Abstract
The enzymes alanine racemase (Alr) and glutamate racemase (MurI) catalyze the formation of the essential building blocks, D-alanine and D-glutamate, respectively, for cell wall biosynthesis. Despite their essential cellular role, very little is known about their regulation in response to various environmental stimuli and conditions where these genes might become non-essential for growth. The aim of this PhD project was to study the regulation and function of Alr and MurI in the fast growing saprophyte Mycobacterium smegmatis. The operon structure of alr and murI was analyzed using reverse transcription-polymerase chain reaction (RT-PCR) using RNA extracted from cells grown in minimal medium under aerobic conditions. This analysis revealed that both operons consisted of six genes in M. smegmatis, the operons were named as the alr operon and murI operon respectively.
The alr operon begins with alr whereas the murI operon starts with MSMEG_4907, which is annotated as Mov34/MPN/PAD-1 family protein. The order of the genes in the alr and murI operons was highly conserved in mycobacteria suggesting a syntenic relationship between species. The promoter of the alr operon was identified within -66 nucleotides (nt) from the start codon of the alr gene. Similarly for the murI operon, a promoter was mapped within -40 nt to -167 nt from the start codon of MSMEG_4907. The transcriptional start site (TSS) of the murI operon was detected at -46 nt upstream of the same start codon. A conserved region (5′-GACAAT-3′) was identified -20 nt upstream of the start codon of alr in M. smegmatis. For the murI operon, on the other hand, a conserved SigH-binding site was detected -6 nt upstream of the TSS. The promoter activity of the operons was not significantly modulated regardless of stimuli (e.g. oxidizing agent, hypoxia, heat-shock, amino acids, antibiotics, and different carbon sources) indicating the expression of the alr and murI operons was constitutive.
Growth kinetics and cell viability experiments revealed that ∆alr is bactericidal and ∆murI is bacteriostatic towards the growth of M. smegmatis. Continued incubation in the absence of D-alanine and D-glutamate gave rise to a revertant phenotype for both the ∆alr and ∆murI mutants. The growth of a putative ∆alr mutant was media-dependent and the mechanism of gaining the revertant phenotype for this strain remains unknown.
On the other hand, a stable growth phenotype of the putative ∆murI revertant suggested that a suppressor mutation in the genome is responsible for its genetic adaptation. Whole genome sequencing on the putative ∆murI suppressor mutants revealed the presence of a SNP, cytosine to thymine (C→T), at position 5863592 on the 3′→5′ DNA strand located in the intergenic region between MSMEG_5795 (4-amino-4-deoxychorismate lyase) and MSMEG_5796 (Glycine cleavage T-protein (aminomethyl transferase)) genes. The MSMEG_5795 gene downstream of the SNP was found to be overexpressed fourteen-fold compared to that of wild-type strain using qPCR suggesting that the SNP affects the promoter region and leads to overexpression. This result was validated using a transcriptional MSMEG_5795-lacZ fusion. Further, the SNP mutated promoter containing MSMEG_5795 gene was found to rescue ∆murI mutant. D-alanine itself was found to rescue the ∆murI mutant. Interestingly, this mutated MSMEG_5795 gene is also able to rescue ∆alr mutant. Taken together these results suggest that MSMEG_5795 has D-amino acid transaminase (D-AAT) activity and can catalyze the synthesis of D-glutamate from D-alanine in M. smegmatis.