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Characterization of LtsA from Rhodococcus erythropolis, an Enzyme with Glutamine Amidotransferase Activity

Proteolysis and Protein Turnover Research Group, Research Institute of Genome-Based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Tsukisamu-Higashi, Toyohira-ku,¹ Laboratory of Molecular Environmental Microbiology, Graduate School of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo,³ Microbial and Genetic Resources Research Group, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba-Higashi, Tsukuba, Japan²

Received 10 October 2004/ Accepted 4 January 2005

The nocardioform actinomycete Rhodococcus erythropolis has a characteristic cell wall structure. The cell wall is composed of arabinogalactan and mycolic acid and is highly resistant to the cell wall-lytic activity of lysozyme (muramidase). In order to improve the isolation of recombinant proteins from R. erythropolis host cells (N. Nakashima and T. Tamura, Biotechnol. Bioeng. 86:136-148, 2004), we isolated two mutants, L-65 and L-88, which are susceptible to lysozyme treatment. The lysozyme sensitivity of the mutants was complemented by expression of Corynebacterium glutamicum ltsA, which codes for an enzyme with glutamine amidotransferase activity that results from coupling of two reactions (a glutaminase activity and a synthetase activity). The lysozyme sensitivity of the mutants was also complemented by ltsA homologues from Bacillus subtilis and Mycobacterium tuberculosis, but the homologues from Streptomyces coelicolor and Escherichia coli did not complement the sensitivity. This result suggests that only certain LtsA homologues can confer lysozyme resistance. Wild-type recombinant LtsA from R. erythropolis showed glutaminase activity, but the LtsA enzymes from the L-88 and L-65 mutants displayed drastically reduced activity. Interestingly, an ltsA disruptant mutant, which expressed the mutated LtsA, changed from lysozyme sensitive to lysozyme resistant when NH₄Cl was added into the culture media. The glutaminase activity of the LtsA mutants inactivated by site-directed mutagenesis was also restored by addition of NH₄Cl, indicating that NH₃ can be used as an amide donor molecule. Taken together, these results suggest that LtsA is critically involved in mediating lysozyme resistance in R. erythropolis cells.