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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...

a b s t r a c tArchaea-specific D-family DNA polymerase forms a heterotetramer consisting of two large polymerase subunits and two small exonuclease subunits. The N-terminal (1-300) domain structure of the large subunit was determined by X-ray crystallography, although $50 N-terminal residues were disordered. The determined structure consists of nine alpha helices and three beta strands. We also identified the DNA-binding ability of the domain by SPR measurement. The N-terminal (1-100) region plays crucial roles in the folding of the large subunit dimer by connecting the $50 N-terminal residues with their own catalytic region (792-1163).Structured summary: DP2 binds to DP2 by molecular sieving (View interaction) DP2 binds to DP2 by fluorescence technology (View interaction) DP2 binds to DP2 by circular dichroism (View interaction)

Antigen-binding fragments (Fab fragments)

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