Bacillus Halodurans C-125 Research Articles

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

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.

A kinetic study on pH-activity relationship of XynA from alkaliphilic Bacillus halodurans C-125 using aryl-xylobiosides

Xylanase A from alkaliphilic Bacillus halodurans C-125 was expressed in Escherichia coli and purified by affinity and anion exchange chromatographies. It exhibited a strong substrate inhibition using xylan as the substrate. Its K i value increased with an increase in pH. The effect of pH on the enzyme activity was determined using two aryl-xylobiosides as substrates, and it was found that the enzyme had a flat k cat-pH curve in the pH range of 5.8–8.8. This range was different from that obtained with 0.45% xylan as previously reported (Honda, H. et al., Agric. Biol. Chem., 49, 3165–3169, 1985). The substrate inhibition was presumed to cause the difference. It has been clarified that the use of aryl-xylobiosides as substrates yields more accurate kinetic results than that of xylan.

Employing chimeric xylanases to identify regions of an alkaline xylanase participating in enzyme activity at basic pH

The xylanase A (XynA) from the alkaliphilic Bacillus halodurans C-125 and the xylanase B (XynB) from Clostridium stercorarium F9 were subdivided into four fragments at highly homologous regions present in their primary structures: an amino-terminal region (A or a), a region containing the putative proton donor (P or p), a region containing the putative catalytic nucleophile (N or n), and a carboxyl-terminal region (C or c). Six chimeric xylanases were constructed by the selective substitution of the four fragments using an overlapping PCR technique. Two of the six xylanases, APnc and Apnc (regions originating from XynA are denoted by upper case letters and those from XynB are denoted by lower case letters), were produced in Escherichia coli while the other four xylanases were obtained only as inclusion bodies. The APnc and Apnc chimeric enzymes were purified by column chromatography using Ni-NTA agarose and DEAE-Toyopearl. The respective pH and temperature stabilities of the purified enzymes were observed from pH 5.6 to 11.6 and up to 45°C for APnc, and from pH 5.6 to 11.2 and up to 45°C for Apnc. Thus, these enzymes were slightly less stable than the parental xylanases. An assessment of the pH-activity relationships for the chimeric xylanases employed p-nitrophenyl-β- d-xylobioside as the sustrate in determinations of the k cat values. The p K a1 values for the APnc and Apnc chimeric enzymes were 4.3 and 4.2, respectively, which were almost identical to those for the parental xylanases. In contrast, the p K a2 values obtained for APnc and Apnc were 9.1 and 8.5, respectively; these values fall between those for the parental xylanases, XynA (9.4) and XynB (7.8). These results indicate that the main regions necessary to maintain the high p K a2 value of XynA locate in the A and P sections.

A globin-coupled oxygen sensor from the facultatively alkaliphilic Bacillus halodurans C-125.

We have recently discovered heme-containing signal transducers from the archaeon Halobacterium salinarum (HemAT-Hs) and the gram-positive bacterium Bacillus subtilis (HemAT-Bs). These proteins bind diatomic oxygen and trigger aerotactic responses. We identified that HemAT oxygen-sensing domains contain a globin-coupled sensor (GCS) motif, which exists as a two-domain transducer, having no similarity to the PAS domain (Period circadian protein, Ah receptor nuclear translocator protein, Single-minded protein) superfamily transducers. Using the GCS motif, we predicted that a 439-amino-acid protein annotated as a methyl-accepting chemotaxis protein (MCP) in the facultatively alkaliphilic bacterium Bacillus halodurans is a globin-coupled oxygen sensor. We cloned, expressed, and purified GCS(Bh), and performed its spectral analysis. GCS(Bh), binds heme and shows myoglobin-like spectra. This suggests that GCS(Bh) acts as an oxygen sensor and transmits a conformational signal through a linked signaling domain to trigger an aerotactic response in B. halodurans.

Analysis of the genome of an alkaliphilic Bacillus strain from an industrial point of view.

Bacillus species and other microbes with pH optima for growth higher than pH 9 are defined as alkaliphiles. A large number of alkaliphilic Bacillus strains producing useful enzymes, have been isolated from various environments. Some of these enzymes, such as proteases and cellulases from alkaliphilic Bacillus strains, have been commercialized and have brought great advantages to industry and domestic life. To support further development of the enzyme industry, we initiated analysis of the genome of Bacillus halodurans C-125, which is 4.25Mb in size, and constructed a physical and genetic map for comparison with the Bacillus subtilis chromosome. Systematic sequencing of the whole genome of Bacillus halodurans C-125 has been automated since the beginning of May 1998, and sequencing of 98% of the whole genome has been done so far. Through genome analysis, it became apparent that the genome organization of alkaliphilic Bacillus halodurans C-125 is totally different from that of B. subtilis orthologues.

An automated system for genome analysis to support microbial whole-genome shotgun sequencing.

We developed a semi-automated genome analysis system called GAMBLER in order to support the current whole-genome sequencing project focusing on alkaliphilic Bacillus halodurans C-125. GAMBLER was designed to reduce the human intervention required and to reduce the complications in annotating thousands of ORFs in the microbial genome. GAMBLER automates three major routines: analyzing assembly results provided by genome assembler software, assigning ORFs, and homology searching. GAMBLER is equipped with an interface for convenience of annotation. All processes and options are manipulatable through a WWW browser that enables scientists to share their genome analysis results without choosing computer platforms.

Replication origin region of the chromosome of alkaliphilic Bacillus halodurans C-125.

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.