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Applied and Environmental Microbiology, May 2003, p. 2491-2497, Vol. 69, No. 5
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.5.2491-2497.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Characterization of Poly-{gamma}-Glutamate Hydrolase Encoded by a Bacteriophage Genome: Possible Role in Phage Infection of Bacillus subtilis Encapsulated with Poly-{gamma}-Glutamate

Keitarou Kimura and Yoshifumi Itoh*

Division of Applied Microbiology, National Food Research Institute, Tsukuba 305-8642, Japan

Received 20 September 2002/ Accepted 7 February 2003

Some Bacillus subtilis strains, including natto (fermented soybeans) starter strains, produce a capsular polypeptide of glutamate with a {gamma}-linkage, called poly-{gamma}-glutamate ({gamma}-PGA). We identified and purified a monomeric 25-kDa degradation enzyme for {gamma}-PGA (designated {gamma}-PGA hydrolase, PghP) from bacteriophage {Phi}NIT1 in B. subtilis host cells. The monomeric PghP internally hydrolyzed {gamma}-PGA to oligopeptides, which were then specifically converted to tri-, tetra-, and penta-{gamma}-glutamates. Monoiodoacetate and EDTA both inhibited the PghP activity, but Zn2+ or Mn2+ ions fully restored the enzyme activity inhibited by the chelator, suggesting that a cysteine residue(s) and these metal ions participate in the catalytic mechanism of the enzyme. The corresponding pghP gene was cloned and sequenced from the phage genome. The deduced PghP sequence (208 amino acids) with a calculated Mr of 22,939 was not significantly similar to any known enzyme. Thus, PghP is a novel {gamma}-glutamyl hydrolase. Whereas phage {Phi}NIT1 proliferated in B. subtilis cells encapsulated with {gamma}-PGA, phage BS5 lacking PghP did not survive well on such cells. Moreover, all nine phages that contaminated natto during fermentation produced PghP, supporting the notion that PghP is important in the infection of natto starters that produce {gamma}-PGA. Analogous to polysaccharide capsules, {gamma}-PGA appears to serve as a physical barrier to phage absorption. Phages break down the {gamma}-PGA barrier via PghP so that phage progenies can easily establish infection in encapsulated cells.

* Corresponding author. Mailing address: Division of Applied Microbiology, National Food Research Institute, Kannondai 2-1-12, Tsukuba, Ibaraki 305-8642, Japan. Phone: 81-298-38-8075. Fax: 81-298-38-7996. E-mail: yosifumi{at}nfri.affrc.go.jp.

Applied and Environmental Microbiology, May 2003, p. 2491-2497, Vol. 69, No. 5
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.5.2491-2497.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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