The genes required for ␥-polyglutamic acid (PGA) production were cloned from Bacillus subtilis IFO16449, a strain isolated from fermented soybeans. There were four open reading frames in the cloned 4.2-kb DNA fragment, and they were almost identical to those in the ywsC and ywtABC genes of B. subtlis 168. Northern blot analysis showed that the four genes constitute an operon. Three genes, ywsC, ywtA, and ywtB, were disrupted to determine which gene plays a central role in PGA biosynthesis. No PGA was produced in ⌬ywsC and ⌬ywtA strains, indicating that both of these genes are essential for PGA production. To clarify the function of the YwsC protein, histidine-tagged YwsC (YwsC-His) was produced in the ⌬ywsC strain and purified from the lysozyme-treated lysate of the transformant by Ni-nitrilotriacetic acid affinity chromatography. Western blot analysis revealed that the YwsC-His protein consists of two subunits, the 44-kDa and 33-kDa proteins, which are encoded by in-phase overlapping in the ywsC gene.14 C-labeled PGA was synthesized by the purified proteins from L- Some Bacillus strains produce ␥-polyglutamic acid (PGA), an amino acid polymer that consists of only D-glutamic acid or D-and L-glutamic acid polymerized through ␥-glutamyl bonds, as a capsular or an extracellular viscous material (6). PGA was first discovered as a component of the capsule of Bacillus anthracis (19) and Bacillus mesentericus (18) and was isolated from the culture medium of Bacillus subtilis (7). Since then, a number of bacteria producing PGA, including B. subtilis (8,17,21,22), Bacillus licheniformis (9, 42), and Bacillus megaterium (13, 41), have been reported. PGA is a main constituent of the sticky material in natto, a Japanese traditional food made from soybeans that have been steamed and then fermented by B. subtilis (14).Concerning PGA biosynthesis, Makino et al. reported cloning of three genes, capBCA, responsible for capsular PGA biosynthesis from B. anthracis, and the gene products occurred together as membrane-associated proteins in the Escherichia coli transformant (26,27,43). The complete genome sequence of B. subtilis 168, in which ywsC and ywtAB were found to be highly homologous to the capBCA genes of B. anthracis, has been made available in databases (31). Recently, pgsBCA genes for PGA biosynthesis were also cloned from B. subtilis IFO3336, and their sequences were found to be the same as those of the ywsC and ywtAB genes of B. subtilis 168 (2). These three genes seem to be involved in PGA production; however, little is known about the function of each gene product in PGA biosynthesis.In this paper, we describe the cloning and gene disruption of the ywsC and ywtAB genes, which are responsible for PGA production in B. subtilis IFO16449, a strain isolated from natto, and we also describe the characterization of the YwsC 44-kDa and 33-kDa proteins, which catalyze the biosynthesis of PGA from L-glutamate, a crucial enzyme in PGA production. MATERIALS AND METHODSBacterial strains, plasmids, and media. Bacillus subtilis IF...

The ywtD gene, which codes for an enzyme that degrades ␥-polyglutamic acid (PGA), was cloned from Bacillus subtilis IFO16449. The gene is located immediately downstream of ywsC and ywtABC, a PGA operon involved in PGA biosynthesis, and it showed partial similarity to genes coding for DL-endopeptidase, a peptidoglycan-degrading enzyme. The ywtD gene, from which signal sequence is excised, was inserted into pET15b, and the recombinant plasmid was then transformed into Escherichia coli. Histidine-tagged YwtD was purified from sonicated cells of the transformant. The purified YwtD degraded PGA to yield two hydrolyzed products, a high-molecular-mass product (490 kDa with nearly 100% L-glutamic acid) and an 11-kDa product (with D-glutamic acid and L-glutamic acid in an 80:20 ratio). This finding and results of enzymatic analysis of the two products with carboxypeptidase G suggest that YwtD is a novel enzyme cleaving the ␥-glutamyl bond only between D-and L-glutamic acids of PGA, and it may be designated ␥-DL-glutamyl hydrolase.␥-Polyglutamic acid (PGA), an amino acid polymer that consists of only D-glutamic acid or D-and L-glutamic acids polymerized through ␥-glutamyl bonds, is produced by several strains of Bacillus (4,11,19,21). The genes required for PGA biosynthesis have also been cloned as ywsC and ywtABC (capBCA or pgsBCA) from Bacillus anthracis (14) and Bacillus subtilis (2,26,27), and ywsC was found to code for PGA synthetase (EC 6.3.2), which is a crucial enzyme in PGA production, catalyzing the biosynthesis of PGA from L-glutamate in the presence of ATP and Mn 2ϩ ions (26). Sequencing of the complete genome of B. subtilis 168 (12) revealed that there is a gene (ywtD) located just downstream of ywsC and ywtABC that encodes a protein of 413 amino acids with a molecular mass of 45 kDa, and YwtD has been identified as an extracellular protein with a signal peptide of 32 amino acids, though its function remains unknown (6). Furthermore, a homology search revealed that a partial amino acid sequence of YwtD exhibits 40 and 37% identity with those of the catalytic domains of LytF and LytE, DL-endopeptidases that cleave the ␥-glutamyl bond between D-glutamic acid and L-diamino acid of cell wall peptidoglycan (15,20,22), suggesting that YwtD functions as an enzyme that degrades PGA (Fig. 1). In this study, we purified and characterized the YwtD protein, and we demonstrated that YwtD is a novel PGA-hydrolyzing enzyme that cleaves only the ␥-glutamyl bond between D-glutamic acid and L-glutamic acid of PGA.Construction of a plasmid containing ywtD with a histidinetag codon. Plasmid pYWTD for expression of ywtD from which a signal sequence (6) was excised was constructed as follows. A sense primer, 5Ј-CTCGAGGATATACATCAGAATTG-3Ј (XhoI site underlined), and an antisense primer, 5Ј-CTCGAG TTATTGGCACCCGTATACTTCC-3Ј (XhoI site underlined), were designed on the basis of the sequence of the ywtD gene of B. subtilis 168 (11). A DNA fragment was amplified by PCR with the two primers and B. subtilis IFO16449 (26) chromosomal DNA a...

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The methyltransferase RsmG methylates the N7 position of nucleotide G535 in 16S rRNA of Bacillus subtilis (corresponding to G527 in Escherichia coli). Disruption of rsmG resulted in low-level resistance to streptomycin. A growth competition assay revealed that there are no differences in fitness between the rsmG mutant and parent strains under the various culture conditions examined. B. subtilis rsmG mutants emerged spontaneously at a relatively high frequency, 10 ؊6 . Importantly, in the rsmG mutant background, high-level-streptomycinresistant rpsL (encoding ribosomal protein S12) mutants emerged at a frequency 200 times greater than that seen for the wild-type strain. This elevated frequency in the emergence of high-level streptomycin resistance was facilitated by a mutation pattern in rpsL more varied than that obtained by selection of the wild-type strain.

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