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Journal of Bacteriology, January 2002, p. 337-343, Vol. 184, No. 2
0021-9193/01/$04.00+0     DOI: 10.1128/JB.184.2.337-343.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Characterization of the Bacillus subtilis ywsC Gene, Involved in {gamma}-Polyglutamic Acid Production

Yuji Urushibata, Shinji Tokuyama, and Yasutaka Tahara*

Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan

Received 12 July 2001/ Accepted 17 October 2001

The genes required for {gamma}-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 {Delta}ywsC and {Delta}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 {Delta}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. 14C-labeled PGA was synthesized by the purified proteins from L-[14C]-glutamate in the presence of ATP and MnCl2, through an acylphosphate intermediate, indicating that the ywsC gene encodes PGA synthetase (EC 6.3.2), a crucial enzyme in PGA biosynthesis.

* Corresponding author. Mailing address: Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan. Phone: 81 54 238 4878. Fax: 81 54 237 3028. E-mail: acytaha1{at}agr.shizuoka.ac.jp.

Journal of Bacteriology, January 2002, p. 337-343, Vol. 184, No. 2
0021-9193/01/$04.00+0     DOI: 10.1128/JB.184.2.337-343.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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