Characterization of Bacillus subtilis  -glutamyltransferase and its involvement in the degradation of capsule poly- -glutamate

Abstract

During early stationary phase, Bacillus subtilis NAFM5 produces capsular poly(c-glutamic acid) (cPGA, 2610 6 Da), which contains D-and L-glutamate, and then degrades it during late stationary phase. The c-glutamyltransferase (EC 2.3.2.2; GGT) of this strain successively hydrolysed cPGA from the amino-terminal end, to yield both D-and L-glutamate. This enzyme was specifically synthesized during the stationary phase through transcriptional activation of the corresponding ggt gene by the ComQXPA quorum-sensing system. A ggt knockout mutant degraded cPGA into 1610 5 Da fragments, but not any further, indicating that the capsule cPGA is first internally degraded by an endo-type of cPGA hydrolase into 1610 5 Da intermediates, then externally into glutamates via GGT. Due to its inability to generate the glutamates from the capsule, the ggt mutant sporulated more frequently than the wild-type strain. The results show that B. subtilis GGT has a powerful exo-c-glutamyl hydrolase activity that participates in capsule cPGA degradation to supply stationary-phase cells with constituent glutamates.

Poly-γ-glutamic acid: production, properties and applications

“…The function also depends on whether the c-PGA is peptidoglycan bound or released. If the c-PGA is peptidoglycan bound, it may help confer virulence or it can act as a source of glutamate under conditions of starvation (Kimura et al, 2004;Kocianova et al, 2005). If it is released into the environment, c-PGA can help the organism survive under adverse conditions (McLean et al, 1990).…”

“…RacE is a cytosolic enzyme with a high selectivity for glutamic acid and a preference for L-glutamic acid (Thorne et al, 1955;Buescher & Margaritis, 2007). According to Kimura et al (2004), racE was only important for growth in complex medium, whereas yrpC was found to be active when cells were grown on minimal medium. Neither of the two glutamate racemase genes was found to be responsible for the synthesis of c-PGA even though both seemed essential for D-glutamate catabolism (Kimura et al, 2004).…”

“…B. subtilis has two homologues of the glutamate racemase gene, racE/glr and yrpC (Ashiuchi et al, 1998(Ashiuchi et al, , 1999b. The functions of these genes are still uncertain as there are differing reports on the importance of each gene (Ashiuchi et al, 2003b;Kada et al, 2004;Kimura et al, 2004). RacE is a cytosolic enzyme with a high selectivity for glutamic acid and a preference for L-glutamic acid (Thorne et al, 1955;Buescher & Margaritis, 2007).…”

“…In both cases, c-PGA helps the pathogenic bacteria to escape phagocytosis, hence contributing to virulence. In some cases, c-PGA also acts as a source of glutamate when bacteria are starved in the late stationary phase (Kimura et al, 2004).…”

“…c-Glutamyl-transpeptidases (GGT) are enzymes that breakdown the transfer of a cglutamyl group from a donor species to acceptor (peptides and amino acids) species by developing an intermediate c-glutamyl enzyme in a transpeptidation reaction (Morelli et al, 2014). The enzyme has the ability to perform exohydrolase activity towards c-PGA, and the released glutamic acid is used by the bacterium as a source of carbon and nitrogen (Kimura et al, 2004;Morelli et al, 2014).…”