Regulation of poly-gamma-glutamate synthesis and degradation in Bacillus subtlis

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We investigated nutrient factors that affect the synthesis and degradation of extracellular polymer of glutamate, poly-γ-glutamate (PGA), in Bacillus subtilis NAFM5, a derivative of a starter strain for Natto (Japanese fermented soybean food). We also analyzed regulatory systems involved in the regulation of the PGA biosynthetic and degradative enzyme genes. Our major results include :1. Regulation of the capBCAEPGA biosynthetic operon by nutrients.L-Glutamate and glucose stimulated PGA production by 2.5-and 3-fold, but repressed expression of a capB::lacZ fusion to 1/2 and 1/6, respectively. Nutrient-rich GSP medium, which contained soybean protein hydroysates, greatly improved (11 fold of that in minimal medium), whereas LB medium almost prevent capB::lacZ expression and PGA production.2. Regulation of the capBCAE PGA biosynthetic operon by the quorum-sensing system.We found that degQ and degSU genes are essential for PGA synthesis and determined by gel-shift assays that DegU binds to-720 ro-660 region of the capB promoter.3. Regulation of ywtD and ggt.]We found that ggt is under the control of the ComQXPA quorum-sensing system. Genetic study showed that ywrD encodes an end-type of PGA hdrolase. This gene appeared not regulated by the quorum-sensing system.4. Functional analysis of vvzD.Using a knockout mutant of yvzD, we demonstrated that this gene encode a deanchoring enzyme detaching cell-wall associated PGA. Thus, at least three genes, yvzD, ywtD and ggt, are involved in PGA degradation. YvzD is also not controlled by the quorum-sensing system.