Nudix hydrolases with Coenzyme A (CoA) and acyl-CoA pyrophosphatase activities confer growth advantage to Mycobacterium smegmatis.

Nudix hydrolase family proteins hydrolyse toxic by-products of cellular metabolism such as mutagenic nucleoside triphosphates, sugar nucleotides and signalling molecules. We studied the substrate specificities of Nudix hydrolases encoded by rv3672c and rv3040c from Mycobacterium tuberculosis and their respective homologues, msmeg_6185 and msmeg_2327 from M. smegmatis. The rv3672c- and msmeg_6185-encoded proteins (Rv3672 and MSMEG_6185, respectively) showed CoA pyrophosphatase (CoAse) activity that converted acyl-CoA to adenosine-3',5'-diphosphate (3', 5'-ADP) and 4-acyl phosphopantetheine. The efficiencies of Rv3672 and MSMEG_6185 in hydrolysing CoA derivatives were found to be higher than those of the Rv3040 and MSMEG_2327 (encoded by rv3040c and msmeg_2327, respectively). Further, amongst the substrates tested, Rv3672 and MSMEG_6185 used CoA and oxidized CoA as the most preferred substrates. Use of the M. smegmatis model showed that the expression of msmeg_6185 occurs in the log and stationary phases but declines during the late stationary phase and becomes undetectable during hypoxia. The co-culture competition experiments performed between the wild-type and Δmsmeg_6185 strains of M. smegmatis in different carbon sources revealed that the presence of msmeg_6185 provided growth fitness advantage to M. smegmatis, irrespective of the carbon source, implicating its function in regulation for the optimal physiological levels of acyl-CoAs in the cell.

Rapid formylation of the cellular initiator tRNA population makes a crucial contribution to its exclusive participation at the step of initiation.

Initiator tRNAs (i-tRNAs) possess highly conserved three consecutive GC base pairs (GC/GC/GC, 3GC pairs) in their anticodon stems. Additionally, in bacteria and eukaryotic organelles, the amino acid attached to i-tRNA is formylated by Fmt to facilitate its targeting to 30S ribosomes. Mutations in GC/GC/GC to UA/CG/AU in i-tRNACUA/3GC do not affect its formylation. However, the i-tRNACUA/3GC is non-functional in initiation. Here, we characterised an Escherichia coli strain possessing an amber mutation in its fmt gene (fmtam274), which affords initiation with i-tRNACUA/3GC. Replacement of fmt with fmtam274 in the parent strain results in production of truncated Fmt, accumulation of unformylated i-tRNA, and a slow growth phenotype. Introduction of i-tRNACUA/3GC into the fmtam274 strain restores accumulation of formylated i-tRNAs and rescues the growth defect of the strain. We show that i-tRNACUA/3GC causes a low level suppression of am274 in fmtam274. Low levels of cellular Fmt lead to compromised efficiency of formylation of i-tRNAs, which in turn results in distribution of the charged i-tRNAs between IF2 and EF-Tu allowing the plasmid borne i-tRNACUA/3GC to function at both the initiation and elongation steps. We show that a speedy formylation of i-tRNA population is crucial for its preferential binding (and preventing other tRNAs) into the P-site.

Utilisation of 10-formyldihydrofolate as substrate by dihydrofolate reductase (DHFR) and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) tranformylase/IMP cyclohydrolase (PurH) in Escherichia coli.

Dihydrofolate reductase (DHFR) and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/IMP cyclohydrolase (PurH) play key roles in maintaining folate pools in cells, and are targets of antimicrobial and anticancer drugs. While the activities of bacterial DHFR and PurH on their classical substrates (DHF and 10-CHO-THF, respectively) are known, their activities and kinetic properties of utilisation of 10-CHO-DHF are unknown. We have determined the kinetic properties (kcat/Km) of conversion of 10-CHO-DHF to 10-CHO-THF by DHFR, and to DHF by PurH. We show that DHFR utilises 10-CHO-DHF about one third as efficiently as it utilises DHF. The 10-CHO-DHF is also utilised (as a formyl group donor) by PurH albeit slightly less efficiently than 10-CHO-THF. The utilisation of 10-CHO-DHF by DHFR is ~50 fold more efficient than its utilisation by PurH. A folate deficient Escherichia coli (∆pabA) grows well when supplemented with adenine, glycine, thymine and methionine, the metabolites that arise from the one-carbon metabolic pathway. Notably, when the ∆pabA strain harboured a folate transporter, it grew in the presence of 10-CHO-DHF alone, suggesting that it (10-CHO-DHF) can enter one-carbon metabolic pathway to provide the required metabolites. Thus, our studies reveal that both DHFR and PurH could utilise 10-CHO-DHF for folate homeostasis in E. coli.

Species-Specific Interactions of Arr with RplK Mediate Stringent Response in Bacteria.

Bacteria respond to stressful growth conditions through a conserved phenomenon of stringent response mediated by synthesis of stress alarmones ppGpp and pppGpp [referred to as (p)ppGpp]. (p)ppGpp synthesis is known to occur by ribosome-associated RelA. In addition, a dual-function protein, SpoT (with both synthetase and hydrolase activities), maintains (p)ppGpp homeostasis. The presence of (p)ppGpp is also known to contribute to antibiotic resistance in bacteria. Mycobacterium smegmatis possesses Arr, which inactivates rifampin by its ADP ribosylation. Arr has been shown to be upregulated in response to stress. However, the roles Arr might play during growth have remained unclear. We show that Arr confers growth fitness advantage to M. smegmatis even in the absence of rifampin. Arr deficiency in M. smegmatis resulted in deficiency of biofilm formation. Further, we show that while Arr does not interact with the wild-type Escherichia coli ribosomes, it interacts with them when the E. coli ribosomal protein L11 (a stringent response regulator) is replaced with its homolog from M. smegmatis The Arr interaction with E. coli ribosomes occurs even when the N-terminal 33 amino acids of its L11 protein were replaced with the corresponding sequence of M. smegmatis L11 (Msm-EcoL11 chimeric protein). Interestingly, Arr interaction with the E. coli ribosomes harboring M. smegmatis L11 or Msm-EcoL11 results in the synthesis of ppGpp in vivo Our study shows a novel role of antibiotic resistance gene arr in stress response.IMPORTANCEMycobacterium smegmatis, like many other bacteria, possesses an ADP-ribosyltransferase, Arr, which confers resistance to the first-line antituberculosis drug, rifampin, by its ADP ribosylation. In this report, we show that in addition to its known property of conferring resistance to rifampin, Arr confers growth fitness advantage to M. smegmatis even when there is no rifampin in the growth medium. We then show that Arr establishes species-specific interactions with ribosomes through the N-terminal sequence of ribosomal protein L11 (a stringent response regulator) and results in ppGpp (stress alarmone) synthesis. Deficiency of Arr in M. smegmatis results in deficiency of biofilm formation. Arr protein is physiologically important both in conferring antibiotic resistance as well as in mediating stringent response.