| 1. |
Martínez MA,
Delgado OD,
Breccia JD,
Baigorí MD,
Siñeriz F,
( 2002 ) Revision of the taxonomic position of the xylanolytic Bacillus sp. MIR32 reidentified as Bacillus halodurans and plasmid-mediated transformation of B. halodurans. PMID : 12382115 : DOI : 10.1007/s00792-002-0269-4 DOI : 10.1007/s00792-002-0269-4 Abstract >>
Bacillus sp. MIR32 has been isolated using xylan as the only carbon source, and one of its xylanolytic enzymes has been extensively studied. Biochemical analysis first related this strain to Bacillus amyloliquefaciens, but further studies based on a comparison of 16S rDNA sequences, G+C content, and DNA-DNA hybridization showed that strain MIR32 should be classified as a member of the species Bacillus halodurans. This change is also supported by the typical phenotype observed and by the results of PCR amplification directed toward spacers in rDNA and tDNA genes, which were assayed and compared with those of B. halodurans DSM 497(T). Although among alkaliphilic bacilli competence development has not been experimentally demonstrated, in this work both B. halodurans MIR32 and DSM 497(T) were transformed according to a simple procedure developed in our laboratory, reaching 10(2)-10(3) stable transformants per microgram of plasmid DNA.
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2. |
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. 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.
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3. |
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. 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.
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4. |
Nakasone K,
Masui N,
Takaki Y,
Sasaki R,
Maeno G,
Sakiyama T,
Hirama C,
Fuji F,
Takami H,
( 2000 ) Characterization and comparative study of the rrn operons of alkaliphilic Bacillus halodurans C-125. PMID : 10972189 : Abstract >>
The ribosomal RNA operons (rrn) of alkaliphilic Bacillus halodurans C-125 were characterized and compared with those of B. subtilis. We isolated clones containing rrn operons from a lambda phage library of the C-125 chromosome, and the complete nucleotide sequence of each was determined. Eight rrn operons were identified by PFGE analysis of the C-125 chromosome digested with I-CeuI. The transcriptional orientation of the rrn operons mapped on the chromosome by Southern hybridization analysis was the same as the direction of replication of the chromosome. These operons were designated as rrnA-H, starting from the oriC locus in clockwise rotation. Sequence and structural analyses of these operons suggested that six of the rrn operons in the C-125 chromosome, rrnA, rrnB, rrnC-rrnD, rrnE, and rrnH, correspond to rrnO, rrnA, rrnJ-rrnW, rrnI, and rrnD in B. subtilis, whereas the other rrn operons (rrnF and rrnG) were specifically observed in C-125. The rrn loci were positioned from 0 degrees to 90 degrees on the physical map, with the oriC locus assigned the position zero degrees. Two ORFs annotated as tnpA and ykfC, whose gene products are likely to act as transposases, were found downstream of these six operons. Comparative analysis of the 16S-23S and 23S-5S ITS (internally transcribed sequence) regions of B. halodurans C-125 and those of B. subtilis revealed that the ITS regions in C-125 were much longer than those in B. subtilis. There was no substantial difference in the length of potential promoter sequences in B. halodurans and B. subtilis.
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5. |
Masui N,
Nakasone K,
Horikoshi K,
( 1999 ) Replication origin region of the chromosome of alkaliphilic Bacillus halodurans C-125. PMID : 10427704 : Abstract >>
An 18.5-kb DNA fragment containing the oriC region of the chromosome of the alkaliphilic Bacillus halodurans C-125 was obtained by PCR and sequenced. Sixteen open reading frames (ORFs) were identified in this region. A sequencing similarity search using the BSORF database found that ORF1 to 13 all had significant similarities to gene products of Bacillus subtilis. Three other ORFs (ORF14-16) of unknown function were positioned down-stream of gyrB instead of rrnO, which is found in the same region in the case of B. subtilis. The ORF organization from gidA to gyrA was the same as that of B. subtilis. The gene organization and the location of the DnaA-box region were also similar to those of the chromosomes of other bacteria, such as Escherichia coli and Pseudomonas putida. There were two DnaA-box clusters (Box-region C and R) with a consensus sequence TTATCCACA on both sides of the dnaA gene but another DnaA box cluster (Box-region L) which is found in the region between thdF and jag in B. subtilis was not found in the corresponding region in the case of alkaliphilic Bacillus halodurans C-125.
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6. |
Sakiyama T,
Maeno G,
Nakasone K,
Takaki Y,
Takami H,
( 1999 ) Genetic analysis of the chromosome of alkaliphilic Bacillus halodurans C-125. PMID : 10484179 : Abstract >>
Seventeen Sse8387I linking clones isolated from the chromosome of Bacillus halodurans C-125 for the purpose of constructing a physical map were sequenced and analyzed by comparison with the BSORF database and the nonredundant protein databank. The orientations of Sse8387I or AscI linking clones serving to join adjacent fragments were determined by southern blot analysis using specific DNA probes. One-third of the open reading frames (ORFs) identified in the Sse8387I linking clones showed no significant similarity to any protein so far reported. The ORFs showing significant similarities to those of Bacillus subtilis were mapped in the chromosome of strain C-125, and the locations of the putative genes on the map were not well conserved between B. halodurans C-125 and B. subtilis.
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7. |
Horikoshi K,
Inoue A,
Nakasone K,
Hirama C,
( 1999 ) Sequence analysis of a 32-kb region including the major ribosomal protein gene clusters from alkaliphilic Bacillus sp. strain C-125. PMID : 10192928 : DOI : 10.1271/bbb.63.452 Abstract >>
Forty-one open reading frames (ORFs) were identified in a 32-kb DNA fragment of alkaliphilic Bacillus sp. C-125. A similarity search using the BSORF database found 37 ORFs with significant sequence similarity to B. subtilis RNA polymerase subunits, elongation factor G, elongation factor Tu, and ribosomal proteins. Each ORF product showed more than 70% identity to those of B. subtilis. Gene organization in the region of str, S10, spc, and the alpha cluster was highly conserved among three strains, C-125, B. subtilis, and B. stearothermophilus.
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8. |
Horikoshi K,
Inoue A,
Takaki Y,
Fuji F,
Masui N,
Nakamura Y,
Hirama C,
( 1999 ) Sequencing of three lambda clones from the genome of alkaliphilic Bacillus sp. strain C-125. PMID : 10086842 : Abstract >>
The nucleotide sequences of three independent fragments (designated no. 3, 4, and 9; each 15-20 kb in size) of the genome of alkaliphilic Bacillus sp. C-125 cloned in a lambda phage vector have been determined. Thirteen putative open reading frames (ORFs) were identified in sequenced fragment no. 3 and 11 ORFs were identified in no. 4. Twenty ORFs were also identified in fragment no. 9. All putative ORFs were analyzed in comparison with the BSORF database and non-redundant protein databases. The functions of 5 ORFs in fragment no. 3 and 3 ORFs in fragment no. 4 were suggested by their significant similarities to known proteins in the database. Among the 20 ORFs in fragment no. 9, the functions of 11 ORFs were similarly suggested. Most of the annotated ORFs in the DNA fragments of the genome of alkaliphilic Bacillus sp. C-125 were conserved in the Bacillus subtilis genome. The organization of ORFs in the genome of strain C-125 was found to differ from the order of genes in the chromosome of B. subtilis, although some gene clusters (ydh, yqi, yer, and yts) were conserved as operon units the same as in B. subtilis.
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9. |
Inoue A,
Nakamura Y,
Fuji F,
Masui N,
Takaki Y,
( 1999 ) An improved physical and genetic map of the genome of alkaliphilic Bacillus sp. C-125. PMID : 10086841 : Abstract >>
Among alkaliphilic bacteria reported so far, Bacillus sp. C-125 is the strain most thoroughly characterized physiologically, biochemically, and genetically. A physical map of the chromosome of this strain was constructed to facilitate further genome analysis, and the genome size was revised from 3.7 to 4.25Mb. Complete digestion of the chromosomal DNA with two rare cut restriction endonucleases, AscI and Sse8387I, each yielded 20 fragments ranging in size from 20 to 600 kb. Seventeen linking clones were isolated in each instance to join the adjacent AscI or Sse8387I fragments in the chromosomal map. All AscI linking clones isolated were sequenced and analyzed by comparison with the BSORF database to map the genes in the chromosome of strain C-125. Several ORFs showing significant similarities to those of B. subtilis in the AscI linking clones were positioned on the physical map. The oriC region of the C-125 chromosome was identified by southern blot analysis with a DNA probe containing the gyrB region.
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10. |
Solbak AI,
Richardson TH,
McCann RT,
Kline KA,
Bartnek F,
Tomlinson G,
Tan X,
Parra-Gessert L,
Frey GJ,
Podar M,
Luginbühl P,
Gray KA,
Mathur EJ,
Robertson DE,
Burk MJ,
Hazlewood GP,
Short JM,
Kerovuo J,
( 2005 ) Discovery of pectin-degrading enzymes and directed evolution of a novel pectate lyase for processing cotton fabric. PMID : 15618218 : DOI : 10.1074/jbc.M411838200 Abstract >>
There is a growing need in the textile industry for more economical and environmentally responsible approaches to improve the scouring process as part of the pretreatment of cotton fabric. Enzymatic methods using pectin-degrading enzymes are potentially valuable candidates in this effort because they could reduce the amount of toxic alkaline chemicals currently used. Using high throughput screening of complex environmental DNA libraries more than 40 novel microbial pectate lyases were discovered, and their enzymatic properties were characterized. Several candidate enzymes were found that possessed pH optima and specific activities on pectic material in cotton fibers compatible with their use in the scouring process. However, none exhibited the desired temperature characteristics. Therefore, a candidate enzyme was selected for evolution. Using Gene Site Saturation Mutagenesistrade mark technology, 36 single site mutants exhibiting improved thermotolerance were produced. A combinatorial library derived from the 12 best performing single site mutants was then generated by using Gene Reassemblytrade mark technology. Nineteen variants with further improved thermotolerance were produced. These variants were tested for both improved thermotolerance and performance in the bioscouring application. The best performing variant (CO14) contained eight mutations and had a melting temperature 16 degrees C higher than the wild type enzyme while retaining the same specific activity at 50 degrees C. Optimal temperature of the evolved enzyme was 70 degrees C, which is 20 degrees C higher than the wild type. Scouring results obtained with the evolved enzyme were significantly better than the results obtained with chemical scouring, making it possible to replace the conventional and environmentally harmful chemical scouring process.
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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. 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.
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12. |
Ruijssenaars HJ,
Hartmans S,
( 2004 ) A cloned Bacillus halodurans multicopper oxidase exhibiting alkaline laccase activity. PMID : 15293032 : DOI : 10.1007/s00253-004-1571-0 Abstract >>
The gene product of open reading frame bh2082 from Bacillus halodurans C-125 was identified as a multicopper oxidase with potential laccase activity. A homologue of this gene, lbh1, was obtained from a B. halodurans isolate from our culture collection. The encoded gene product was expressed in Escherichia coli and showed laccase-like activity, oxidising 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid), 2,6-dimethoxyphenol and syringaldazine (SGZ). The pH optimum of Lbh1 with SGZ is 7.5-8 (at 45 degrees C) and the laccase activity is stimulated rather than inhibited by chloride. These unusual properties make Lbh1 an interesting biocatalyst in applications for which classical laccases are unsuited, such as biobleaching of kraft pulp for paper production.
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13. |
Visser DF,
Hennessy F,
Rashamuse K,
Louw ME,
Brady D,
( 2010 ) Cloning, purification and characterisation of a recombinant purine nucleoside phosphorylase from Bacillus halodurans Alk36. PMID : 20063024 : DOI : 10.1007/s00792-009-0297-4 PMC : PMC2832885 Abstract >>
A purine nucleoside phosphorylase from the alkaliphile Bacillus halodurans Alk36 was cloned and overexpressed in Escherichia coli. The enzyme was purified fivefold by membrane filtration and ion exchange. The purified enzyme had a V (max) of 2.03 x 10(-9) s (-1) and a K (m) of 206 microM on guanosine. The optimal pH range was between 5.7 and 8.4 with a maximum at pH 7.0. The optimal temperature for activity was 70 degrees C and the enzyme had a half life at 60 degrees C of 20.8 h.
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14. |
Rajan LA,
Joseph TC,
Thampuran N,
James R,
Chinnusamy V,
Bansal KC,
( 2008 ) Characterization and phylogenetic analysis of ectoine biosynthesis genes from Bacillus halodurans. PMID : 18629475 : DOI : 10.1007/s00203-008-0397-z Abstract >>
Ectoine, a cyclic tetrahydropyrimidine (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid), is a natural compound, which serves as a protective substance in many bacterial cells. In this study, the putative ectABC gene cluster from Bacillus halodurans was heterologously expressed in E. coli and the production of ectoine was confirmed by HPLC analysis. The activity of the enzymes coded by the ectA, B and C genes were found to be higher in induced transgenic cells compared to the uninduced cells. Phylogenetic analysis revealed sequence identities ranging from 36-73% for ectA gene, 55-81% for ectB gene and 55-80% for ectC gene indicating that the enzymes are evolutionarily well conserved.
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15. |
Koeppel A,
Perry EB,
Sikorski J,
Krizanc D,
Warner A,
Ward DM,
Rooney AP,
Brambilla E,
Connor N,
Ratcliff RM,
Nevo E,
Cohan FM,
( 2008 ) Identifying the fundamental units of bacterial diversity: a paradigm shift to incorporate ecology into bacterial systematics. PMID : 18272490 : DOI : 10.1073/pnas.0712205105 PMC : PMC2268166 Abstract >>
The central questions of bacterial ecology and evolution require a method to consistently demarcate, from the vast and diverse set of bacterial cells within a natural community, the groups playing ecologically distinct roles (ecotypes). Because of a lack of theory-based guidelines, current methods in bacterial systematics fail to divide the bacterial domain of life into meaningful units of ecology and evolution. We introduce a sequence-based approach ("ecotype simulation") to model the evolutionary dynamics of bacterial populations and to identify ecotypes within a natural community, focusing here on two Bacillus clades surveyed from the "Evolution Canyons" of Israel. This approach has identified multiple ecotypes within traditional species, with each predicted to be an ecologically distinct lineage; many such ecotypes were confirmed to be ecologically distinct, with specialization to different canyon slopes with different solar exposures. Ecotype simulation provides a long-needed natural foundation for microbial ecology and systematics.
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16. |
Murakami S,
Nishimoto H,
Toyama Y,
Shimamoto E,
Takenaka S,
Kaulpiboon J,
Prousoontorn M,
Limpaseni T,
Pongsawasdi P,
Aoki K,
( 2007 ) Purification and characterization of two alkaline, thermotolerant alpha-amylases from Bacillus halodurans 38C-2-1 and expression of the cloned gene in Escherichia coli. PMID : 17928706 : DOI : 10.1271/bbb.60666 Abstract >>
A newly isolated strain, 38C-2-1, produced alkaline and thermotolerant alpha-amylases and was identified as Bacillus halodurans. The enzymes were purified to homogeneity and named alpha-amylase I and II. These showed molecular masses of 105 and 75 kDa respectively and showed maximal activities at 50-60 degrees C and pH 10-11, and 42 and 38% relative activities at 30 degrees C. These results indicate that the enzymes are thermotolerant. The enzyme activity was not inhibited by a surfactant or a bleaching reagent used in detergents. A gene encoding alpha-amylase I was cloned and named amyI. Production of AmyI with a signal peptide repressed the growth of an Escherichia coli transformant. When enzyme production was induced by the addition of isopropyl beta-D(-)-thiogalactopyranoside in the late exponential growth phase, the highest enzyme yield was observed. It was 45-fold that of the parent strain 38C-2-1.
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17. |
Kumar V,
Satyanarayana T,
( 2015 ) Generation of xylooligosaccharides from microwave irradiated agroresidues using recombinant thermo-alkali-stable endoxylanase of the polyextremophilic bacterium Bacillus halodurans expressed in Pichia pastoris. PMID : 25553569 : DOI : 10.1016/j.biortech.2014.12.049 Abstract >>
The recombinant Pichia pastoris harboring the endoxylanase gene (TSEV1xyl) of Bacillus halodurans TSEV1 yielded a high titer of extracellular xylanase (502��23 U ml(-1)) on induction with methanol. The purified recombinant xylanase (TSEV1xyl) displayed optimal activity at 80�XC and pH 9.0. The glycosylated recombinant xylanase exhibited higher thermostability (T1/2 of 45 min at 80�XC) than the native enzyme (T1/2 of 35 min at 80�XC). The agroresidues subjected to pretreatment (soaking in alkali followed by microwave irradiation) liberated xylooligosaccharides (XOS) upon hydrolysis with the recombinant xylanase. The removal of unhydrolyzed agroresidues, xylanase and xylose from the hydrolysate by two-step ultrafiltration led to the purification of XOS as confirmed by TLC as well as HPLC analysis.
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18. |
( 1998 ) Cloning and expression of the gene encoding RNA polymerase alpha subunit from alkaliphilic Bacillus sp. strain C-125. PMID : 9835038 : DOI : 10.1111/j.1574-6968.1998.tb13283.x Abstract >>
The rpoA gene, encoding the alpha subunit of RNA polymerase, was isolated from alkaliphilic Bacillus sp. strain C-125 by the PCR method. A 3-kb HindIII fragment containing the complete rpoA gene was cloned and sequenced. The alpha subunit gene was found to encode a protein consisting of 314 amino acid residues with a molecular mass of 34,805 Da. Compared with the amino acid sequences of other known eubacterial RNA polymerase alpha subunits, the gene has 84% identity to that of B. subtilis, while showing 48% and 47% identity to that of Streptomyces coelicolor and Escherichia coli, respectively. Six conserved regions, which are observed in the case of other eubacteria, were found in the RNA polymerase alpha subunit of this strain. Five of them are located in the N-terminal domain involved in assembly of the core enzyme, while one is located in the C-terminal domain, which interacts with several transcriptional factors and a specific DNA element. By means of recombinant plasmids, a hexahistidine-tagged derivative of the RNA polymerase alpha subunit of strain C-125 and two deletion derivatives (C- and N-terminal domains) of this protein were overexpressed in E. coli cells and purified to near homogeneity.
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