrillin by Western blot assay, furthermore, their DNA sequences displayed a high degree of homology with one another. Moreover, S. oralis recombinant GAPDH inhibited coaggregation between P. gingivalis and the aforementioned five streptococcal strains in a dose-dependent manner. These results suggest that GAPDHs of various plaque-forming streptococci may be involved in their attachment to P. gingivalis fimbriae and that they may contribute to P. gingivalis colonization.
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10. |
Hill JE,
Penny SL,
Crowell KG,
Goh SH,
Hemmingsen SM,
( 2004 ) cpnDB: a chaperonin sequence database. 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.
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11. |
Stapleton P,
Pike R,
Mullany P,
Lucas V,
Roberts G,
Rowbury R,
Wilson M,
Richards H,
( 2004 ) Mercuric resistance genes in gram-positive oral bacteria. PMID : 15251199 : DOI : 10.1016/j.femsle.2004.05.041 Abstract >>
Mercury-resistant bacteria isolated from the oral cavities of children carried one of two types of merA gene that appear to have evolved from a common ancestor. Streptococcus oralis, Streptococcus mitis and a few other species had merA genes that were very similar to merA of Bacillus cereus strain RC607. Unlike the B. cereus RC607 merA gene, however, the streptococcal merA genes were not carried on Tn5084-like transposons. Instead, comparisons with microbial genomic sequences suggest the merA gene is located on a novel type II transposon. Coagulase-negative staphylococci and Streptococcus parasanguis had identical merA genes that represent a new merA variant.
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12. |
Liang X,
Chen YY,
Ruiz T,
Wu H,
( 2011 ) New cell surface protein involved in biofilm formation by Streptococcus parasanguinis. PMID : 21576336 : DOI : 10.1128/IAI.00029-11 PMC : PMC3147580 Abstract >>
Dental biofilm formation is critical for maintaining the healthy microbial ecology of the oral cavity. Streptococci are predominant bacterial species in the oral cavity and play important roles in the initiation of plaque formation. In this study, we identified a new cell surface protein, BapA1, from Streptococcus parasanguinis FW213 and determined that BapA1 is critical for biofilm formation. Sequence analysis revealed that BapA1 possesses a typical cell wall-sorting signal for cell surface-anchored proteins from Gram-positive bacteria. No functional orthologue was reported in other streptococci. BapA1 possesses nine putative pilin isopeptide linker domains which are crucial for pilus assembly in a number of Gram-positive bacteria. Deletion of the 3' portion of bapA1 generated a mutant that lacks surface-anchored BapA1 and abolishes formation of short fibrils on the cell surface. The mutant failed to form biofilms and exhibited reduced adherence to an in vitro tooth model. The BapA1 deficiency also inhibited bacterial autoaggregation. The N-terminal muramidase-released-protein-like domain mediated BapA1-BapA1 interactions, suggesting that BapA1-mediated cell-cell interactions are important for bacterial autoaggregation and biofilm formation. Furthermore, the BapA1-mediated bacterial adhesion and biofilm formation are independent of a fimbria-associated serine-rich repeat adhesin, Fap1, demonstrating that BapA1 is a new streptococcal adhesin.
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13. |
Maeda Y,
Murayama M,
Goldsmith CE,
Coulter WA,
Mason C,
Millar BC,
Dooley JS,
Lowery CJ,
Matsuda M,
Rendall JC,
Elborn JS,
Moore JE,
( 2011 ) Molecular characterization and phylogenetic analysis of quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC and parE gene loci in viridans group streptococci isolated from adult patients with cystic fibrosis. PMID : 21193474 : DOI : 10.1093/jac/dkq485 Abstract >>
Ciprofloxacin is the most frequently used member of the fluoroquinolones during initial eradication therapy of Pseudomonas aeruginosa, as well as during acute pulmonary exacerbations. However, its long-term effect on the susceptibility of the commensal flora within the cystic fibrosis (CF) airways has not yet been examined. The aim of this study was therefore to examine the consequence of oral ciprofloxacin usage on the resistance of the commensal viridans group streptococci (VGS), in terms of MICs and mutational analysis of the quinolone resistance-determining regions (QRDRs). The MICs of ciprofloxacin, efflux activities and amino acid substitutions in the QRDRs for 190 isolates of VGS, originating from the sputa of adult CF patients who had been exposed constantly to ciprofloxacin, were examined. VGS organisms included Streptococcus salivarius, Streptococcus mitis, Streptococcus sanguinis, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus infantis, Streptococcus gordonii, Streptococcus anginosus, Streptococcus cristatus, Streptococcus australis and Streptococcus mutans. Ciprofloxacin susceptibility was determined by broth microdilution and QRDRs within the gyrA, gyrB, parC and parE gene loci were explored using sequence analysis. Twenty-seven (14.2%) streptococcal isolates were resistant to ciprofloxacin (MICs ?8 mg/L) and 21 (11.1%) had reduced susceptibility (MICs 4 mg/L). As a comparator, clinically non-significant and non-invasive VGS organisms were examined in 12 consecutive non-CF patients in the community, where no resistance to ciprofloxacin was observed. Five novel QRDR PCR assays were developed to elucidate mutations within the CF VGS population, where there were six positions, which corresponded to previously reported quinolone resistance responsible mutations, and eight novel potential QRDR resistance mutations. Double mutations in gyrA and parC/parE led to MICs of 16 to >64 mg/L, while single mutations in parC or parE resulted in MICs of 8-32 mg/L and 8 mg/L, respectively. The mean homologies of each species to Streptococcus pneumoniae R6 were: gyrA, 70.3%-95%; gyrB, 69.6%-96.2%; parC, 76.1%-94.8%; and parE, 70.7%-94.7%. The close relatives of S. pneumoniae, S. mitis and S. oralis, showed high similarity for all four genes (more than 86%). Treatment of P. aeruginosa with oral ciprofloxacin in patients with CF may concurrently reduce antibiotic susceptibility in the commensal VGS flora, where these organisms may potentially act as a reservoir of fluoroquinolone resistance gene determinants for newly acquired and antibiotic-susceptible pathogens, particularly the Streptococcus milleri group.
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14. |
Itzek A,
Gillen CM,
Fulde M,
Friedrichs C,
Rodloff AC,
Chhatwal GS,
Nitsche-Schmitz DP,
( 2010 ) Contribution of plasminogen activation towards the pathogenic potential of oral streptococci. PMID : 21072208 : DOI : 10.1371/journal.pone.0013826 PMC : PMC2972214 Abstract >>
Oral streptococci are a heterogeneous group of human commensals, with a potential to cause serious infections. Activation of plasminogen has been shown to increase the virulence of typical human pathogenic streptococci such as S. pneumoniae. One important factor for plasminogen activation is the streptococcal �\-enolase. Here we report that plasminogen activation is also common in oral streptococci species involved in clinical infection and that it depends on the action of human plasminogen activators. The ability to activate plasminogen did not require full conservation of the internal plasminogen binding sequence motif FYDKERKVY of �\-enolase that was previously described as crucial for increased plasminogen binding, activation and virulence. Instead, experiments with recombinant �\-enolase variants indicate that the naturally occurring variations do not impair plasminogen binding. In spite of these variations in the internal plasminogen binding motif oral streptococci showed similar activation of plasminogen. We conclude that the pathomechanism of plasminogen activation is conserved in oral streptococci that cause infections in human. This may contribute to their opportunistic pathogenic character that is unfurled in certain niches.
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15. |
Wu R,
Zhou M,
Wu H,
( 2010 ) Purification and characterization of an active N-acetylglucosaminyltransferase enzyme complex from Streptococci. PMID : 20971868 : DOI : 10.1128/AEM.01434-10 PMC : PMC3008268 Abstract >>
A new family of bacterial serine-rich repeat glycoproteins can function as adhesins required for biofilm formation and pathogenesis in streptococci and staphylococci. Biogenesis of these proteins depends on a gene cluster coding for glycosyltransferases and accessory secretion proteins. Previous studies show that Fap1, a member of this family from Streptococcus parasanguinis, can be glycosylated by a protein glycosylation complex in a recombinant heterogeneous host. Here we report a tandem affinity purification (TAP) approach used to isolate and study protein complexes from native streptococci. This method demonstrated that a putative glycosyltransferase (Gtf2), which is essential for Fap1 glycosylation, readily copurified with another glycosyltransferase (Gtf1) from native S. parasanguinis. This result and the similar isolation of a homologous two-protein complex from Streptococcus pneumoniae indicate the biological relevance of the complexes to the glycosylation in streptococci. Furthermore, novel N-acetylglucosaminyltransferase activity was discovered for the complexes. Optimal activity required heterodimer formation and appears to represent a novel type of glycosylation.
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16. |
Ferrándiz MJ,
Ardanuy C,
Liñares J,
Balsalobre L,
García MT,
de la Campa AG,
( 2011 ) New species genetic approach to identify strains of mitis group streptococci that are donors of rifampin resistance to Streptococcus pneumoniae. PMID : 21041504 : DOI : 10.1128/AAC.00856-10 PMC : PMC3019657 Abstract >>
Eight rifampin-resistant streptococci of the mitis group were identified at the species level by using a concatenated 16S rRNA gene-sodA-rpoB-hlpA sequence. Characterization of their rpoB alleles showed single amino acid changes involved in rifampin resistance. Comparison of RpoB sequences from pneumococcal recombinant isolates, viridans isolates, and type strains revealed a species-specific amino acid signature, which allowed it to be ascertained that recombinant RpoBs were originated in genetic interchanges with Streptococcus mitis and Streptococcus oralis.
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17. |
Zhou M,
Li Y,
Peng Z,
Taylor JD,
Lee WC,
Bodey A,
Murray JW,
Alguel Y,
Bergeron J,
Bardiaux B,
Sawyer E,
Isaacson R,
Tagliaferri C,
Cota E,
Nilges M,
Simpson P,
Ruiz T,
Wu H,
Matthews S,
Ramboarina S,
Garnett JA,
( 2010 ) Structural insights into serine-rich fimbriae from Gram-positive bacteria. PMID : 20584910 : DOI : 10.1074/jbc.M110.128165 PMC : PMC2952246 Abstract >>
The serine-rich repeat family of fimbriae play important roles in the pathogenesis of streptococci and staphylococci. Despite recent attention, their finer structural details and precise adhesion mechanisms have yet to be determined. Fap1 (Fimbriae-associated protein 1) is the major structural subunit of serine-rich repeat fimbriae from Streptococcus parasanguinis and plays an essential role in fimbrial biogenesis, adhesion, and the early stages of dental plaque formation. Combining multidisciplinary, high resolution structural studies with biological assays, we provide new structural insight into adhesion by Fap1. We propose a model in which the serine-rich repeats of Fap1 subunits form an extended structure that projects the N-terminal globular domains away from the bacterial surface for adhesion to the salivary pellicle. We also uncover a novel pH-dependent conformational change that modulates adhesion and likely plays a role in survival in acidic environments.
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18. |
Zhou M,
Zhu F,
Dong S,
Pritchard DG,
Wu H,
( 2010 ) A novel glucosyltransferase is required for glycosylation of a serine-rich adhesin and biofilm formation by Streptococcus parasanguinis. PMID : 20164186 : DOI : 10.1074/jbc.M109.066928 PMC : PMC2852953 Abstract >>
Fap1-like serine-rich glycoproteins are conserved in streptococci, staphylococci, and lactobacilli, and are required for bacterial biofilm formation and pathogenesis. Glycosylation of Fap1 is mediated by a gene cluster flanking the fap1 locus. The key enzymes responsible for the first step of Fap1 glycosylation are glycosyltransferases Gtf1 and Gtf2. They form a functional enzyme complex that catalyzes the transfer of N-acetylglucosamine (GlcNAc) residues to the Fap1 polypeptide. However, until now nothing was known about the subsequent step in Fap1 glycosylation. Here, we show that the second step in Fap1 glycosylation is catalyzed by nucleotide-sugar synthetase-like (Nss) protein. The nss gene located upstream of fap1 is also highly conserved in streptococci and lactobacilli. Nss-deficient mutants failed to catalyze the second step of Fap1 glycosylation in vivo in Streptococcus parasanguinis and in a recombinant Fap1 glycosylation system. Nss catalyzed the direct transfer of the glucosyl residue to the GlcNAc-modified Fap1 substrate in vitro, demonstrating that Nss is a glucosyltransferase. Thus we renamed Nss as glucosyltransferase 3 (Gtf3). A gtf3 mutant exhibited a biofilm defect. Taken together, we conclude that this new glucosyltransferase mediates the second step of Fap1 glycosylation and is required for biofilm formation.
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19. |
Olson AB,
Sibley CD,
Schmidt L,
Wilcox MA,
Surette MG,
Corbett CR,
( 2010 ) Development of real-time PCR assays for detection of the Streptococcus milleri group from cystic fibrosis clinical specimens by targeting the cpn60 and 16S rRNA genes. PMID : 20164275 : DOI : 10.1128/JCM.02082-09 PMC : PMC2849594 Abstract >>
Cystic fibrosis (CF) is a multiorgan disease, with the majority of mortalities resulting from pulmonary failure due to repeated pulmonary exacerbations. Recently, members of the Streptococcus anginosus group (S. anginosus, S. constellatus, and S. intermedius), herein referred to as the "Streptococcus milleri group" (SMG) have been implicated as important etiological pathogens contributing to pulmonary exacerbations in CF patients. This is partly due to better microbiological detection of the SMG species through the development of a novel specific medium termed "McKay agar." McKay agar demonstrated that SMG has been an underreported respiratory pathogen contributing to lung exacerbations. Our aim was to develop a real-time PCR assay to expedite the detection of SMG within diagnostic samples. The cpn60 gene was chosen as a target, with all three members amplified using a single hybridization probe set. SMG strain analysis showed that speciation based on melting curve analysis allowed for the majority of the S. constellatus (96%), S. intermedius (94%), and S. anginosus (60%) strains to be correctly identified. To increase specificity for S. anginosus, two 16S rRNA real-time PCR assays were developed targeting the 16S rRNA gene. The 16s_SA assay is specific for S. anginosus (100%), while the 16s_SCI assay is specific for S. constellatus and S. intermedius (100%). These assays can detect <10 genome equivalents in pure culture and >10(4) genome equivalents in sputum samples, making this a great tool for assessment of the presence of SMG in complex polymicrobial samples. Novel molecular methods were developed providing detection ability for SMG, an emerging opportunistic pathogen.
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20. |
Glazunova OO,
Raoult D,
Roux V,
( 2009 ) Partial sequence comparison of the rpoB, sodA, groEL and gyrB genes within the genus Streptococcus. PMID : 19620365 : DOI : 10.1099/ijs.0.005488-0 Abstract >>
Phylogenetic analysis and species identification of members of the genus Streptococcus were carried out using partial sequence comparison of the 16S rRNA gene (1468-1478 bp), rpoB, encoding the beta subunit of RNA polymerase (659-680 bp), sodA, encoding the manganese-dependent superoxide dismutase (435-462 bp), groEL, encoding the 60 kDa heat-shock protein (757 bp), and gyrB, encoding the Beta subunit of DNA gyrase (458-461 bp). For the first time, most species within the genus Streptococcus were represented in the study (65 strains, representing 58 species and nine subspecies). Phylogenies inferred from rpoB, sodA, gyrB and groEL sequence comparisons were more discriminative than those inferred from 16S rRNA gene sequence comparison, and showed common clusters. The minimal interspecies divergence was 0.3, 2.7, 0, 2.5 and 3.4 % for the 16S rRNA gene, rpoB, sodA, gyrB and groEL, respectively. In general, groEL partial gene sequence comparison represented the best tool for identifying species and subspecies and for phylogenetic analysis.
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21. |
Glazunova OO,
Raoult D,
Roux V,
( 2010 ) Partial recN gene sequencing: a new tool for identification and phylogeny within the genus Streptococcus. PMID : 19880633 : DOI : 10.1099/ijs.0.018176-0 Abstract >>
Partial sequences of the recN gene (1249 bp), which encodes a recombination and repair protein, were analysed to determine the phylogenetic relationship and identification of streptococci. The partial sequences presented interspecies nucleotide similarity of 56.4-98.2 % and intersubspecies similarity of 89.8-98 %. The mean DNA sequence similarity of recN gene sequences (66.6 %) was found to be lower than those of the 16S rRNA gene (94.1 %), rpoB (84.6 %), sodA (74.8 %), groEL (78.1 %) and gyrB (73.2 %). Phylogenetically derived trees revealed six statistically supported groups: Streptococcus salivarius, S. equinus, S. hyovaginalis/S. pluranimalium/S. thoraltensis, S. pyogenes, S. mutans and S. suis. The 'mitis' group was not supported by a significant bootstrap value, but three statistically supported subgroups were noted: Streptococcus sanguinis/S. cristatus/S. sinensis, S. anginosus/S. intermedius/S. constellatus (the 'anginosus' subgroup) and S. mitis/S. infantis/S. peroris/S. oralis/S. oligofermentans/S. pneumoniae/S. pseudopneumoniae. The partial recN gene sequence comparison highlighted a high percentage of divergence between Streptococcus dysgalactiae subsp. dysgalactiae and S. dysgalactiae subsp. equisimilis. This observation is confirmed by other gene sequence comparisons (groEL, gyrB, rpoB and sodA). A high percentage of similarity was found between S. intermedius and S. constellatus after sequence comparison of the recN gene. To study the genetic diversity among the 'anginosus' subgroup, recN, groEL, sodA, gyrB and rpoB sequences were determined for 36 clinical isolates. The results that were obtained confirmed the high genetic diversity within this group of streptococci.
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22. |
Tazumi A,
Maeda Y,
Goldsmith CE,
Coulter WA,
Mason C,
Millar BC,
McCalmont M,
Rendall J,
Elborn JS,
Matsuda M,
Moore JE,
( 2009 ) Molecular characterization of macrolide resistance determinants [erm(B) and mef(A)] in Streptococcus pneumoniae and viridans group streptococci (VGS) isolated from adult patients with cystic fibrosis (CF). PMID : 19584106 : DOI : 10.1093/jac/dkp213 Abstract >>
Although long-term use of azithromycin has shown a significant clinical improvement for patients with cystic fibrosis (CF), its long-term effect on the susceptibility of commensal flora within CF airways has not yet been examined. We therefore suggest that long-term use of azithromycin increases macrolide resistance in commensal streptococci. Erythromycin susceptibility in naturally colonizing viridans group streptococci (VGS) was characterized, as well as macrolide resistance gene determinants through sequence analysis, in pneumococci (n = 15) and VGS [n = 84; i.e. Streptococcus salivarius (n = 30), Streptococcus mitis (n = 17), Streptococcus sanguinis (n = 11), Streptococcus oralis (n = 10), Streptococcus parasanguinis (n = 6), Streptococcus gordonii (n = 3), Streptococcus infantis (n = 3), Streptococcus cristatus (n = 2), Streptococcus anginosus (n = 1) and Streptococcus australis (n = 1)] isolated from sputum from 24 adult CF patients, who were on oral azithromycin therapy for at least the previous 7 months. Almost three-quarters of isolates (74; 74.7%) were resistant to erythromycin, whilst a further 15 (15.2%) had reduced susceptibility, leaving only 10 (10.1%) isolates susceptible to erythromycin. The majority (89.8%) were not susceptible to erythromycin, as demonstrated by possession of the erm(B) gene in 25/99 (25.3%), the mef(A) gene in 1/99 (1.0%), the mef(E) gene in 75/99 (75.8%) and both erm(B) and mef(E) genes simultaneously in 11/99 (11.1%). These results indicate that genotypic resistance for macrolides is common in VGS in adult CF patients, with efflux being over three times more frequent. Long-term treatment with azithromycin in CF patients may reduce antibiotic susceptibility in commensal VGS, where these organisms may potentially act as a reservoir of macrolide resistance determinants for newly acquired and antibiotic-susceptible pathogens.
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23. |
Warburton P,
Roberts AP,
Allan E,
Seville L,
Lancaster H,
Mullany P,
( 2009 ) Characterization of tet(32) genes from the oral metagenome. PMID : 18955517 : DOI : 10.1128/AAC.00788-08 PMC : PMC2612163 Abstract >>
tet(32) Was identified in three bacterial isolates and in metagenomic DNA from the human oral cavity. The regions immediately flanking the gene were found to have similarities to the mobile elements TnB1230 from Butyrivibrio fibrisolvens, ATE-3 from Arcanobacterium pyogenes, and CTn5 from Clostridium difficile.
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24. |
Bu S,
Li Y,
Zhou M,
Azadin P,
Zeng M,
Fives-Taylor P,
Wu H,
( 2008 ) Interaction between two putative glycosyltransferases is required for glycosylation of a serine-rich streptococcal adhesin. PMID : 18083807 : DOI : 10.1128/JB.01078-07 PMC : PMC2238222 Abstract >>
Fap1, a serine-rich glycoprotein, is essential for fimbrial biogenesis and biofilm formation of Streptococcus parasanguinis (formerly S. parasanguis). Fap1-like proteins are conserved in many streptococci and staphylococci and have been implicated in bacterial virulence. Fap1 contains two serine-rich repeat regions that are modified by O-linked glycosylation. A seven-gene cluster has been identified, and this cluster is implicated in Fap1 biogenesis. In this study, we investigated the initial step of Fap1 glycosylation by using a recombinant Fap1 as a model. This recombinant molecule has the same monosaccharide composition profile as the native Fap1 protein. Glycosyl linkage analyses indicated that N-acetylglucosamine (GlcNAc) is among the first group of sugar residues transferred to the Fap1 peptide. Two putative glycosyltransferases, Gtf1 and Gtf2, were essential for the glycosylation of Fap1 with GlcNAc-containing oligosaccharide(s) in both S. parasanguinis as well as in the Fap1 glycosylation system in Escherichia coli. Yeast two-hybrid analysis as well as in vitro and in vivo glutathione S-transferase pull-down assays demonstrated the two putative glycosyltransferases interacted with each other. The interaction domain was mapped to an N-terminal region of Gtf1 that was required for the Fap1 glycosylation. The data in this study suggested that the formation of the Gtf1 and Gtf2 complex was required for the initiation of the Fap1 glycosylation and that the N-terminal region of Gtf1 was necessary.
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25. |
Denapaite D,
Rieger M,
Köndgen S,
Brückner R,
Ochigava I,
Kappeler P,
Mätz-Rensing K,
Leendertz F,
Hakenbeck R,
( N/A ) Highly Variable Streptococcus oralis Strains Are Common among Viridans Streptococci Isolated from Primates. PMID : 27303717 : DOI : 10.1128/mSphere.00041-15 PMC : PMC4863584 Abstract >>
Viridans streptococci were obtained from primates (great apes, rhesus monkeys, and ring-tailed lemurs) held in captivity, as well as from free-living animals (chimpanzees and lemurs) for whom contact with humans is highly restricted. Isolates represented a variety of viridans streptococci, including unknown species. Streptococcus oralis was frequently isolated from samples from great apes. Genotypic methods revealed that most of the strains clustered on separate lineages outside the main cluster of human S. oralis strains. This suggests that S. oralis is part of the commensal flora in higher primates and evolved prior to humans. Many genes described as virulence factors in Streptococcus pneumoniae were present also in other viridans streptococcal genomes. Unlike in S. pneumoniae, clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) gene clusters were common among viridans streptococci, and many S. oralis strains were type PI-2 (pilus islet 2) variants. S. oralis displayed a remarkable diversity of genes involved in the biosynthesis of peptidoglycan (penicillin-binding proteins and MurMN) and choline-containing teichoic acid. The small noncoding cia-dependent small RNAs (csRNAs) controlled by the response regulator CiaR might contribute to the genomic diversity, since we observed novel genomic islands between duplicated csRNAs, variably present in some isolates. All S. oralis genomes contained a �]-N-acetyl-hexosaminidase gene absent in S. pneumoniae, which in contrast frequently harbors the neuraminidases NanB/C, which are absent in S. oralis. The identification of S. oralis-specific genes will help us to understand their adaptation to diverse habitats. IMPORTANCE Streptococcus pneumoniae is a rare example of a human-pathogenic bacterium among viridans streptococci, which consist of commensal symbionts, such as the close relatives Streptococcus mitis and S. oralis. We have shown that S. oralis can frequently be isolated from primates and a variety of other viridans streptococci as well. Genes and genomic islands which are known pneumococcal virulence factors are present in S. oralis and S. mitis, documenting the widespread occurrence of these compounds, which encode surface and secreted proteins. The frequent occurrence of CRISP-Cas gene clusters and a surprising variation of a set of small noncoding RNAs are factors to be considered in future research to further our understanding of mechanisms involved in the genomic diversity driven by horizontal gene transfer among viridans streptococci.
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26. |
Fenno JC,
LeBlanc DJ,
Fives-Taylor P,
( 1989 ) Nucleotide sequence analysis of a type 1 fimbrial gene of Streptococcus sanguis FW213. PMID : 2572555 : PMC : PMC259863 Abstract >>
A structural gene for type 1 fimbriae of Streptococcus sanguis FW213 was located within a 6-kilobase fragment cloned in Escherichia coli. A 1.6-kilobase internal fragment contains an open reading frame of 927 bases coding for an immunoreactive peptide of 34,349 daltons, which corresponds in size with an observed cytoplasmic form of fimbrial peptide (P. M. Fives-Taylor, F. L. Macrina, T. J. Pritchard, and S. J. Peene, Infect. Immun. 55:123-128, 1987). Disruption of the reading frame by insertional mutagenesis results in loss of immunoreactivity. Consensus sequences for initiation of transcription and translation were identified 5' to the coding region. Transcription terminator-like sequences were found downstream of the coding region. The deduced amino acid sequence of the cloned fimbrial peptide shows a strongly hydrophobic signal sequence at the amino terminus. The carboxyl-terminal region does not include a hydrophobic membrane anchor sequence such as has been reported for other gram-positive surface structures. A hydrophobic region of 12 to 14 amino acids downstream from the putative signal sequence cleavage site exhibits homology with the Streptococcus pyogenes type 6 M protein repetitive region A (S. K. Hollingshead, V. A. Fischetti, and J. R. Scott, J. Biol. Chem., 261:1677-1686, 1986). Functional homology at the level of protein secondary structure with Actinomyces viscosus T14V type 1 fimbriae (M. K. Yeung, B. M. Chassy, and J. O. Cisar, J. Bacteriol., 169:1678-1683, 1987) is proposed.
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27. |
Zhu F,
Zhang H,
Wu H,
( 2015 ) A conserved domain is crucial for acceptor substrate binding in a family of glucosyltransferases. PMID : 25404702 : DOI : 10.1128/JB.02267-14 PMC : PMC4285981 Abstract >>
Serine-rich repeat glycoproteins (SRRPs) are highly conserved in streptococci and staphylococci. Glycosylation of SRRPs is important for bacterial adhesion and pathogenesis. Streptococcus agalactiae is the leading cause of bacterial sepsis and meningitis among newborns. Srr2, an SRRP from S. agalactiae strain COH1, has been implicated in bacterial virulence. Four genes (gtfA, gtfB, gtfC, and gtfD) located downstream of srr2 share significant homology with genes involved in glycosylation of other SRRPs. We have shown previously that gtfA and gtfB encode two glycosyltransferases, GtfA and GtfB, that catalyze the transfer of GlcNAc residues to the Srr2 polypeptide. However, the function of other glycosyltransferases in glycosylation of Srr2 is unknown. In this study, we determined that GtfC catalyzed the direct transfer of glucosyl residues to Srr2-GlcNAc. The GtfC crystal structure was solved at 2.7 ? by molecular replacement. Structural analysis revealed a loop region at the N terminus as a putative acceptor substrate binding domain. Deletion of this domain rendered GtfC unable to bind to its substrate Srr2-GlcNAc, concurrently abolished the glycosyltransferase activity of GtfC, and also altered glycosylation of Srr2. Furthermore, deletion of the corresponding regions from GtfC homologs also abolished their substrate binding and enzymatic activity, indicating that this region is functionally conserved. In summary, we have determined that GtfC is important for the glycosylation of Srr2 and identified a conserved loop region that is crucial for acceptor substrate binding from GtfC homologs in streptococci. These findings shed new mechanistic insight into this family of glycosyltransferases.
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28. |
Carvalho Mda G,
Pimenta FC,
Moura I,
Roundtree A,
Gertz RE,
Li Z,
Jagero G,
Bigogo G,
Junghae M,
Conklin L,
Feikin DR,
Breiman RF,
Whitney CG,
Beall BW,
( 2013 ) Non-pneumococcal mitis-group streptococci confound detection of pneumococcal capsular serotype-specific loci in upper respiratory tract. PMID : 23825797 : DOI : 10.7717/peerj.97 PMC : PMC3698467 Abstract >>
We performed culture-based and PCR-based tests for pneumococcal identification and serotyping from carriage specimens collected in rural and urban Kenya. Nasopharyngeal specimens from 237 healthy children <5 years old (C-NPs) and combined nasopharyngeal/oropharyngeal specimens from 158 adults (A-NP/OPs, 118 HIV-positive) were assessed using pneumococcal isolation (following broth culture enrichment) with Quellung-based serotyping, real-time lytA-PCR, and conventional multiplexed PCR-serotyping (cmPCR). Culture-based testing from C-NPs, HIV-positive A-NP/OPs, and HIV-negative A-NP/OPs revealed 85.2%, 40.7%, and 12.5% pneumococcal carriage, respectively. In contrast, cmPCR serotypes were found in 93.2%, 98.3%, and 95.0% of these sets, respectively. Two of 16 lytA-negative C-NPs and 26 of 28 lytA-negative A-NP/OPs were cmPCR-positive for 1-10 serotypes (sts) or serogroups (sgs). A-NP/OPs averaged 5.5 cmPCR serotypes/serogroups (5.2 in HIV-positive, 7.1 in HIV-negative) and C-NPs averaged 1.5 cmPCR serotypes/serogroups. cmPCR serotypes/serogroups from lytA-negative A-NP/OPs included st2, st4, sg7F/7A, sg9N/9L, st10A, sg10F/10C/33C, st13, st17F, sg18C/18A/18B/18F, sg22F/22A, and st39. Nine strains of three non-pneumococcal species (S. oralis, S. mitis, and S. parasanguinis) (7 from A-OP, 1 from both A-NP and A-OP, and 1 from C-NP) were each cmPCR-positive for one of 7 serotypes/serogroups (st5, st13, sg15A/15F, sg10F/10C/33C, sg33F/33A/37, sg18C/18A/18B/18F, sg12F/12A/12B/ 44/46) with amplicons revealing 83.6-99.7% sequence identity to pneumococcal references. In total, 150 cmPCR amplicons from carriage specimens were sequenced, including 25 from lytA-negative specimens. Amplicon sequences derived from specimens yielding a pneumococcal isolate with the corresponding serotype were identical or highly conserved (>98.7%) with the reference cmPCR amplicon for the st, while cmPCR amplicons from lytA-negative specimens were generally more divergent. Separate testing of 56 A-OPs and 56 A-NPs revealed that ?94% of the positive cmPCR results from A-NP/OPs were from OP microbiota. In contrast, A-NPs yielded >2-fold more pneumococcal isolates than A-OPs. Verified and suspected non-pneumococcal cmPCR serotypes/serogroups appeared to be relatively rare in C-NPs and A-NPs compared to A-OPs. Our findings indicate that non-pneumococcal species can confound serotype-specific PCR and other sequence-based assays due to evolutionarily conserved genes most likely involved in biosynthesis of surface polysaccharide structures.
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29. |
Naveen Kumar V,
van der Linden M,
Menon T,
Nitsche-Schmitz DP,
( 2014 ) Viridans and bovis group streptococci that cause infective endocarditis in two regions with contrasting epidemiology. PMID : 24220665 : DOI : 10.1016/j.ijmm.2013.10.004 Abstract >>
Viridans group (VGS) or bovis group streptococci (BGS) are the major causes for streptococcal infective endocarditis (IE). However, the causative isolates are not sufficiently characterized. Using multilocus sequence analysis we have examined VGS and BGS (VGS/BGS) isolates that caused IE in southern India and Germany, two distant geographic regions with a contrasting IE epidemiology. Other than in Germany, the majority of patients (68%) in Chennai, southern India had an underlying rheumatic heart disease (RHD). In accord with the high prevalence of RHD in the younger population and with the expansive age structure of India, the median age (24 years) of the VGS/BGS endocarditis patients was lower than in Germany (63 years), where RHD is rare and the age structure is contractive. Both in Germany and in southern India, the majority of cases were caused by mitis group streptococci, however, with considerable differences in the spectra of causative (sub)species. BGS endocarditis was more frequent in Germany. The spectrum of VGS/BGS that cause IE differs considerably between distant geographic regions in which different predisposing conditions prevail. Therefore, improved microbiological diagnosis in IE may facilitate determination of the optimal therapy.
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30. |
Kitten T,
Munro CL,
Zollar NQ,
Lee SP,
Patel RD,
( 2012 ) Oral streptococcal bacteremia in hospitalized patients: taxonomic identification and clinical characterization. PMID : 22189124 : DOI : 10.1128/JCM.06438-11 PMC : PMC3295135 Abstract >>
Oral streptococci have been associated with systemic diseases, including infective endocarditis and neutropenic bacteremia. We analyzed 58 recent oral streptococcal bloodstream isolates, and we obtained clinical and demographic data for source patients. The sodA gene was found to be a better target than the 16S-23S rRNA internal transcribed spacer for DNA sequence-based species identification. Together, Streptococcus mitis and Streptococcus oralis were significantly more likely than the 12 combined remaining species to be isolated from neutropenic patients.
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31. |
Zhu F,
Erlandsen H,
Ding L,
Li J,
Huang Y,
Zhou M,
Liang X,
Ma J,
Wu H,
( 2011 ) Structural and functional analysis of a new subfamily of glycosyltransferases required for glycosylation of serine-rich streptococcal adhesins. PMID : 21653318 : DOI : 10.1074/jbc.M110.208629 PMC : PMC3143663 Abstract >>
Serine-rich repeat glycoproteins (SRRPs) are a growing family of bacterial adhesins found in many streptococci and staphylococci; they play important roles in bacterial biofilm formation and pathogenesis. Glycosylation of this family of adhesins is essential for their biogenesis. A glucosyltransferase (Gtf3) catalyzes the second step of glycosylation of a SRRP (Fap1) from an oral streptococcus, Streptococcus parasanguinis. Although Gtf3 homologs are highly conserved in SRRP-containing streptococci, they share minimal homology with functionally known glycosyltransferases. We report here the 2.3 ? crystal structure of Gtf3. The structural analysis indicates that Gtf3 forms a tetramer and shares significant structural homology with glycosyltransferases from GT4, GT5, and GT20 subfamilies. Combining crystal structural analysis with site-directed mutagenesis and in vitro glycosyltransferase assays, we identified residues that are required for UDP- or UDP-glucose binding and for oligomerization of Gtf3 and determined their contribution to the enzymatic activity of Gtf3. Further in vivo studies revealed that the critical amino acid residues identified by the structural analysis are crucial for Fap1 glycosylation in S. parasanguinis in vivo. Moreover, Gtf3 homologs from other streptococci were able to rescue the gtf3 knock-out mutant of S. parasanguinis in vivo and catalyze the sugar transfer to the modified SRRP substrate in vitro, demonstrating the importance and conservation of the Gtf3 homologs in glycosylation of SRRPs. As the Gtf3 homologs only exist in SRRP-containing streptococci, we conclude that the Gtf3 homologs represent a unique subfamily of glycosyltransferases.
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32. |
Wu R,
Wu H,
( 2011 ) A molecular chaperone mediates a two-protein enzyme complex and glycosylation of serine-rich streptococcal adhesins. PMID : 21862581 : DOI : 10.1074/jbc.M111.239350 PMC : PMC3186434 Abstract >>
Serine-rich repeat glycoproteins identified from streptococci and staphylococci are important for bacterial adhesion and biofilm formation. Two putative glycosyltransferases, Gtf1 and Gtf2, from Streptococcus parasanguinis form a two-protein enzyme complex that is required for glycosylation of a serine-rich repeat adhesin, Fap1. Gtf1 is a glycosyltransferase; however, the function of Gtf2 is unknown. Here, we demonstrate that Gtf2 enhances the enzymatic activity of Gtf1 by its chaperone-like property. Gtf2 interacted with Gtf1, mediated the subcellular localization of Gtf1, and stabilized Gtf1. Deletion of invariable amino acid residues in a conserved domain of unknown function (DUF1975) at the N terminus of Gtf2 had a greater impact on Fap1 glycosylation than deletion of the C-terminal non-DUF1975 residues. The DUF1975 deletions concurrently reduced the interaction between Gtf1 and Gtf2, altered the subcellular localization of Gtf1, and destabilized Gtf1, suggesting that DUF1975 is crucial for the chaperone activity of Gtf2. Homologous GtfA and GtfB from Streptococcus agalactiae rescued the glycosylation defect in the gtf1gtf2 mutant; like Gtf2, GtfB also possesses chaperone-like activity. Taken together, our studies suggest that Gtf2 and its homologs possess the conserved molecular chaperone activity that mediates protein glycosylation of bacterial adhesins.
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33. |
Fenno JC,
Shaikh A,
Spatafora G,
Fives-Taylor P,
( 1995 ) The fimA locus of Streptococcus parasanguis encodes an ATP-binding membrane transport system. PMID : 7596287 : DOI : 10.1111/j.1365-2958.1995.tb02355.x Abstract >>
The gene encoding fimA, a 36 kDa fimbrial adhesion of Streptococcus parasanguis FW213, is highly conserved in all four genetic groups of sanguis streptococci. FimA-like peptides were produced by all strains tested. The nucleotide sequence directly upstream of fimA contains two open reading frames, ORF5 and ORF1, whose deduced protein products are homologous to members of a superfamily of ATP-binding cassette membrane transport proteins, including both prokaryotic and eukaryotic uptake and export systems. The amino acid sequence of FimA contains the consensus prolipoprotein cleavage site (LxxC) common to the 'periplasmic' binding proteins of Gram-positive transport systems. The deduced product of ORF5 is a 28.6 kDa membrane-associated protein that has the consensus binding site for ATP (GxxGxGKS). It shares significant homology with AmiE of Streptococcus pneumoniae as well as with Escherichia coli proteins involved in iron(III) uptake. Allelic-replacement mutagenesis of ORF5 resulted in greatly increased resistance to aminopterin. These data demonstrate functionality with the amiE locus as well. The deduced product of ORF1 is an extremely hydrophobic integral membrane protein of 30.8 kDa with a pattern of six potential membrane-spanning regions, typical of a component of these types of transport system. The nucleotide sequence downstream of fimA, ORF3, encodes a 20 kDa protein having 78% identity with the 20 kDa protein encoded downstream of ssaB, a fimA homologue in S. sanguis 12. It also exhibits significant homology with bacterioferritin co-migratory protein (Bcp) of E. coli K-12. Allelic-replacement mutagenesis in the fimA locus of FW213 showed that (i) expression of fimA was initiated at a site far upstream of the fimA start codon, and (ii) expression of fimA was not linked to expression of ORF3. Northern blots probed with internal fragments of ORF5, ORF1, fimA or ORF3 hybridized to the same transcript of 3.3 kb, which suggested that these loci were transcribed as a polycistronic message. The ORF3 probe also hybridized to a 540 bp transcript consistent with the size of ORF3 alone and supportive of the mutagenesis data of non-linkage. Strains mutated in fimA continued to produce fimbriae, indicating that FimA was not the fimbrial structural subunit. Immunoelectron microscopy revealed FimA was localized at the tips of the fimbriae of FW213. This is the first study that demonstrates that an adhesin which binds a bacterial cell to a substrate is associated with an ATP-binding cassette.
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34. |
Jiang X,
Hall AB,
Arthur TD,
Plichta DR,
Covington CT,
Poyet M,
Crothers J,
Moses PL,
Tolonen AC,
Vlamakis H,
Alm EJ,
Xavier RJ,
( 2019 ) Invertible promoters mediate bacterial phase variation, antibiotic resistance, and host adaptation in the gut. PMID : 30630933 : DOI : 10.1126/science.aau5238 PMC : PMC6543533 Abstract >>
Phase variation, the reversible alternation between genetic states, enables infection by pathogens and colonization by commensals. However, the diversity of phase variation remains underexplored. We developed the PhaseFinder algorithm to quantify DNA inversion-mediated phase variation. A systematic search of 54,875 bacterial genomes identified 4686 intergenic invertible DNA regions (invertons), revealing an enrichment in host-associated bacteria. Invertons containing promoters often regulate extracellular products, underscoring the importance of surface diversity for gut colonization. We found invertons containing promoters regulating antibiotic resistance genes that shift to the ON orientation after antibiotic treatment in human metagenomic data and in vitro, thereby mitigating the cost of antibiotic resistance. We observed that the orientations of some invertons diverge after fecal microbiota transplant, potentially as a result of individual-specific selective forces.
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35. |
Redanz S,
Masilamani R,
Cullin N,
Giacaman RA,
Merritt J,
Kreth J,
( 2018 ) Distinct Regulatory Role of Carbon Catabolite Protein A (CcpA) in Oral Streptococcal spxB Expression. PMID : 29378884 : DOI : 10.1128/JB.00619-17 PMC : PMC5869474 Abstract >>
Pyruvate oxidase (SpxB)-dependent H2O2 production is under the control of carbon catabolite protein A (CcpA) in the oral species Streptococcus sanguinis and Streptococcus gordonii Interestingly, both species react differently to the presence of the preferred carbohydrate source glucose. S. gordonii CcpA-dependent regulation of spxB follows classical carbon catabolite repression. Conversely, spxB expression in S. sanguinis is not influenced by glucose but is repressed by CcpA. Here, we constructed strains expressing the heterologous versions of CcpA or the spxB promoter region to learn if the distinct regulation of spxB expression is transferable from S. gordonii to S. sanguinis and vice versa. While cross-species binding of CcpA to the spxB promoter is conserved in vitro, we were unable to swap the species-specific regulation. This suggests that a regulatory mechanism upstream of CcpA most likely is responsible for the observed difference in spxB expression. Moreover, the overall ecological significance of differential spxB regulation in the presence of various glucose concentrations was tested with additional oral streptococcus isolates and demonstrated that carbohydrate-dependent and carbohydrate-independent mechanisms exist to control expression of spxB in the oral biofilm. Overall, our data demonstrate the unexpected finding that metabolic pathways between two closely related oral streptococcal species can be regulated differently despite an exceptionally high DNA sequence identity.IMPORTANCE Polymicrobial diseases are the result of interactions among the residential microbes, which can lead to a dysbiotic community. Streptococcus sanguinis and Streptococcus gordonii are considered commensal species that are present in the healthy dental biofilm. Both species are able to produce significant amounts of H2O2 via the enzymatic action of the pyruvate oxidase SpxB. H2O2 is able to inhibit species associated with oral diseases. SpxB and its gene-regulatory elements present in both species are highly conserved. Nonetheless, a differential response to the presence of glucose was observed. Here, we investigate the mechanisms that lead to this differential response. Detailed knowledge of the regulatory mechanisms will aid in a better understanding of oral disease development and how to prevent dysbiosis.
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36. |
Wyllie AL,
Pannekoek Y,
Bovenkerk S,
van Engelsdorp Gastelaars J,
Ferwerda B,
van de Beek D,
Sanders EAM,
Trzci?ski K,
van der Ende A,
( 2017 ) Sequencing of the variable region of rpsB to discriminate between Streptococcus pneumoniae and other streptococcal species. PMID : 28931649 : DOI : 10.1098/rsob.170074 PMC : PMC5627049 Abstract >>
The vast majority of streptococci colonizing the human upper respiratory tract are commensals, only sporadically implicated in disease. Of these, the most pathogenic is Mitis group member, Streptococcus pneumoniae Phenotypic and genetic similarities between streptococci can cause difficulties in species identification. Using ribosomal S2-gene sequences extracted from whole-genome sequences published from 501 streptococci, we developed a method to identify streptococcal species. We validated this method on non-pneumococcal isolates cultured from cases of severe streptococcal disease (n = 101) and from carriage (n = 103), and on non-typeable pneumococci from asymptomatic individuals (n = 17) and on whole-genome sequences of 1157 pneumococcal isolates from meningitis in the Netherlands. Following this, we tested 221 streptococcal isolates in molecular assays originally assumed specific for S. pneumoniae, targeting cpsA, lytA, piaB, ply, Spn9802, zmpC and capsule-type-specific genes. Cluster analysis of S2-sequences showed grouping according to species in line with published phylogenies of streptococcal core genomes. S2-typing convincingly distinguished pneumococci from non-pneumococcal species (99.2% sensitivity, 100% specificity). Molecular assays targeting regions of lytA and piaB were 100% specific for S. pneumoniae, whereas assays targeting cpsA, ply, Spn9802, zmpC and selected serotype-specific assays (but not capsular sequence typing) showed a lack of specificity. False positive results were over-represented in species associated with carriage, although no particular confounding signal was unique for carriage isolates.
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37. |
( 1998 ) Isolation and characterization of Fap1, a fimbriae-associated adhesin of Streptococcus parasanguis FW213. PMID : 9632253 : DOI : 10.1046/j.1365-2958.1998.00805.x Abstract >>
An adhesin of Streptococcus parasanguis FW213, a primary colonizer of the tooth surface, has been purified from the culture medium by immunoaffinity chromatography. The purified protein has a molecular mass of 200 kDa and stains positively for carbohydrate. The amino-terminal sequence indicated that this protein represented a unique streptococcal surface protein. Immunogold labelling of the bacterium indicated that this protein was associated with fimbriae and designated Fap1 (fimbriae-associated protein). A polymerase chain reaction (PCR) product based on the amino terminus of Fap1 was used to probe an FW213 genomic library. A 9 kb fragment containing the fap1 gene was isolated and 2.5 kb have been sequenced. Generation of fap1 mutants by a single cross-over (Campbell insertion) or a non-polar allelic exchange abolished the expression of Fap1. The inactivation of fap1 resulted in a dramatic reduction in the expression of the long peritrichous fimbriae and adhesion to saliva-coated hydroxylapatite (SHA). Northern blots probed with an internal gene fragment of fap1 hybridized to a 9 kb transcript, which suggests that fap1 is transcribed as a polycistronic message. These data demonstrate that Fap1 is a unique streptococcal adhesin that is involved in the assembly of S. parasanguis FW213 fimbriae and adhesion to SHA.
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( 1998 ) Identification of streptococci to species level by sequencing the gene encoding the manganese-dependent superoxide dismutase. PMID : 9431917 : PMC : PMC124804 Abstract >>
We have used a PCR assay based on the use of degenerate primers in order to characterize an internal fragment (sodA(int)) representing approximately 85% of the genes encoding the manganese-dependent superoxide dismutase in various streptococcal type strains (S. acidominimus, S. agalactiae, S. alactolyticus, S. anginosus, S. bovis, S. constellatus, S. canis, S. cricetus, S. downei, S. dysgalactiae, S. equi subsp. equi, S. equi subsp. zooepidemicus, S. equinus, S. gordonii, S. iniae, S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguis, S. pneumoniae, S. porcinus, S. pyogenes, S. salivarius, S. sanguis, S. sobrinus, S. suis, S. thermophilus, and S. vestibularis). Phylogenetic analysis of these sodA(int) fragments yields an evolutionary tree having a topology similar to that of the tree constructed with the 16S rRNA sequences. We have shown that clinical isolates could be identified by determining the positions of their sodA(int) fragments on the phylogenetic tree of the sodA(int) fragments of the type species. We propose this method for the characterization of strains that cannot be assigned to a species on the basis of their conventional phenotypic reactions.
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