Abstract
A gene named ltsA was earlier identified in Rhodococcus and Corynebacterium species while screening for mutations leading to increased cell susceptibility to lysozyme. The encoded protein belonged to a huge family of glutamine amidotransferases whose members catalyze amide nitrogen transfer from glutamine to various specific acceptor substrates. We here describe detailed physiological and biochemical investigations demonstrating the specific role of LtsA protein from Corynebacterium glutamicum (LtsACg) in the modification by amidation of cell wall peptidoglycan diaminopimelic acid (DAP) residues. A morphologically altered but viable ΔltsA mutant was generated, which displays a high susceptibility to lysozyme and β-lactam antibiotics. Analysis of its peptidoglycan structure revealed a total loss of DAP amidation, a modification that was found in 80% of DAP residues in the wild-type polymer. The cell peptidoglycan content and cross-linking were otherwise not modified in the mutant. Heterologous expression of LtsACg in Escherichia coli yielded a massive and toxic incorporation of amidated DAP into the peptidoglycan that ultimately led to cell lysis. In vitro assays confirmed the amidotransferase activity of LtsACg and showed that this enzyme used the peptidoglycan lipid intermediates I and II but not, or only marginally, the UDP-MurNAc pentapeptide nucleotide precursor as acceptor substrates. As is generally the case for glutamine amidotransferases, either glutamine or NH4(+) could serve as the donor substrate for LtsACg. The enzyme did not amidate tripeptide- and tetrapeptide-truncated versions of lipid I, indicating a strict specificity for a pentapeptide chain length.
Highlights
Diaminopimelic acid (DAP) is mainly found in amidated form in cell wall peptidoglycan of Corynebacteriales
We here describe detailed physiological and biochemical investigations demonstrating the specific role of LtsA protein from Corynebacterium glutamicum (LtsACg) in the modification by amidation of cell wall peptidoglycan diaminopimelic acid (DAP) residues
The peptidoglycan of the parental strain was digested readily by mutanolysin but was not hydrolyzed, or only very slowly, by lysozyme, whereas both enzymes hydrolyzed the peptidoglycan from the ⌬ltsA strain quite efficiently. These results showed that the presence of DAPNH2 residues in the peptidoglycan inhibited in some way the hydrolase activity of lysozyme, a finding perfectly consistent with the higher resistance of the C. glutamicum wild-type cells toward this enzyme
Summary
Diaminopimelic acid (DAP) is mainly found in amidated form in cell wall peptidoglycan of Corynebacteriales. The observation that ltsA from R. erythropolis and C. glutamicum could not complement an asparagine-requiring mutant of E. coli (in which the two genes encoding asparagine synthases AsnA (the L-aspartate:ammonia ligase) and AsnB were inactivated) further supported this assumption [19, 20] Both LtsARe and AsnBEc displayed ATP-dependent glutaminase activity in vitro, the subsequent step of transfer of the amino group from glutamine to aspartic acid was only observed for AsnBEc. By contrast, LtsARe displayed a synthetase activity when lysozyme-treated cell wall extracts were added to the incubation mixture, suggesting the presence of a glutamine amino group acceptor in the cell wall of this species, which was not further identified [20]. In vitro enzymatic assays were developed that clearly demonstrated the glutamine amidotransferase activity of LtsACg and further specified its substrate specificity
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