An unusual mutation results in the replacement of diaminopimelate with lanthionine in the peptidoglycan of a mutant strain of Mycobacterium smegmatis.

Mycobacterial peptidoglycan contains L-alanyl-D-iso-glutaminyl-meso-diaminopimelyl-D-alanyl-D-alanine peptides, with the exception of the peptidoglycan of Mycobacterium leprae, in which glycine replaces the L-alanyl residue. The third-position amino acid of the peptides is where peptidoglycan cross-linking occurs, either between the meso-diaminopimelate (DAP) moiety of one peptide and the penultimate D-alanine of another peptide or between two DAP residues. We previously described a collection of spontaneous mutants of DAP-auxotrophic strains of Mycobacterium smegmatis that can grow in the absence of DAP. The mutants are grouped into seven classes, depending on how well they grow without DAP and whether they are sensitive to DAP, temperature, or detergent. Furthermore, the mutants are hypersusceptible to beta-lactam antibiotics when grown in the absence of DAP, suggesting that these mutants assemble an abnormal peptidoglycan. In this study, we show that one of these mutants, M. smegmatis strain PM440, utilizes lanthionine, an unusual bacterial metabolite, in place of DAP. We also demonstrate that the abilities of PM440 to grow without DAP and use lanthionine for peptidoglycan biosynthesis result from an unusual mutation in the putative ribosome binding site of the cbs gene, encoding cystathionine beta-synthase, an enzyme that is a part of the cysteine biosynthetic pathway.

Use of a novel allele of the Escherichia coli aacC4 aminoglycoside resistance gene as a genetic marker in mycobacteria.

The aacC4 gene from Escherichia coli can be expressed in mycobacteria and confers resistance to apramycin. However, the major limitation of the aacC4 gene as a genetic tool is that the gene also confers resistance to kanamycin and gentamicin, two antibiotics commonly used for selection in mycobacterial genetics, thus reducing the utility of the aacC4 gene in the mycobacterial field. To overcome this problem we constructed, by chemical mutagenesis, a mutant allele of the E. coli aacC4 gene that still confers resistance to apramycin but has a reduced ability to confer resistance to kanamycin and gentamicin. We then constructed a variety of E. coli-mycobacteria shuttle plasmids containing this mutant allele.

Extragenic suppression of the requirement for diaminopimelate in diaminopimelate auxotrophs of Mycobacterium smegmatis.

Mycobacteria, like many prokaryotes, have a peptidoglycan with peptides composed of L-alanine (or glycine), D-iso-glutamine, meso-diaminopimelate, and D-alanine. We sought to study mycobacterial peptidoglycan biosynthesis by constructing diaminopimelate (DAP) auxotrophs of Mycobacterium smegmatis and then isolating spontaneous mutants of these auxotrophs that can grow in the absence of DAP. Here we report the isolation and characterization of seven classes of spontaneous M. smegmatis mutants with extragenic mutations that can suppress the DAP requirement of DAP auxotrophs.