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Cyclic‐di‐nucleotide‐based secondary messengers regulate various physiological functions, including stress responses in bacteria. Cyclic diadenosine monophosphate (c‐di‐AMP) has recently emerged as a crucial second messenger with implications in processes including osmoregulation, antibiotic resistance, biofilm formation, virulence, DNA repair, ion homeostasis, and sporulation, and has potential therapeutic applications. The contrasting activities of the enzymes diadenylate cyclase (DAC) and phosphodiesterase (PDE) determine the equilibrium levels of c‐di‐AMP. Although c‐di‐AMP is suspected of playing an essential role in the pathophysiology of bacterial infections and in regulating host‐pathogen interactions, the mechanisms of its regulation remain relatively unexplored in mycobacteria. In this report, we biochemically and structurally characterize the c‐di‐AMP synthase (MsDisA) from Mycobacterium smegmatis. The enzyme activity is regulated by pH and substrate concentration; conditions of significance in the homoeostasis of c‐di‐AMP levels. Substrate binding stimulates conformational changes in the protein, and pApA and ppApA are synthetic intermediates detectable when enzyme efficiency is low. Unlike the orthologous Bacillus subtilis enzyme, MsDisA does not bind to, and its activity is not influenced in the presence of DNA. Furthermore, we have determined the cryo‐EM structure of MsDisA, revealing asymmetry in its structure in contrast to the symmetric crystal structure of Thermotoga maritima DisA. We also demonstrate that the N‐terminal minimal region alone is sufficient and essential for oligomerization and catalytic activity. Our data shed light on the regulation of mycobacterial DisA and possible future directions to pursue. PDB Code(s): EMDB-33540;PDB-7Y0D [ABSTRACT FROM AUTHOR]

Rifampicin and its derivatives are at the forefront of the current standard chemotherapeutic regimen for active tuberculosis; they act by inhibiting the transcription activity of prokaryotic RNA polymerase. Rifampicin is believed to interact with the [beta] subunit of RNA polymerase. However, it has been observed that protein--protein interactions with RNA polymerase core enzyme lead to its reduced susceptibility to rifampicin. This mechanism became more diversified with the discovery of RbpA, a novel RNA polymerase-binding protein, in Streptomyces coelicolor that could mitigate the effect of rifampicin on RNA polymerase activity. MsRbpA is a homologue of RbpA in Mycobacterium smegmatis. On deciphering the role of MsRbpA in M. smegmatis we found that it interacts with RNA polymerase and increases the rifampicin tolerance levels, both in vitro and in vivo. It interacts with the [beta] subunit of RNA polymerase. However, it was found to be incapable of rescuing rifampicin-resistant RNA polymerases in the presence of rifampicin at the respective [IC.sub.50]. DOI 10.1099/mic.0.033670-0

Cyclic di-GMP (c-di-GMP) plays an important role in bacterial adaptation to enable survival in changing environments. It orchestrates various pathways involved in biofilm formation, changes in the cell surface, host colonization and virulence. In this article, we report the presence of c-di-GMP in Mycobacterium smegmatis, and its role in the long-term survival of the organism. M. smegmatis has a single bifunctional protein with both GGDEF and EAL domains, which show diguanylate cyclase (DGC) and phosphodiesterase (PDE)-A activity, respectively, in vitro. We named this protein MSDGC-1. Deletion of the gene encoding MSDGC-1 did not affect growth and biofilm formation in M. smegmatis, but long-term survival under conditions of nutritional starvation was affected. Most of the proteins that contain GGDEF and EAL domains have been demonstrated to have either DGC or PDE-A activity. To gain further insight into the regulation of the protein, we cloned the individual domains, and tested their respective activities. MSDGC-1, the full-length protein, is required for activity, as its GGDEF and EAL domains are inactive when separated.

Anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, [[sigma].sup.70], of E. coli. Though both factors are known to interact with the C-terminal region of [[sigma].sup.70], the physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to the inhibition of E. coli cell growth to different amounts. It was found that AsiA is the more potent inhibitor of in vivo transcription and thus causes higher inhibition of E. coli cell growth. Measurements of affinity constants by surface plasmon resonance experiments showed that Rsd and AsiA bind to [[sigma].sup.70] with similar affinity. Data obtained from in vivo and in vitro binding experiments clearly demonstrated that the major difference between AsiA and Rsd is the ability of AsiA to form a stable ternary complex with RNA polymerase. The binding patterns of AsiA and Rsd with [[sigma].sup.70] studied by using the yeast two-hybrid system revealed that region 4 of [[sigma].sup.70] is involved in binding to both of these anti-sigma factors; however, Rsd interacts with other regions of [[sigma].sup.70] as well. Taken together, these results suggest that the higher inhibition of E. coli growth by AsiA expression is probably due to the ability of the AsiA protein to trap the holoenzyme RNA polymerase rather than its higher binding affinity to [[sigma].sup.70].

The survival of a bacterium with a depleted oxygen or nutrient supply is important for its long-term persistence inside the host under stressful conditions. We studied a gene, dps, from Mycobacterium smegmatis, encoding a protein, Dps (for DNA binding protein from starved cells), which is overexpressed under oxidative and nutritional stresses and provides bimodal protection to the bacterial DNA. Characterization of the dps promoter in vivo is therefore important. We cloned a 1-kb putative promoter region of the dps gene of M. smegmatis in an Escherichia coli-Mycobacterium shuttle vector, pSD5B, immediately upstream of the lacZ gene. Promoter activities were assayed in vivo both in solid medium and in liquid cultures by quantitative [beta]-galactosidase activity measurements. To characterize the minimal promoter region, a 200-bp fragment from the whole 1-kb sequence was further cloned in the same vector, and in a similar way, [beta]-galactosidase activity was quantitated. Primer extension analysis was performed to determine the +1 transcription start site of the gene. Point mutations were inserted in the putative promoter sequences in the -10 and -20 regions, and the promoter sequence was confirmed. The promoter was not recognized by purified M. smegmatis core RNA polymerase reconstituted with purified Mycobacterium tuberculosis [[sigma].sup.A] or [[sigma].sup.B] during multiple- and single-round in vitro transcription assays. Promoter-specific in vivo pull-down assays with an immobilized 1-kb DNA fragment containing the dps promoter established that extracellular function sigma factors were associated with this starvation-inducible promoter. Single-round transcription at the dps promoter further supported the idea that only core RNA polymerase reconstituted with [[sigma].sup.F] or [[sigma].sup.H] can generate proper transcripts.

The [omega] subunit, the smallest subunit of bacterial RNA polymerase, is known to be involved in maintaining the conformation of the [beta]' subunit and aiding its recruitment to the rest of the core enzyme assembly in Escherichia coli. It has recently been shown in Mycobacterium smegmatis, by creating a deletion mutation of the rpoZ gene encoding [omega], that the physiological role of the [omega] subunit also includes providing physical protection to [beta]'. Interestingly, the mutant had altered colony morphology. This paper demonstrates that the mutant mycobacterium has pleiotropic phenotypes including reduced sliding motility and defective biofilm formation. Analysis of the spatial arrangement of biofilms by electron microscopy suggests that the altered phenotype of the mutant arises from a deficiency in generation of extracellular matrix. Complementation of the mutant strain with a copy of the wild-type rpoZ gene integrated in the bacterial chromosome restored both sliding motility and biofilm formation to the wild-type state, unequivocally proving the role of [omega] in the characteristics observed for the mutant bacterium. Analysis of the cell wall composition demonstrated that the mutant bacterium had an identical glycopeptidolipid profile to the wild-type, but failed to synthesize the short-chain mycolic acids characteristic of biofilm growth in M. smegmatis.

A deletion mutation in the gene rpoZ of Mycobacterium smegmatis causes reduced growth rate and a change in colony morphology. During purification of RNA polymerase from the mutant strain, the [beta]' subunit undergoes fragmentation but the fragments remain associated with the enzyme and maintain it in an active state until the whole destabilized assembly breaks down in the final step of purification. Complementation of the mutant strain with an integrated copy of the wild-type rpoZ brings back the wild-type colony morphology and improves the growth rate and activity of the enzyme, and the integrity of the [beta]' subunit remains unaffected.

The presence of a polar species of glycopeptidolipid (GPL) in carbon-starved Mycobacterium smegmatis has been reported previously. In this study, the complete structure of this GPL is established with the help of MALDI-TOF (matrix assisted laser desorption/ionization time of flight) and ESI (electrospray ionization) -MS, [sup.13]C-SEFT (spin echo Fourier transform) -NMR spectroscopy, and HPLC analysis. In the molecule, two units of a 3,4-di-Q-methyl derivative of rhamnose are attached to L-alaninol via a 1 [right arrow] 2 linkage. Various methyl derivatives of rhamnose and 6-deoxytalose were synthesized as standards to establish this structure. The accumulation of this polar GPL in M. smegmatis is sigB dependent, as a SigB-overproducing strain of M. smegmatis shows the presence of this spot in the exponential phase, and a sigB-knockout strain of M. smegmatis does not show the presence of any polar GPLs.