Chemical analysis of poly-gamma-glutamic acid produced by plasmid-free Bacillus subtilis (natto): Evidence that plasmids are not involved in poly-gamma-glutamic acid production.

It has been postulated that the psf gene on a small plasmid, pUH1 (5.8 kb), regulates positively the synthesis of capsular poly-gamma-glutamic acid (gammaPGA) in Bacillus subtilis (natto) Asahikawa. We found that this strain harbored a second plasmid, named pNAGL1 (ca. 50 kb), in addition to pUH1. The growth conditions that cure pUH1 or pNAGL1 were established. The plasmid-free NAF4 strain derived from B. subtilis (natto) Asahikawa was found to produce gammaPGA which was the same as the parent strain in terms of quantity and chemical properties having the same molecular mass and content of D-glutamic acid. Furthermore, as in the case of the parent cells, the D-glutamic acid in gammaPGA, which is known to increase up to ca. 80% of the total glutamic acid as Mn(2+) ion concentration increases in growth medium, was found to make up 80% of the total glutamic acid of the gammaPGA produced by NAF4 cells grown in the presence of 0.1 mm MnCl(2). Thus, these results led us to conclude that the plasmids do not encode any gene important for gammaPGA production.