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Japan Marine Science and Technology Center Frontier Research System for Extremophiles BH sdhC - succinate dehydrogenase (EC ...) cytochrome b BH BH - unknown conserved protein in others BH polA - DNA polymerase I (EC ...) BH BH + unknown conserved protein BH BH - unknown conserved protein in B. subtilis BH BH - glycine cleavage system protein H BH BH - epidermal surface antigen BH BH + unknown BH BH + unknown BH BH - unknown conserved protein BH BH - unknown conserved protein BH BH - unknown conserved protein BH hag - flagellin BH BH - oligopeptide ABC transporter BH BH - unknown conserved protein in others BH flhP - flagellar hook-basal body protein BH mbl - cell shape determining protein (MreB-like protein) BH atpD - ATP synthase (EC ...) beta subunit BH atpE - ATP synthase (EC ...) subunit c BH BH + ABC transporter (substrate-binding protein) BH BH - PTS system, mannitol-specific enzyme II, BC component BH BH - methyl-accepting chemotaxis protein BH lctE - L-lactate dehydrogenase (EC ...) BH BH - unknown conserved protein in B. subtilis BH soj - centromere-like function BH rpmH - ribosomal protein L nificantly upregulated in alkaline cultures. The mRNA expression of selected genes were confirmed by Northern hybridization, indicated that the results from the two methods are consistent with each other. Since the exact functions of many genes are still unknown, it is difficult to judge the physiological significance of this observation now. Further investigation is necessary. One interesting point is that a human epidermal surface antigen (ESA) homologue BH was observed to be significantly upregulated. Northern hybridization analysis showed that the expression of BH was pH-dependent, and the highest level of expression was observed at pH and pH. ESA gene is thought to play important roles in intercellular epidermal adhesion and is localized to human chromosome in the same region as the gene for neurofibromatosis. It is conserved in all mammals, but is not present in amphibians or insects. This is the first finding that the expression of prokaryotic ESA gene was induced by alkaline pH. The functions and mechanisms of this gene in alkaline adaptation remain to be further studied. It is well known that sodium ions are indispensable for growth and motility of alkaliphilic Bacillus. We 73
JAMSTEC 2002 Annual Report Frontier Research System for Extremophiles also carried out DNA chip analysis to explore the gene expression patterns in different concentrations of NaCl. The results showed that a lot of genes encoding proteins related to cell membrane and cellular transport systems were significantly induced by Na + ion. Another characterisitic of B. halodurans C- is the alkali-dependent swimming motility. This alkalidependent swimming motility was not found from other alkaliphilic Bacillus such as Oceanobacillus iheyensis HTE. We made several mutants that showed alkali-independent swimming motility, and then systematically examined the mRNA profiles with the DNA chips. Significant alterations of several genes encoding proteins such as flagellin and glycolate oxidase were found between wild type and mutant. It was suggested that these genes may be involved in alkali-dependent swimming motility. 1.6. Proteomic analysis of pH-dependent gene expression in facultative alkaliphilic Bacillus halodurans C-125 The complete identification of coding sequences in a number of model species has led to announce the beginning of the post-sequencing era; rapid advances in genomic sequencing technology and bioinformatics have established the field of genomics to investigate the expression profile of the genes identified in the genome through mRNA display. Since recent studies have demonstrated, however, a lack of correlation between the transcriptional profiles and the actual protein levels in the cells, the comprehensive analysis of the gene products is indispensable to link genomic data to biological function. Analysis of the genome of Bacillus halodurans C- was initiated in as a standard model for facultative alkaliphilic Bacillus strains, and the systemic sequencing of the whole genome of B. halodurans C- finished in . The B. halodurans C- is the first industrial strain whose whole genomic sequence has been determined. The objective of proteomics is a large-scale, comprehensive characterization of the proteins and protein interactions in the cells. The proteomic analysis with sensitive mass spectrometry subsequent to high-resolution two-dimensional electrophoresis (-DE) was used to identify the proteins involved in the mechanisms of adaptation to alkaline environment in B. halodurans C-. The total proteins and membrane protein fractions of the B.halodurans C- cells grown to the mid- exponential phase at pH . and pH . were subjected to -DE. A pH gradient of to was chosen for the isoelectrofocusing (IEF) due to the stability and reproducibility of the gradient in this pH range. The results of the -DE are shown in Fig.. Approximately protein spots could be resolved by LabScan v. software (Amersham Biosciences) on a gel stained with Coomasie Brilliant Blue (CBB) R-. The amounts of the protein in spots out of the were more than twofold higher in pH. culture than pH . cultures. All proteins were identified by N-terminal amino acid sequencing and LC/MS/MS. Eight proteins of interest were pI kDa 4.0 94 A 67 43 30 20 14 94 67 43 30 20 14 C 7.0 4.0 7.0 Fig. 3 Two-dimensional gel electrophoresis of B. halodurans C-125 mid-exponential phase cell proteins. A total of 150 µg of total proteins (A, pH 7.0 and B, pH10.0) and membrane proteins (C, pH 7.0 and D, pH10.0) were loaded for each gel. Prior to electrophoresis, samples were incubated in SDS buffer at room temperature for 15 min. After electrophoresis, gels were stained with Coomassie blue R- 250. Spots whose density was increased in the growth of pH 10.0 are marked with circles. B D 74
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