Genetic Analysis of Peptidoglycan Biosynthesis in Mycobacteria: Characterization of a ddlA Mutant of Mycobacterium smegmatis

Abstract

A temperature-sensitive mutant of Mycobacterium smegmatis was characterized that contains a mutation in ddlA, the gene encoding D-alanine:D-alanine ligase. Enzymatic assays using recombinant proteins and Dcycloserine susceptibility indicate that the A365V mutation in the SMEG35 DdlA protein causes a reduction in enzymatic activity in vitro and in vivo.A nearly universal component of bacterial cell walls is peptidoglycan, a macromolecule that is composed of polysaccharide chains that are cross-linked by short peptide bridges. Peptidoglycan gives the bacterial cell its characteristic shape and prevents the cell from lysing due to high internal osmotic pressure. Our understanding of how peptidoglycan is synthesized in bacteria is derived mostly from work done with Escherichia coli in which a number of temperature-sensitive mutants have been isolated that are defective in the biosynthesis of peptidoglycan at 42°C (10,11,13,14,20). Two hallmarks of these mutants are cell lysis at the nonpermissive temperature and suppression of the temperature-sensitive phenotype on media containing osmotic stabilizers (10,11,13,14,20).We previously described the generation of a bank of temperature-sensitive mutants of Mycobacterium smegmatis mc 2 155 (2, 4). One of the mutants, SMEG35, exhibits the two phenotypic characteristics associated with E. coli mutants defective in peptidoglycan biosynthesis. First, SMEG35 cells grown at 30°C to an optical density at 600 nm of 0.5 and then shifted to 42°C lyse after one doubling time, as evidenced by a visual clearing of the culture and the appearance of flocculent material (data not shown). Second, the temperature-sensitive phenotype of SMEG35 can be suppressed on growth medium containing either 0.5 M sucrose or 0.2 M NaCl (data not shown).The bacterial strains and plasmids used in this study are listed in Table 1. To identify the mutated gene, SMEG35 was complemented with an M. smegmatis genomic cosmid library as previously described (2). A sublibrary was constructed from the complementing cosmid pAEB222 and the E. coli-mycobacterial shuttle vector pMD30 as described previously (2). The nucleotide sequence was determined for the insert of the smallest complementing subclone pAEB224 with an ABI310 automated sequencer (PE Biosystems, Foster City, Calif.) and genespecific primers. The presence of a mutation in the ddlA gene of SMEG35 was confirmed by sequencing of the mutant allele. The gene was amplified from the SMEG35 genome with Pfu DNA polymerase (Stratagene) and the primers 5Ј-TTGTGACTGCCCC GAACC-3Ј (forward) and 5Ј-CGAAAAACCCGTCGAGCC-3Ј (reverse) in a PCR mixture supplemented with 5% formamide. Sequence analysis revealed a single mutation at bp 1095 of ddlA that changes a C to a T on the top strand. This mutation results in an alanine-to-valine substitution at amino acid 365 of Ddl, close to the C terminus of the protein. The alanine that is mutated in the SMEG35 Ddl is a nonconserved amino acid residue that does not correspond to any of the amino acids that were previously shown to be...

Peptidoglycan precursor synthesis along the sidewall of pole-growing mycobacteria

“…We reasoned that a more precise gauge for MurF-dependent incorporation of the intact probe would be whether it could support the replication of a strain unable to ligate d -alanine to itself. Therefore, we confirmed that alkDADA rescues the growth of a temperature-sensitive ddlA mutant (47) at the non-permissive temperature ( Figure 4B ).…”

Mycobacterium smegmatis d -Alanine Racemase Mutants Are Not Dependent on d -Alanine for Growth

“…In this context, D-alanine ligase is an attractive candidate since this enzyme activity is also inhibited by DCS (13). More importantly, the construction of a conditionally lethal mutant bank led to the isolation of a thermosensitive mutant impaired in this gene function (3). Furthermore, DCS hypersusceptibility may reflect an alteration of the peptidoglycan structure of TAM23 cells, as a direct consequence of the inactivation of the alr gene.…”

Mutation in a d-alanine-d-alanine ligase of Azospirillum brasilense Cd results in an overproduction of exopolysaccharides and a decreased tolerance to saline stress

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