Bh3216

Yvrl

Yvrl

B. halodurans

II. subtilis

Fig. 6.5 Dendrograms of members of sigma factors. These unrooted distance dendrograms were generated from the multiple sequence alignment of each paralogue and ortho-logue between the B. halodurans and B. subtilis genomes. Bar = 0.1 Knuc unit. (Reproduced with permission from H. Takami et al., Nucleic Acids Research, 28, 4317, Oxford Univ. Press(2000))

B. halodurans

II. subtilis o70 family ECF family

Fig. 6.5 Dendrograms of members of sigma factors. These unrooted distance dendrograms were generated from the multiple sequence alignment of each paralogue and ortho-logue between the B. halodurans and B. subtilis genomes. Bar = 0.1 Knuc unit. (Reproduced with permission from H. Takami et al., Nucleic Acids Research, 28, 4317, Oxford Univ. Press(2000))

rans and B. subtilis. As shown in Fig. 6.5, out of eleven sigma factors identified in B. halodurans, belonging to the extracytoplasmic function (ECF) family, sw is also found in B. subtilis but the other ten (BH640, BH672, BH1615, BH2026, BH3117, BH3216, BH3223, BH3380, BH3632 and BH3882) are unique to B. halodurans (Takami et al. 2000). These unique sigma factors may play a role in the special physiological mechanisms by which B. halodurans is able to live in an alkaline environment, because it is well known that ECF sigma factors are present in a wide variety of bacteria and serve to control the uptake or secretion of specific molecules or ions and to control responses to a variety of extracellular stress signals.

Seventy-nine tRNA species, organized into 11 clusters involving 71 genes plus 8 single genes, were identified (Table 6.2). Of the 11 clusters, 6 were organized in association with ribosomal RNA (rRNA) operons. Eight rRNA operons are present in the C-125 genome and their organisation is the same as that in B. subtilis (tRNA-16S-23S-5S, 16S-tRNA-23S-5S, and 16S-23S-5S-tRNA). With respect to tRNA synthetases, the C-125 genome lacks the glutaminyl-tRNA synthetase gene (glnS), one of two threonyl-tRNA synthetase gene (thrZ), and one of two tyrosyl-tRNA synthetase genes (tyrS). The B. subtilis genome has all of these tRNA synthetase genes except for the glutaminyl-tRNA gene. It is likely that glutamyl-tRNA synthetase aminoacy-lates tRNAGln with glutamate followed by transamidation by Glu-tRNA ami-dotransferase in both Bacillus species.

6.3.3 Competence and Sporulation

Of 20 genes related to competence in B. subtilis, thirteen genes (tin A, comC, comEA, comEB, comEC, comER, comFA, comFC, comGA, comGB, comGC, comGD, and mecA) mainly expressed in the late stage of competence were identified in the B. halodurans genome, but Takami et al. (2000) could not find any of the genes expressed in the early stage of competence. Among six genes whose products are known to serve as components of DNA transport machinery, only three genes (comGB, romGC, and comGD), but not the others well conserved in B. subtilis, were identified in B. halodurans C-125. Actually, competence has not been demonstrated in C-125 experimentally, although Takami et al. attempted to use standard and modified methods by changing some conditions such as pH, temperature and medium for transformation. It has become clear that this is due to lack of some of the necessary genes, especially those expressed in the early stage such as comS, srfA, and rapC. Only 68 genes related to sporulation were identified in the C-125 genome, in contrast with 138 genes found in the B. subtilis genome. Although the minimum set of genes for sporulation was well conserved, the same as in the case of B. subtilis, the C-125 genome lacks some genes encoding key regulatory proteins (the response regulator for aspartate phosphatase and the phosphatase regulator) and the spore coat protein for sporulation conserved in the B. subtilis genome. In particular, the rap (rapA-K) and phr (phrA, phrC, phrE-G, phrl, and phrK) genes were not found in the C-125 genome, suggesting that C-125 may have another type(s) of regulatory genes for control of sporulation in a manner the same as or different from that in B. subtilis, because sporulation has been observed in B. halodurans.

6.3.4 Cell Walls

The peptidoglycan of alkaliphilic B. halodurans C-125 appears to be similar to that of neutrophilic B. subtilis. However, the cell wall components in C-125 are characterized by an excess of hexosamines and amino acids compared to that of B. subtilis. Glucosamine, muramic acid, D- and I-alanine, D-glutamic acid, meso-diaminopimelic acid and acetic acid were found in cell wall hydrolysates (Horikoshi 1999a). Although some variation was found in the amide content of the peptidoglycan isolated from alkaliphilic Bacillus halodurans C-125, the pattern of variation was similar to that known to occur in B. subtilis. All genes related to peptidoglycan biosynthesis such as mraY, murC-G, cwlA, ddlA and glnA confirmed to be present in the B. subtilis genome, were also conserved in the C-125 genome. A bacitracin-resistance gene found in the B. subtilis genome is duplicated in the C-125 genome (BH474 and BH1538). On the other hand, although tagH and tagG genes were identified in B. halodurans C-125, 13 other genes for teichoic acid biosynthesis found in II. subtilis (dltA-E, ggaA, ggaB, tagA-G, tagE, tagE and tagO) are missing in the B. halodurans genome. B. halodurans C-125 also lacks six genes (tuaB-tuaFand tuaH) for teichuronic acid biosynthesis, all except tuaA and luaG, in comparison with those of B. subtilis (Takami et al. 2000). In addition to peptidoglycan, the cell wall of alkaliphilic B. halodurans is known to contain certain acidic polymers, such as galacturonic acid, glutamic acid, aspartic acid and phosphoric acid. The negative charges on acidic nonpeptidoglycan components may give the cell surface the ability to absorb sodium and hydronium ions and to repel hydroxide ions, and, as a consequence, may contribute to allowing the cells to grow in alkaline environments. A mutant defective in poly-y-L-glutamic acid synthesis (Accession Numbers AB071407-AB071409) which was called tupA by Aono et al. (1999) grows very slowly at alkaline pH. Further details are discussed in Section 3.2.2. Takami et al.'s study (2000) has made it clear that B. halodurans C-125 has no paralogue of poly-y-L-glutamic acid synthesis gene (pig 1-3) in the genome and the orthologue of these genes have not been found in other microorganisms except that of Oceanobacillus iheyensis HTE831 (Fig. 6.6)

6.3.5 Membrane Transport and Energy Generation

B. halodurans C-125 requires Na + for growth under alkaline conditions. The presence of sodium ions in the surrounding environment has been proved to be essential for effective solute transport through the cytoplasmic membrane of C-125 cells. According to the chemiosmotic theory, a protonmotive force is generated across the cytoplasmic membrane by the electron transport chain or by extrusion of H + derived from ATP metabolism through the action of ATPase. Takami et al. (2000) identified four types of ATPases (preprotein translocase subunit, class III heat-shock ATP-depen-dent protease, heavy metal-transporting ATPase and cation-transporting ATPase). These ATPases are well conserved between B. halodurans and B. subtilis.

Through a series of analyses such as a BLAST2 search, clustering analysis by the single linkage method examining all CDSs identified in the B. halodurans C-125 and B. subtilis genomes (8166 CDSs), and multiple alignment, 18 CDSs were grouped into the category of antiporter- and transporter-related protein genes in the C-125 genome. In this analysis, five CDSs were found to be candidates for Na +/H * antiporter genes (BH1316, BH1319, BH2844, BH2964 and BH3946). However, no gene encoding antibiotic-resistance proteins in the C-125 were found, whereas the B. subtilis genome has nine different ones. Eleven genes for multidrug-resistant proteins were identified in the C-125 genome, 6 fewer than in B. subtilis. A non-alkaliphilic mutant strain (mutant 38154) derived from B. halodurans C-125, which is useful as a host for cloning genes related to alkaliphily has been isolated and characterized (Kudo et al. 1990). A 3.7-kb DNA fragment (pALK fragment) from the parent strain restored the growth of mutant 38154 under alkaline pH conditions. This fragment was found to contain CDS BH1319 which is one of the Na + / H " antiporter genes in B.

Bacillus halodurans C-125

transcription!

rr*ula,c>r "'"bhwi BH.WiJ BH.16S5 liH.lfo? liU.WW BH.Wil UH.IMi.l

HH-1W7 RHW4B HII.VW) HM.1M3 RH365-1 BHJftffi HH.W5K RHlftftd KH3«i2 Hmwu

ftjfH ItigG utaCt

ftjfH ItigG utaCt

OH1«» OKP9II OBJ« 12 UKW 11829)6 OB29IH OK 2420 OB2922 OB292S OH 2927

DHNU OB29I5 OH29I9 (11(2921 Oli292.1 OB29'(i 0«»»

1>H»I7 OB2924

OH1«» OKP9II OBJ« 12 UKW 11829)6 OB29IH OK 2420 OB2922 OB292S OH 2927

DHNU OB29I5 OH29I9 (11(2921 Oli292.1 OB29'(i 0«»»

1>H»I7 OB2924

Was this article helpful?

0 0

Post a comment