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1. Ivey  DM, Krulwich  TA,     ( 1992 )

Two unrelated alkaliphilic Bacillus species possess identical deviations in sequence from those of other prokaryotes in regions of F0 proposed to be involved in proton translocation through the ATP synthase.

Research in microbiology 143 (5)
PMID : 1448623  :  
Abstract >>
The a and c subunits of two unrelated alkaliphilic Bacillus species contain unusual motifs in regions previously implicated by others in H(+)-coupled oxidative phosphorylation. The facultative alkaliphile B. firmus OF4 apparently does not contain genes encoding an alternative F0, supporting other evidence that a single species of proton-translocating F1F0-ATPase catalyses oxidative phosphorylation both at low and high pH. The unusual F0 sequence motifs may be part of the adaptation of the extreme alkaliphiles to growth at very high pH.
KeywordMeSH Terms
2. Ivey  DM, Guffanti  AA, Shen  Z, Kudyan  N, Krulwich  TA,     ( 1992 )

The cadC gene product of alkaliphilic Bacillus firmus OF4 partially restores Na+ resistance to an Escherichia coli strain lacking an Na+/H+ antiporter (NhaA).

Journal of bacteriology 174 (15)
PMID : 1321115  :   DOI  :   10.1128/jb.174.15.4878-4884.1992     PMC  :   PMC206298    
Abstract >>
A 5.6-kb fragment of alkaliphilic Bacillus firmus OF4 DNA was isolated by screening a library of total genomic DNA constructed in pGEM3Zf(+) for clones that reversed the Na+ sensitivity of Escherichia coli NM81, in which the gene encoding an Na+/H+ antiporter (NhaA) is deleted (E. Padan, N. Maisler, D. Taglicht, R. Karpel, and S. Schuldiner, J. Biol. Chem. 264:20297-20302, 1989). The plasmid, designated pJB22, contained two genes that apparently encode transposition functions and two genes that are apparent homologs of the cadA and cadC genes of cadmium resistance-conferring plasmid pI258 of Staphylococcus aureus. E. coli NM81 transformed with pJB22 had enhanced membrane Na+/H+ antiporter activity that was cold labile and that decreased very rapidly following isolation of everted vesicles. Subclones of pJB22 containing cadC as the only intact gene showed identical complementation patterns in vivo and in vitro. The cadC gene product of S. aureus has been proposed to act as an accessory protein for the Cd2+ efflux ATPase (CadA) (K. P. Yoon and S. Silver, J. Bacteriol. 173:7636-7642, 1991); perhaps the alkaliphile CadC also binds Na+ and enhances antiporter activity by delivering a substrate to an integral membrane antiporter. A 6.0-kb fragment overlapping the pJB22 insert was isolated to complete the sequence of the cadA homolog. A partial sequence of a region approximately 2 kb downstream of the cadA locus shares sequence similarity with plasmids from several gram-positive bacteria. These results suggest that the region of alkaliphile DNA containing the cadCA locus is present on a transposon that could reside on a heretofore-undetected endogenous plasmid.
KeywordMeSH Terms
3. Chen  W, He  B, Lou  X, Guan  F, Ye  C,     ( 1998 )

[Sequencing of a beta-amylase gene from Bacillus firmus].

Wei sheng wu xue bao = Acta microbiologica Sinica 38 (2)
PMID : 12549376  :  
Abstract >>
The gene encoding a beta-amylase from Bacillus firmus 725 was sequenced. The sequenced DNA of 2012 bp contains one open reading frame of 1406 nucleotides without a translation stop codon. The deduced amino acid sequence homology with those known bacterial and some plant beta-amylase was 98% for Bacillus polymyxa 72, 98% for Bacillus polymyxa ATCC8523, 82% for Bacillus circulans, 54% for Clostridium thermosulfurogenes, 49% for Bacillus cereus BQ10-S1, 50% for Bacillus cereus var. mycoides, 36% for barley, and 36% for soybean Eleven well-conserved regions were found among the amino acid sequences of the nine beta-amylases.
KeywordMeSH Terms
4. Takami  H, Han  CG, Takaki  Y, Ohtsubo  E,     ( 2001 )

Identification and distribution of new insertion sequences in the genome of alkaliphilic Bacillus halodurans C-125.

Journal of bacteriology 183 (14)
PMID : 11418576  :   DOI  :   10.1128/JB.183.14.4345-4356.2001     PMC  :   PMC95325    
Abstract >>
Fifteen kinds of new insertion sequences (ISs), IS641 to IS643, IS650 to IS658, IS660, IS662, and IS663, and a group II intron (Bh.Int) were identified in the 4,202,352-bp genome of alkaliphilic Bacillus halodurans C-125. Out of 120 ISs identified in the C-125 genome, 29 were truncated, indicating the occurrence of internal rearrangements of the genome. The ISs other than IS650, IS653, IS660, and IS663 generated a 2- to 9-bp duplication of the target site sequence, and the ISs other than IS650, IS653, and IS657 carry 14- to 64-bp inverted repeats. Sequence analysis revealed that six kinds of ISs (IS642, IS643, IS654, IS655, IS657, and IS658) belong to a separate IS family (IS630, IS21, IS256, IS3, IS200/IS605, and IS30, respectively) as a new member. Also, IS651 and IS652 were characterized as new members of the ISL3 family. Significant similarity was found between the transposase (Tpase) sequences between IS650 and IS653 (78.2%), IS651 and IS652 (56.3%), IS656 and IS662 (71.0%), and IS660 and IS663 (44.5%), but the others showed no similarity to one another. Tpases in 28 members of IS651 in the C-125 genome were found to have become diversified. Most of the IS elements widely distributed throughout the genome were inserted in noncoding regions, although some genes, such as those coding for an ATP-binding cassette transporter/permease, a response regulator, and L-indole 2-dehydrogenase, have been mutated through the insertion of IS elements. It is evident, however, that not all IS elements have transposed and caused rearrangements of the genome in the past 17 years during which strain C-125 was subcultured under neutral and alkaline conditions.
KeywordMeSH Terms
DNA Transposable Elements
DNA, Bacterial
Genome, Bacterial
5. Takami  H, Nakasone  K, Takaki  Y, Maeno  G, Sasaki  R, Masui  N, Fuji  F, Hirama  C, Nakamura  Y, Ogasawara  N, Kuhara  S, Horikoshi  K,     ( 2000 )

Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis.

Nucleic acids research 28 (21)
PMID : 11058132  :   DOI  :   10.1093/nar/28.21.4317     PMC  :   PMC113120    
Abstract >>
The 4 202 353 bp genome of the alkaliphilic bacterium Bacillus halodurans C-125 contains 4066 predicted protein coding sequences (CDSs), 2141 (52.7%) of which have functional assignments, 1182 (29%) of which are conserved CDSs with unknown function and 743 (18. 3%) of which have no match to any protein database. Among the total CDSs, 8.8% match sequences of proteins found only in Bacillus subtilis and 66.7% are widely conserved in comparison with the proteins of various organisms, including B.subtilis. The B. halodurans genome contains 112 transposase genes, indicating that transposases have played an important evolutionary role in horizontal gene transfer and also in internal genetic rearrangement in the genome. Strain C-125 lacks some of the necessary genes for competence, such as comS, srfA and rapC, supporting the fact that competence has not been demonstrated experimentally in C-125. There is no paralog of tupA, encoding teichuronopeptide, which contributes to alkaliphily, in the C-125 genome and an ortholog of tupA cannot be found in the B.subtilis genome. Out of 11 sigma factors which belong to the extracytoplasmic function family, 10 are unique to B. halodurans, suggesting that they may have a role in the special mechanism of adaptation to an alkaline environment.
KeywordMeSH Terms
Genome, Bacterial
6. Gilmour  R, Messner  P, Guffanti  AA, Kent  R, Scheberl  A, Kendrick  N, Krulwich  TA,     ( 2000 )

Two-dimensional gel electrophoresis analyses of pH-dependent protein expression in facultatively alkaliphilic Bacillus pseudofirmus OF4 lead to characterization of an S-layer protein with a role in alkaliphily.

Journal of bacteriology 182 (21)
PMID : 11029415  :   DOI  :   10.1128/jb.182.21.5969-5981.2000     PMC  :   PMC94729    
Abstract >>
The large majority of proteins of alkaliphilic Bacillus pseudofirmus OF4 grown at pH 7.5 and 10.5, as studied by two-dimensional gel electrophoresis analyses, did not exhibit significant pH-dependent variation. A new surface layer protein (SlpA) was identified in these studies. Although the prominence of some apparent breakdown products of SlpA in gels from pH 10.5-grown cells led to discovery of the alkaliphile S-layer, the largest and major SlpA forms were present in large amounts in gels from pH 7.5-grown cells as well. slpA RNA abundance was, moreover, unchanged by growth pH. SlpA was similar in size to homologues from nonalkaliphiles but contained fewer Arg and Lys residues. An slpA mutant strain (RG21) lacked an exterior S-layer that was identified in the wild type by electron microscopy. Electrophoretic analysis of whole-cell extracts further indicated the absence of a 90-kDa band in the mutant. This band was prominent in wild-type extracts from both pH 7.5- and 10.5-grown cells. The wild type grew with a shorter lag phase than RG21 at either pH 10.5 or 11 and under either Na(+)-replete or suboptimal Na(+) concentrations. The extent of the adaptation deficit increased with pH elevation and suboptimal Na(+). By contrast, the mutant grew with a shorter lag and faster growth rate than the wild type at pH 7. 5 under Na(+)-replete and suboptimal Na(+) conditions, respectively. Logarithmically growing cells of the two strains exhibited no significant differences in growth rate, cytoplasmic pH regulation, starch utilization, motility, Na(+)-dependent transport of alpha-aminoisobutyric acid, or H(+)-dependent synthesis of ATP. However, the capacity for Na(+)-dependent pH homeostasis was diminished in RG21 upon a sudden upward shift of external pH from 8. 5 to 10.5. The energy cost of retaining the SlpA layer at near-neutral pH is apparently adverse, but the constitutive presence of SlpA enhances the capacity of the extremophile to adjust to high pH.
KeywordMeSH Terms
7. Lagunavicius  A, Vilkaitis  G, Sasnauskas  G, Sapranauskas  R,     ( 2000 )

Novel subtype of type IIs restriction enzymes. BfiI endonuclease exhibits similarities to the EDTA-resistant nuclease Nuc of Salmonella typhimurium.

The Journal of biological chemistry 275 (40)
PMID : 10880511  :   DOI  :   10.1074/jbc.M003350200    
Abstract >>
The type IIs restriction enzyme BfiI recognizes the non-palindromic nucleotide sequence 5'-ACTGGG-3' and cleaves complementary DNA strands 5/4 nucleotides downstream of the recognition sequence. The genes coding for the BfiI restriction-modification (R-M) system were cloned/sequenced and biochemical characterization of BfiI restriction enzyme was performed. The BfiI R-M system contained three proteins: two N4-methylcytosine methyltransferases and a restriction enzyme. Sequencing of bisulfite-treated methylated DNA indicated that each methyltransferase modifies cytosines on opposite strands of the recognition sequence. The N-terminal part of the BfiI restriction enzyme amino acid sequence revealed intriguing similarities to an EDTA-resistant nuclease of Salmonella typhimurium. Biochemical analyses demonstrated that BfiI, like the nuclease of S. typhimurium, cleaves DNA in the absence of Mg(2+) ions and hydrolyzes an artificial substrate bis(p-nitrophenyl) phosphate. However, unlike the nonspecific S. typhimurium nuclease, BfiI restriction enzyme cleaves DNA specifically. We propose that the DNA-binding specificity of BfiI stems from the C-terminal part of the protein. The catalytic N-terminal subdomain of BfiI radically differs from that of type II restriction enzymes and is presumably similar to the EDTA-resistant nonspecific nuclease of S. typhimurium; therefore, BfiI did not require metal ions for catalysis. We suggest that BfiI represents a novel subclass of type IIs restriction enzymes that differs from the archetypal FokI endonuclease by the fold of its cleavage domain, the domain location, and reaction mechanism.
KeywordMeSH Terms
Bacterial Proteins
Micrococcal Nuclease
8. Guffanti  AA, Krulwich  TA,     ( 1999 )

Sequence analysis and functional studies of a chromosomal region of alkaliphilic Bacillus firmus OF4 encoding an ABC-type transporter with similarity of sequence and Na+ exclusion capacity to the Bacillus subtilis NatAB transporter.

Extremophiles : life under extreme conditions 3 (2)
PMID : 10356997  :  
Abstract >>
A 14.1-kb DNA fragment was cloned from a lambda library containing inserts of DNA from alkaliphilic Bacillus firmus OF4 on the basis of its hybridization to a probe from a previously sequenced alkaliphile homolog of the natA gene from Bacillus subtilis. Sequence analysis of the entire fragment revealed that, as in B. subtilis, the natA gene was part of a putative gene locus encoding an ABC-type transporter. In the alkaliphile, the transporter involved three genes, designated natCAB, that are part of a larger operon of unknown function. This is in contrast to the two-gene natAB operon and to another homolog from B. subtilis, the yhaQP genes. Like natAB, however, the alkaliphile natCAB catalyzes Na+ extrusion as assessed in a mutant of Escherichia coli that is deficient in Na+ extrusion. The full 14.1-kb fragment of alkaliphile DNA sequenced in this study contained several probable operons as well as likely monocistronic units. Among the 17 predicted ORFs apart from natCAB were acsA, a homolog of a halobacterial gene encoding acetylCoA synthetase; sspA, a homolog of a small acid-soluble spore protein; and malK, an ATP-binding component that was unaccompanied by candidates for other mal transport genes but was able to complement a malK-deficient mutant of E. coli. No strong candidates for genes encoding a secondary Na+/H+ antiporter were found in the fragment, either from the sequence analysis or from analyses of complementation of E. coli mutants by subclones of the 14.1-kb piece. There were a total of 12 ORFs whose closest and significant homologs were genes from B. subtilis; of these, one-third were in apparently different contexts, as assessed by the sequence of the neighboring genes, than the B. subtilis homologs.
KeywordMeSH Terms
Amino Acid Transport Systems, Neutral
Escherichia coli Proteins
9. Ivey  DM, Guffanti  AA, Bossewitch  JS, Padan  E, Krulwich  TA,     ( 1991 )

Molecular cloning and sequencing of a gene from alkaliphilic Bacillus firmus OF4 that functionally complements an Escherichia coli strain carrying a deletion in the nhaA Na+/H+ antiporter gene.

The Journal of biological chemistry 266 (34)
PMID : 1660475  :  
Abstract >>
A gene has been cloned from a DNA library from alkaliphilic Bacillus firmus OF4 that functionally complements a mutant strain of Escherichia coli, NM81, that carries a deletion for one of that strain's Na+/H+ antiporter genes (delta nhaA). The cloned alkaliphile gene restored to NM81 the ability to grow at pH 7.5 in the presence of 0.6 M NaCl and on 100 mM Li+ in the presence of melibiose, and concomitantly led to an increase in the membrane associated Na+/H+ antiport activity. The biologically active alkaliphile DNA was identified as an incomplete open reading frame, the sequence of which would encode a hydrophobic protein. The insert was used to isolate clones containing the complete open reading frame, which would be predicted to encode a protein with a molecular weight of 42,960 and multiple membrane spanning regions. When the open reading frame was expressed under the control of the T7 promoter, the gene product was localized in the membrane. Southern analysis indicated no homology between the alkaliphile gene, which we propose to call nhaC, and the nhaA gene of Escherichia coli, nor with other genes in digests of DNA from E. coli, Bacillus subtilis, or Bacillus alcalophilus. Although there was also no significant similarity between the deduced protein products of the alkaliphile gene and the nhaA gene of E. coli, there was a small region of significant similarity between the deduced alkaliphile gene product and the protein encoded by a human Na+/H+ antiporter gene (Sardet, C., Franchi, A., and Pouyssegur, J. (1989) Cell 56, 271-280).
KeywordMeSH Terms
Genes, Bacterial
Mutation
10. Grazulis  S, Manakova  E, Roessle  M, Bochtler  M, Tamulaitiene  G, Huber  R, Siksnys  V,     ( 2005 )

Structure of the metal-independent restriction enzyme BfiI reveals fusion of a specific DNA-binding domain with a nonspecific nuclease.

Proceedings of the National Academy of Sciences of the United States of America 102 (44)
PMID : 16247004  :   DOI  :   10.1073/pnas.0507949102     PMC  :   PMC1266039    
Abstract >>
Among all restriction endonucleases known to date, BfiI is unique in cleaving DNA in the absence of metal ions. BfiI represents a different evolutionary lineage of restriction enzymes, as shown by its crystal structure at 1.9-A resolution. The protein consists of two structural domains. The N-terminal catalytic domain is similar to Nuc, an EDTA-resistant nuclease from the phospholipase D superfamily. The C-terminal DNA-binding domain of BfiI exhibits a beta-barrel-like structure very similar to the effector DNA-binding domain of the Mg(2+)-dependent restriction enzyme EcoRII and to the B3-like DNA-binding domain of plant transcription factors. BfiI presumably evolved through domain fusion of a DNA-recognition element to a nonspecific nuclease akin to Nuc and elaborated a mechanism to limit DNA cleavage to a single double-strand break near the specific recognition sequence. The crystal structure suggests that the interdomain linker may act as an autoinhibitor controlling BfiI catalytic activity in the absence of a specific DNA sequence. A psi-blast search identified a BfiI homologue in a Mesorhizobium sp. BNC1 bacteria strain, a plant symbiont isolated from an EDTA-rich environment.
KeywordMeSH Terms
11. Takami  H, Matsuki  A, Takaki  Y,     ( 2004 )

Wide-range distribution of insertion sequences identified in B. halodurans among bacilli and a new transposon disseminated in alkaliphilic and thermophilic bacilli.

DNA research : an international journal for rapid publication of reports on genes and genomes 11 (3)
PMID : 15368891  :   DOI  :   10.1093/dnares/11.3.153    
Abstract >>
All of the insertion sequences (ISs) except for IS663 and a group II intron identified in the alkaliphilic Bacillus halodurans C-125 genome were also detected in nine other strains of the same species by PCR and Southern blot analysis. The transposase of IS 653 identified in the genomes of the 10 strains of B. halodurans was found to have become the most diversified of all ISs identified in the genomes of 10 strains. A new IS element designated IS661 belonging to the IS1380 family with inverted repeats (IRs) 17 bp in length was present within IS658 identified in the genome of B. halodurans A59. In addition, a new transposon designated Tn3271bh was identified within the IS642 element in the A59 genome, which is similar to a transposon identified in thermophilic Geobacillus stearothermophilus T-6. The new transposon, Tn3271bh, generated an 8-bp duplication of the target site sequence and carries a 21-bp IR. On the other hand, all kinds of ISs except for IS643 and IS658 were distributed in the genome of obligately alkaliphilic Bacillus alcalophilus. Three ISs (IS652, IS653, and IS660) and a group II intron (Bh.Int) were widely dispersed in other Bacillus species without a correlation with the phylogenetic placement based on 16S rDNA sequences.
KeywordMeSH Terms
DNA Transposable Elements
12. Chang  P, Tsai  WS, Tsai  CL, Tseng  MJ,     ( 2004 )

Cloning and characterization of two thermostable xylanases from an alkaliphilic Bacillus firmus.

Biochemical and biophysical research communications 319 (3)
PMID : 15184083  :   DOI  :   10.1016/j.bbrc.2004.05.078    
Abstract >>
Two genes encoding thermostable xylanases, named xyn10A and xyn11A, from an alkaliphilic Bacillus firmus were cloned and expressed in Escherichia coli. The E. coli harboring either gene showed clear zone with Congo red clearance assay on xylan plate. The Xyn10A and Xyn11A have molecular weights of 45 and 23kDa, respectively, and both show activities on xylan-zymogram. The xyn10A encodes 396 amino acid residues and is very similar to an alkaliphilic xylanase A from alkaliphilic Bacillus halodurans. The Xyn11A contains 210 amino acid residues and only one amino acid different from an endo-beta-1,4-xylanase from B. halodurans. From alignment of the amino acid sequences with other xylanases, Xyn10A and Xyn11A belong to family 10 and 11 glycosyl hydrolases, respectively. Both show activities over the pH range of 4-11 at 37 degrees C and over 80% activities at 70 degrees C. Interestingly both still retain over 70% activities after 16h preincubation at 62 degrees C.
KeywordMeSH Terms
Hot Temperature
13. 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
14. Ivey  DM, Krulwich  TA,     ( 1990 )

Sequence of the gene encoding the ATP synthase beta subunit from alkaliphilic Bacillus firmus RAB.

Nucleic acids research 18 (5)
PMID : 2181405  :   DOI  :   10.1093/nar/18.5.1296     PMC  :   PMC330458    
Abstract >>
N/A
KeywordMeSH Terms
15. Ki  JS, Zhang  W, Qian  PY,     ( 2009 )

Discovery of marine Bacillus species by 16S rRNA and rpoB comparisons and their usefulness for species identification.

Journal of microbiological methods 77 (1)
PMID : 19166882  :   DOI  :   10.1016/j.mimet.2009.01.003    
Abstract >>
Systematic studies of the Bacillus group have been biased towards terrestrial and pathogenic isolates, and relatively few studies have examined Bacillus species from marine environments. Here we took twenty Bacillus strains from diverse marine environments and sequenced their 16S rRNA. Using molecular comparisons, we separated the strains into thirteen Bacillus genotypes and identified 9 species: B. aquaemaris. B. badius, B. cereus group, B. firmus, B. halmapalus, B. hwajinpoensis, B. litoralis, B. sporothermodurans, B. vietnamensis, and three indistinguishable Bacilli. In addition, we sequenced the DNA-directed RNA polymerase beta subunit (rpoB) gene and assessed its discriminative power in identifying Bacilli. Phylogenetic trees of Bacillus rpoB genes separated each Bacillus according to their taxonomic positions and were supported statistically. The resolution of Bacillus on the rpoB phylogenetic tree was approximately 4.5 times greater than on the 16S rRNA phylogenetic tree. These results demonstrate that the polymorphism of the Bacillus rpoB gene can be used to identify Bacillus species, providing an improved identification scheme for Bacillus species.
KeywordMeSH Terms
16. Steiger  S, Perez-Fons  L, Cutting  SM, Fraser  PD, Sandmann  G,     ( 2015 )

Annotation and functional assignment of the genes for the C30 carotenoid pathways from the genomes of two bacteria: Bacillus indicus and Bacillus firmus.

Microbiology (Reading, England) 161 (Pt 1)
PMID : 25326460  :   DOI  :   10.1099/mic.0.083519-0    
Abstract >>
Bacillus indicus and Bacillus firmus synthesize C30 carotenoids via farnesyl pyrophosphate, forming apophytoene as the first committed step in the pathway. The products of the pathways were methyl 4'-[6-O-acyl-glycosyl)oxy]-4,4'-diapolycopen-4-oic acid and 4,4'-diapolycopen-4,4'-dioic acid with putative glycosyl esters. The genomes of both bacteria were sequenced, and the genes for their early terpenoid and specific carotenoid pathways annotated. All genes for a functional 1-deoxy-d-xylulose 5-phosphate synthase pathway were identified in both species, whereas genes of the mevalonate pathway were absent. The genes for specific carotenoid synthesis and conversion were found on gene clusters which were organized differently in the two species. The genes involved in the formation of the carotenoid cores were assigned by functional complementation in Escherichia coli. This bacterium was co-transformed with a plasmid mediating the formation of the putative substrate and a second plasmid with the gene of interest. Carotenoid products in the transformants were determined by HPLC. Using this approach, we identified the genes for a 4,4'-diapophytoene synthase (crtM), 4,4'-diapophytoene desaturase (crtNa), 4,4'-diapolycopene ketolase (crtNb) and 4,4'-diapolycopene aldehyde oxidase (crtNc). The three crtN genes were closely related and belonged to the crtI gene family with a similar reaction mechanism of their enzyme products. Additional genes encoding glycosyltransferases and acyltransferases for the modification of the carotenoid skeleton of the diapolycopenoic acids were identified by comparison with the corresponding genes from other bacteria.
KeywordMeSH Terms
Biosynthetic Pathways
Genes, Bacterial
Genome, Bacterial
17. Rajesh  PS, Ravishankar Rai  V,     ( N/A )

Quorum quenching activity in cell-free lysate of endophytic bacteria isolated from Pterocarpus santalinus Linn., and its effect on quorum sensing regulated biofilm in Pseudomonas aeruginosa PAO1.

Microbiological research 169 (7��8��)
PMID : 24268182  :   DOI  :   10.1016/j.micres.2013.10.005    
Abstract >>
Quorum sensing mechanism allows the microorganisms to resist the antibiotic treatment by forming biofilms. Quorum quenching is one of the mechanisms to control the development of drug resistance in microbes. Endophyte bacteria are beneficial to plant growth as they support the immune system against the pathogen attack. The endophytic bacteria present in Pterocarpus santalinus were screened for the presence of N-acyl homoserine lactones (AHLs) degrading bacteria using biosensor strains and further confirmed by quantifying the violacein production. Cell-free lysate of endophytic bacteria, Bacillus firmus PT18 and Enterobacter asburiae PT39 exhibited potent AHL degrading ability by inhibiting about 80% violacein production in biosensor strain. Furthermore, when the cell-free lysate was applied to Pseudomonas aeruginosa PAO1 and PAO1-JP2 biofilm it resulted in significant (p<0.01) inhibition of biofilm formation. The biofilm inhibition was confirmed by visualization of biofilm slides under fluorescence microscopy, which showed decrease in total biomass formation in treated slides. Isolation and amplification of the gene (aiiA) indicated that the presence of AHL lactonase in cell-free lysate and sequence alignment indicated that AiiA contains a "HXHXDH" zinc-binding motif that is being conserved in several groups of metallohydrolases. Therefore, the study shows the potential of AHLs degradation by AHL lactonase present in cell-free lysate of isolated endophytic bacteria and inhibition of quorum sensing regulated biofilm formation in P. aeruginosa PAO1.
KeywordMeSH Terms
AHL lactonase
Bacillus firmus
Biofilm inhibition
Enterobacter asburiae
Quorum quenching
AHL lactonase
Bacillus firmus
Biofilm inhibition
Enterobacter asburiae
Quorum quenching
Biofilms
Quorum Sensing
18. Golovenko  D, Manakova  E, Zakrys  L, Zaremba  M, Sasnauskas  G, Gražulis  S, Siksnys  V,     ( 2014 )

Structural insight into the specificity of the B3 DNA-binding domains provided by the co-crystal structure of the C-terminal fragment of BfiI restriction enzyme.

Nucleic acids research 42 (6)
PMID : 24423868  :   DOI  :   10.1093/nar/gkt1368     PMC  :   PMC3973309    
Abstract >>
The B3 DNA-binding domains (DBDs) of plant transcription factors (TF) and DBDs of EcoRII and BfiI restriction endonucleases (EcoRII-N and BfiI-C) share a common structural fold, classified as the DNA-binding pseudobarrel. The B3 DBDs in the plant TFs recognize a diverse set of target sequences. The only available co-crystal structure of the B3-like DBD is that of EcoRII-N (recognition sequence 5'-CCTGG-3'). In order to understand the structural and molecular mechanisms of specificity of B3 DBDs, we have solved the crystal structure of BfiI-C (recognition sequence 5'-ACTGGG-3') complexed with 12-bp cognate oligoduplex. Structural comparison of BfiI-C-DNA and EcoRII-N-DNA complexes reveals a conserved DNA-binding mode and a conserved pattern of interactions with the phosphodiester backbone. The determinants of the target specificity are located in the loops that emanate from the conserved structural core. The BfiI-C-DNA structure presented here expands a range of templates for modeling of the DNA-bound complexes of the B3 family of plant TFs.
KeywordMeSH Terms
19. Steiger  S, Sandmann  G, Fraser  PD,     ( 2012 )

Biosynthesis of a novel C30 carotenoid in Bacillus firmus isolates.

Journal of applied microbiology 113 (4)
PMID : 22738026  :   DOI  :   10.1111/j.1365-2672.2012.05377.x    
Abstract >>
Pigmented Bacillus spp. with probiotic properties have been isolated. In the yellow-/orange-coloured strains, the carotenoid pigments present have been characterized. In contrast, the carotenoids present in the Bacillus isolates coloured red await identification. The present article reports progress on the elucidation of the pigment biosynthetic pathway in these red-pigmented Bacillus firmus strains. A combination of UV/Vis, chromatographic and mass spectrometry (MS) has revealed the properties of the predominant pigment and the end-point carotenoid of the pathway to be methyl 4,4'-diapolycopene-dioate after transmethylation. The diglycosyl ester of 4,4'-diapolycopene-dioate persists in vivo prior to chemical treatment. Different mutants and inhibitor treatment were employed to establish the C30 biosynthesis pathway with all precursors and intermediates to 4,4'-diapolycopene-dioate detected, which include 4,4'-diapophytene and all desaturation intermediates to 4,4'-diapolycopene and 4,4'-diapolycopene-dialdehyde. To cultures synthesizing the 4,4'-diapolycopene-dioate derivative and those in which its formation was inhibited, oxidative stress was induced by peroxide treatment. Conditions that decreased the growth rate of the pigmented cells by only 30% caused a complete growth inhibition of the culture devoid of the 4,4'-diapolycopene-dioate derivative. This finding demonstrates the diversity of C30 carotenoid biosynthesis in Bacillus species and the antioxidative function of the 4,4'-diapolycopene-dioate derivative in B. firmus cells. It could be shown that the C30 4,4'-diapolycopene-dioate derivatives protect pigmented B. firmus from peroxidative reactions. Under oxidative conditions, this can be an ecological advantage over nonpigmented (=noncarotenogenic) strains that are equally abundant.
KeywordMeSH Terms
Biosynthetic Pathways
20. Ivey  DM,     ( 1990 )

Nucleotide sequence of a gene from alkaliphilic Bacillus firmus RAB that is homologous to the fpg gene of Escherichia coli.

Nucleic acids research 18 (19)
PMID : 2216780  :   DOI  :   10.1093/nar/18.19.5882     PMC  :   PMC332331    
Abstract >>
N/A
KeywordMeSH Terms
Escherichia coli Proteins
21.     ( 1997 )

Role of the nhaC-encoded Na+/H+ antiporter of alkaliphilic Bacillus firmus OF4.

Journal of bacteriology 179 (12)
PMID : 9190799  :   DOI  :   10.1128/jb.179.12.3851-3857.1997     PMC  :   PMC179192    
Abstract >>
Application of protoplast transformation and single- and double-crossover mutagenesis protocols to alkaliphilic Bacillus firmus OF4811M (an auxotrophic strain of B. firmus OF4) facilitated the extension of the sequence of the previously cloned nhaC gene, which encodes an Na+/H+ antiporter, and the surrounding region. The nhaC gene is part of a likely 2-gene operon encompassing nhaC and a small gene that was designated nhaS; the operon is preceded by novel direct repeats. The predicted alkaliphile NhaC, based on the extended sequence analysis, would be a membrane protein with 462 amino acid residues and 12 transmembrane segments that is highly homologous to the deduced products of homologous genes of unknown function from Bacillus subtilis and Haemophilus influenzae. The full-length version of nhaC complemented the Na+-sensitive phenotype of an antiporter-deficient mutant strain of Escherichia coli but not the alkali-sensitive growth phenotypes of Na+/H+-deficient mutants of either alkaliphilic B. firmus OF4811M or B. subtilis. Indeed, NhaC has no required role in alkaliphily, inasmuch as the nhaC deletion strain of B. firmus OF4811M, N13, grew well at pH 10.5 at Na+ concentrations equal to or greater than 10 mM. Even at lower Na+ concentrations, N13 exhibited only a modest growth defect at pH 10.5. This was accompanied by a reduced capacity to acidify the cytoplasm relative to the medium compared to the wild-type strain or to N13 complemented by cloned nhaC. The most notable deficiency observed in N13 was its poor growth at pH 7.5 and Na+ concentrations up to 25 mM. During growth at pH 7.5, NhaC is apparently a major component of the relatively high affinity Na+/H+ antiport activity available to extrude the Na+ and to confer some initial protection in the face of a sudden upshift in external pH, i.e., before full induction of additional antiporters. Consistent with the inference that NhaC is a relatively high affinity, electrogenic Na+/H+ antiporter, N13 exhibited a defect in diffusion potential-energized efflux of 22Na+ from right-side-out membrane vesicles from cells that were preloaded with 2 mM Na+ and energized at pH 7.5. When the experiment was conducted with vesicles loaded with 25 mM Na+, comparable efflux was observed in preparations from all the strains.
KeywordMeSH Terms
22. Smith  RL, Thompson  LJ, Maguire  ME,     ( 1995 )

Cloning and characterization of MgtE, a putative new class of Mg2+ transporter from Bacillus firmus OF4.

Journal of bacteriology 177 (5)
PMID : 7868596  :   DOI  :   10.1128/jb.177.5.1233-1238.1995     PMC  :   PMC176728    
Abstract >>
The MM281 strain of Salmonella typhimurium which possesses mutations in each its three known Mg2+ transport systems and requires 100 mM Mg2+ for growth was used to screen a genomic library from the gram-positive alkaliphilic bacterium Bacillus firmus OF4 for clones that could restore the ability to grow without Mg2+ supplementation. Of the clones obtained, five also conferred sensitivity to Co2+, similar to the phenotype of mutants with mutations in the S. typhimurium corA Mg2+ transport locus. All five contained identical inserts by restriction analysis. Using 63Ni2+ as a surrogate for the unavailable 28Mg2+, the plasmid insert was shown to restore cation uptake with properties similar but not identical to those of the S. typhimurium CorA Mg2+ transporter. Sequence analysis of one clone identified a single open reading frame with multiple possible initiation sites. Deletion and mutation analysis identified a minimum open reading frame of 939 bp encoding a polypeptide with a predicted molecular mass of 34 kDa. Disruption of the open reading frame eliminated cation influx activity and restored resistance to Co2+. This putative transporter, designated MgtE, has no sequence similarity to any known protein including CorA and appears to represent a new class of Mg2+ transport system.
KeywordMeSH Terms
Bacterial Proteins
Cation Transport Proteins
23. Hicks  DB,     ( 1995 )

Purification of three catalase isozymes from facultatively alkaliphilic Bacillus firmus OF4.

Biochimica et biophysica acta 1229 (3)
PMID : 7748885  :   DOI  :   10.1016/0005-2728(95)00016-c    
Abstract >>
Cell extracts of facultatively alkaliphilic B. firmus OF4 were assayed for catalase activity and their catalase isozyme content was analyzed on native polyacrylamide gels stained for catalase activity. pH-10.5-grown cells had about twice the specific catalase activity of pH-7.5-grown cells. The higher activity, however, did not confer resistance to exogenous hydrogen peroxide challenge relative to pH-7.5-grown cells, and in fact, the pH-10.5-grown cells were much more sensitive to the challenge. Electrophoresis resolved three catalase isozymes in cell extracts. The isozymes, labeled I-III in order of decreasing electrophoretic mobility, were purified and their Nterminal amino acid sequences were obtained. Isozyme III corresponded to the product of a cloned gene fragment that had been shown to possess substantial sequence similarity to the KatE (HP-II) catalase of E. coli (Quirk, P.G., Krulwich, T.A. and Hicks, D.B. (1993) Biophys J. 64, 164A) and which had similar biochemical properties to HP-II, i.e., it was a chlorin-containing enzyme expressed only in stationary phase. Isozyme II, a protoheme enzyme, was responsible for the higher activity of alkaline-grown cells and was induced in cells treated with hydrogen peroxide or ascorbate. It showed sequence similarity to katA of Bacillus subtilis (Bol, D. and Yasbin, R. (1991) Gene 109, 31-37). Isozyme I was the only isozyme that exhibited detectable levels of peroxidase activity in addition to catalase activity, resembling a catalase enzyme purified from a different alkaliphile, Bacillus YN-2000 (Yumoto, I., Fukumori, Y. and Yamanaka, T. (1990) J. Biochem. 108, 583-587), to which it showed some sequence similarity.
KeywordMeSH Terms
24. ?zdemir  F, Arslan  S,     ( 2019 )

Molecular Characterization and Toxin Profiles of Bacillus spp. Isolated from Retail Fish and Ground Beef.

Journal of food science 84 (3)
PMID : 30690739  :   DOI  :   10.1111/1750-3841.14445    
Abstract >>
Bacillus species are common in the environment due to their spore-forming ability and nutritional versatility and cause food contamination. Bacilli play a significant role in foodborne illnesses and food spoilage. In this study, 52 Bacillus isolates from retail fish and ground beef were identified and differentiated based on 16S rRNA, gyrB, and rpoB gene sequencing. The presence of genes encoding emetic toxin (ces), hemolytic enterotoxin hemolysin BL (hbl), nonhemolytic enterotoxin (nhe) and cytotoxin K (cytK1) was assessed in all Bacillus isolates. The ability of the Bacillus isolates to produce several extracellular enzymes that contribute to pathogenicity and food spoilage was investigated. The 16S rRNA, rpoB, and gyrB gene sequence similarities of the Bacillus isolates tested were 96.1%, 83.2%, and 77.5%, respectively. The gyrB gene demonstrated a higher degree of sequence variation than the 16S rRNA and rpoB genes. The prevalence of Bacillus isolates producing at least two of the genes of the HBL and NHE complexes was 23.1% and 15.4%, respectively. Of the B. cereus isolates, 10 (41.7%) possessed two or more enterotoxin genes. None of the isolates carried the ces and cytK1 genes. All isolates were positive for the production of enzymes such as protease, lipase, gelatinase, and DNase. However, only 92.3% of the tested isolates were positive for amylase. In conclusion, our results revealed that the presence of genes involved in toxin production and enzyme production in meat-originated B. cereus and other Bacillus isolates may cause spoilage of food and pose a health risk for consumers. PRACTICAL APPLICATION: Bacillus species can be found in various foods due to their ubiquitous nature. Bacillus spp., especially B. cereus, are associated with food poisoning and other infections in humans. Toxins and many extracellular enzymes produced by Bacillus spp. are the causative agents of foodborne outbreaks, food spoilage, and low-quality food with significantly reduced edibility. This study highlights the characterization of Bacillus spp. and presence of potentially pathogenic Bacillus species in meats.
KeywordMeSH Terms
16S rRNA
Bacillus spp
extracellular enzymes
housekeeping genes
toxin genes
16S rRNA
Bacillus spp
extracellular enzymes
housekeeping genes
toxin genes
16S rRNA
Bacillus spp
extracellular enzymes
housekeeping genes
toxin genes
16S rRNA
Bacillus spp
extracellular enzymes
housekeeping genes
toxin genes
16S rRNA
Bacillus spp
extracellular enzymes
housekeeping genes
toxin genes
16S rRNA
Bacillus spp
extracellular enzymes
housekeeping genes
toxin genes
16S rRNA
Bacillus spp
extracellular enzymes
housekeeping genes
toxin genes
16S rRNA
Bacillus spp
extracellular enzymes
housekeeping genes
toxin genes
16S rRNA
Bacillus spp
extracellular enzymes
housekeeping genes
toxin genes
Bacillus
Food Microbiology
Genotype
25. Mendis  HC, Thomas  VP, Schwientek  P, Salamzade  R, Chien  JT, Waidyarathne  P, Kloepper  J, De La Fuente  L,     ( 2018 )

Strain-specific quantification of root colonization by plant growth promoting rhizobacteria Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 in non-sterile soil and field conditions.

PloS one 13 (2)
PMID : 29447287  :   DOI  :   10.1371/journal.pone.0193119     PMC  :   PMC5814090    
Abstract >>
Bacillus amyloliquefaciens QST713 and B. firmus I-1582 are bacterial strains which are used as active ingredients of commercially-available soil application and seed treatment products Serenade? and VOTiVO?, respectively. These bacteria colonize plant roots promoting plant growth and offering protection against pathogens/pests. The objective of this study was to develop a qPCR protocol to quantitate the dynamics of root colonization by these two strains under field conditions. Primers and TaqMan? probes were designed based on genome comparisons of the two strains with publicly-available and unpublished bacterial genomes of the same species. An optimized qPCR protocol was developed to quantify bacterial colonization of corn roots after seed treatment. Treated corn seeds were planted in non-sterile soil in the greenhouse and grown for 28 days. Specific detection of bacteria was quantified weekly, and showed stable colonization between ~104-105 CFU/g during the experimental period for both bacteria, and the protocol detected as low as 103 CFU/g bacteria on roots. In a separate experiment, streptomycin-resistant QST713 and rifampicin-resistant I-1582 strains were used to compare dilution-plating on TSA with the newly developed qPCR method. Results also indicated that the presence of natural microflora and another inoculated strain does not affect root colonization of either one of these strains. The same qPCR protocol was used to quantitate root colonization by QST713 and I-1582 in two corn and two soybean varieties grown in the field. Both bacteria were quantitated up to two weeks after seeds were planted in the field and there were no significant differences in root colonization in either bacteria strain among varieties. Results presented here confirm that the developed qPCR protocol can be successfully used to understand dynamics of root colonization by these bacteria in plants growing in growth chamber, greenhouse and the field.
KeywordMeSH Terms
Bacillus firmus
Soil
Bacillus amyloliquefaciens
26.     ( 2012 )

phoR sequences as a phylogenetic marker to differentiate the species in the Bacillus subtilis group.

Canadian journal of microbiology 58 (11)
PMID : 23145827  :   DOI  :   10.1139/w2012-106    
Abstract >>
Bacillus subtilis and its closely related species are indistinguishable from one another by morphological characteristics and 16S rDNA sequences. In this study, the partial phoR sequence was tested to determine the phylogenetic relationship of species in the B. subtilis group. Degenerate primers were developed according to the relatively conserved nucleotide sequences of phoR and the linked gene phoP in the B. subtilis group. The primers amplified a 1100 bp phoR fragment from strains representative of 6 species in the B. subtilis group. Based on the sequenced fragments, 26 type strains comprising these 6 species were clearly distinguished. At the intraspecies level, the phoR sequence similarities were 90%-100%, but at the interspecies level, the phoR sequence similarities were 32.8%-75%. Compared with the gyrB sequence, the phoR sequences showed a larger divergence especially at the interspecies levels. Therefore, the phoR sequence may be an efficient alternative marker for phylogenetic and taxonomic analysis of species in the B. subtilis group. Twenty-three Bacillus undomesticated isolates were tested for identification and phylogenetic analysis based on the phoR and gyrB sequences. The 23 isolates could be clearly delineated into 4 distinct groups, 10 as B. subtilis, 3 as B. mojavensis, 2 as B. atrophaeus, and 8 as B. amyloliquefaciens.
KeywordMeSH Terms
Phylogeny
27.     ( 1997 )

Diverse genes of alkaliphilic Bacillus firmus OF4 that complement K+-uptake-deficient Escherichia coli include an ftsH homologue.

Extremophiles : life under extreme conditions 1 (1)
PMID : 9680333  :  
Abstract >>
Seven clones isolated from libraries of DNA from alkaliphilic Bacillus firmus OF4 restored the growth of a K+-uptake-deficient Escherichia coli mutant on only 10 mM K+. None of the clones contained genes with apparent homology to known K+ transport systems in other organisms. Based on sequence homologies, the newly isolated alkaliphile loci included: ftsH; a dipeptide transport system; a gerC locus with hydrophobic open reading frames not found in the comparable locus of Bacillus subtilis; a sugar phosphotransferase enzyme; and a capBC homologue. The ftsH gene provided a new and striking example of a recognized property of extracellular and external regions of polytopic alkaliphile proteins: a significant paucity of basic amino acid residues relative to neutrophile counterparts. The alkaliphile ftsH gene was able to complement a mutant of E. coli with a temperature-sensitive ftsH gene product.
KeywordMeSH Terms

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