A Mutation in the Corynebacterium glutamicum ltsA Gene Causes Susceptibility to Lysozyme, Temperature-Sensitive Growth, and l -Glutamate Production

Abstract

The Corynebacterium glutamicum mutant KY9714, originally isolated as a lysozyme-sensitive mutant, does not grow at 37°C. Complementation tests and DNA sequencing analysis revealed that a mutation in a single gene of 1,920 bp, ltsA (lysozyme and temperature sensitive), was responsible for its lysozyme sensitivity and temperature sensitivity. The ltsA gene encodes a protein homologous to the glutamine-dependent asparagine synthetases of various organisms, but it could not rescue the asparagine auxotrophy of an Escherichia coli asnA asnB double mutant. Replacement of the N-terminal Cys residue (which is conserved in glutamine-dependent amidotransferases and is essential for enzyme activity) by an Ala residue resulted in the loss of complementation in C. glutamicum. The mutant ltsA gene has an amber mutation, and the disruption of the ltsA gene caused lysozyme and temperature sensitivity similar to that in the KY9714 mutant. L-Glutamate production was induced by elevating growth temperature in the disruptant. These results indicate that the ltsA gene encodes a novel glutamine-dependent amidotransferase that is involved in the mechanisms of formation of rigid cell wall structure and in the L-glutamate production of C. glutamicum.Coryneform bacteria are rod-shaped, nonsporulating, grampositive bacteria that are widely distributed in nature. The nonpathogenic species Corynebacterium glutamicum, Brevibacterium lactofermentum, and Brevibacterium flavum are used for fermentative production of amino acids such as L-glutamic acid (16), L-lysine (21), and L-threonine (26). Recently, B. lactofermentum and B. flavum were reclassified as C. glutamicum (18).C. glutamicum was originally isolated as a glutamate-producing bacterium (16,30). Induction of glutamate excretion by C. glutamicum requires biotin limitation (27) or treatment with penicillin (22) or a fatty acid ester surfactant (28). Since these treatments correlated with alterations in cell surface structure, it had been thought that glutamate leaks passively through a membrane. Recently, several findings that do not agree with the leakage model have been reported (9,12,15). However, the mechanism of the glutamate production and excretion of C. glutamicum is still unknown.Coryneform bacteria have thick cell walls consisting of two layers. The inner layer, which consists mainly of peptidoglycan, invaginates to form the septum. The outer layer, which consists mainly of mycolic acid, breaks after cell division, resulting in postfission movement and the cell arrangements characteristic of these bacteria. C. glutamicum shows high tolerance to lysis by lytic enzymes, such as egg white lysozyme, that catalyze hydrolysis of the ␤-1,4 glycoside bond between the N-acetylglucosamine and N-acetylmuramic acid of peptidoglycan. This is probably because the outer layer, mainly consisting of mycolic acid, gives enough resistance against cell lysis to this bacterium and/or functions as a permeability barrier.As the first step in analyzing the cell wall structure of coryneform bacteria and...

Hydrolysis of peptidoglycan is modulated by amidation of meso‐diaminopimelic acid and Mg2+ in Bacillus subtilis

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Advances in the Development of Genetic Tools for the Genus Rhodococcus

“…erythropolis L-88 and L-65 were reported to be lysozyme-sensitive mutants, and it was shown that ltsA was responsible for the mutant phenotype in both the mutants 39) . The gene was homologous to ltsA from Corynebacterium glutamicum, which has been known to produce a lysozymesensitive phenotype on mutation 40) , and was shown to be . It has been suggested that the function of ltsA is conserved among the genera Mycobacterium, Corynebacterium, Rhodococcus, and Bacillus 39) .…”

ACorynebacterium glutamicum rnhA recGDouble Mutant Showing Lysozyme- sensitivity, Temperature-sensitive Growth, and UV-Sensitivity

“…Since the ltsA mutant showed swollen morphology at the restrictive temperature, ltsA is appeared to be involved in the formation of cell surface structure. 12) Mutations in ltsA induced L-glutamate production by C. glutamicum. 12,13) Its function, however, still remains to be elucidated.…”

“…12) Mutations in ltsA induced L-glutamate production by C. glutamicum. 12,13) Its function, however, still remains to be elucidated.…”

“…In a previous paper, we reported the isolation of a novel gene, ltsA, which is probably responsible for the synthesis or maintenance of cell surface of C. glutamicum. 12) LtsA protein shows high homology with the asparagine synthetase B (AsnB) of Escherichia coli, but has no asparagine synthesis activity in vivo. Since the ltsA mutant showed swollen morphology at the restrictive temperature, ltsA is appeared to be involved in the formation of cell surface structure.…”