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Nature Mar 2023Diverse aerobic bacteria use atmospheric H as an energy source for growth and survival. This globally significant process regulates the composition of the atmosphere,...
Diverse aerobic bacteria use atmospheric H as an energy source for growth and survival. This globally significant process regulates the composition of the atmosphere, enhances soil biodiversity and drives primary production in extreme environments. Atmospheric H oxidation is attributed to uncharacterized members of the [NiFe] hydrogenase superfamily. However, it remains unresolved how these enzymes overcome the extraordinary catalytic challenge of oxidizing picomolar levels of H amid ambient levels of the catalytic poison O and how the derived electrons are transferred to the respiratory chain. Here we determined the cryo-electron microscopy structure of the Mycobacterium smegmatis hydrogenase Huc and investigated its mechanism. Huc is a highly efficient oxygen-insensitive enzyme that couples oxidation of atmospheric H to the hydrogenation of the respiratory electron carrier menaquinone. Huc uses narrow hydrophobic gas channels to selectively bind atmospheric H at the expense of O, and 3 [3Fe-4S] clusters modulate the properties of the enzyme so that atmospheric H oxidation is energetically feasible. The Huc catalytic subunits form an octameric 833 kDa complex around a membrane-associated stalk, which transports and reduces menaquinone 94 Å from the membrane. These findings provide a mechanistic basis for the biogeochemically and ecologically important process of atmospheric H oxidation, uncover a mode of energy coupling dependent on long-range quinone transport, and pave the way for the development of catalysts that oxidize H in ambient air.
Topics: Cryoelectron Microscopy; Hydrogen; Hydrogenase; Oxidation-Reduction; Oxygen; Vitamin K 2; Atmosphere; Mycobacterium smegmatis; Hydrogenation
PubMed: 36890228
DOI: 10.1038/s41586-023-05781-7 -
Disease Markers 2022Sigma factor B (SigB), an alternative sigma factor (ASF), is very similar to primary sigma factor SigA ( ) but dispensable for growth in both (Msmeg) and (Mtb). It is...
Sigma factor B (SigB), an alternative sigma factor (ASF), is very similar to primary sigma factor SigA ( ) but dispensable for growth in both (Msmeg) and (Mtb). It is involved in general stress responses including heat, oxidative, surface, starvation stress, and macrophage infections. Despite having an extremely short half-life, SigB tends to operate downstream of at least three stress-responsive extra cytoplasmic function (ECF) sigma factors (SigH, SigE, SigL) and SigF involved in multiple signaling pathways. There is very little information available regarding the regulation of SigB sigma factor and its interacting protein partners. Hence, we cloned the SigB gene into pET28a vector and optimized its expression in three different strains of , viz., (BL21 (DE3), C41 (DE3), and CodonPlus (DE3)). We also optimized several other parameters for the expression of recombinant SigB including IPTG concentration, temperature, and time duration. We achieved the maximum expression of SigB at 25°C in the soluble fraction of the cell which was purified by affinity chromatography using Ni-NTA and further confirmed by Western blotting. Further, structural characterization demonstrates the instability of SigB in comparison to SigA that is carried out using homology modeling and structure function relationship. We have done protein-protein docking of RNA polymerase (RNAP) of Msmeg and SigB. This effort provides a platform for pulldown assay, structural, and other studies with the recombinant protein to deduce the SigB interacting proteins, which might pave the way to study its signaling networks along with its regulation.
Topics: Bacterial Proteins; Escherichia coli; Gene Expression Regulation, Bacterial; Humans; Immunoglobulin A, Secretory; Mycobacterium smegmatis; Sigma Factor
PubMed: 35634445
DOI: 10.1155/2022/7475704 -
Scientific Reports May 2023There exists decades-old evidence that some mycobacteria, including Mycobacterium avium and Mycobacterium smegmatis, produce hydrazidase, an enzyme that can hydrolyze...
There exists decades-old evidence that some mycobacteria, including Mycobacterium avium and Mycobacterium smegmatis, produce hydrazidase, an enzyme that can hydrolyze the first-line antitubercular agent isoniazid. Despite its importance as a potential resistance factor, no studies have attempted to reveal its identity. In this study, we aimed to isolate and identify M. smegmatis hydrazidase, characterize it, and evaluate its impact on isoniazid resistance. We determined the optimal condition under which M. smegmatis produced the highest amount of hydrazidase, purified the enzyme by column chromatography, and identified it by peptide mass fingerprinting. It was revealed to be PzaA, an enzyme known as pyrazinamidase/nicotinamidase whose physiological role remains unknown. The kinetic constants suggested that this amidase with broad substrate specificity prefers amides to hydrazides as a substrate. Notably, of the five tested compounds, including amides, only isoniazid served as an efficient inducer of pzaA transcription, as revealed by quantitative reverse transcription PCR. Moreover, high expression of PzaA was confirmed to be beneficial for the survival and growth of M. smegmatis in the presence of isoniazid. Thus, our findings suggest a possible role for PzaA, and other hydrazidases yet to be identified, as an intrinsic isoniazid resistance factor of mycobacteria.
Topics: Isoniazid; Mycobacterium; Antitubercular Agents; Mycobacterium smegmatis; Amides; Mycobacterium tuberculosis
PubMed: 37210419
DOI: 10.1038/s41598-023-35213-5 -
Biochemical and Biophysical Research... Jun 2021The Kemp elimination reaction, involving the ring-opening of benzoxazole and its derivatives under the action of natural enzymes or chemical catalysts, has been of...
The Kemp elimination reaction, involving the ring-opening of benzoxazole and its derivatives under the action of natural enzymes or chemical catalysts, has been of interest to researchers since its discovery. Because this reaction does not exist in all currently known metabolic pathways, the computational design of Kemp eliminases has provided valuable insights into principles of enzymatic catalysis. However, it was discovered that the naturally occurring promiscuous enzymes ydbC, xapA and ketosteroid isomerase also can catalyze Kemp elimination. Here, we report the crystal structure of ketosteroid isomerase (KSI) from Mycobacterium smegmatis MC2 155. MsKSI crystallizes in the P222 space group with two molecules in an asymmetric unit, and ultracentrifugation data confirms that it forms a stable dimer in solution, consistent with the 1.9 Å-resolution structure. Our assays confirm that MsKSI accelerates the Kemp elimination of 5-nitrobenzoxazole (5NBI) with an optimal pH of 5.5. A 2.35 Å resolution crystal structure of the MsKSI-5NBI complex reveals that the substrate 5NBI is bound in the active pocket of the enzyme composed of hydrophobic residues. In addition, the Glu127 residue is proposed to play an important role as a general base in proton transfer and breaking weak O-N bonds to open the five-membered ring. This work provides a starting point for exploring the artificial modification of MsKSI using the natural enzyme as the backbone.
Topics: Bacterial Proteins; Biocatalysis; Crystallography, X-Ray; Models, Molecular; Mycobacterium smegmatis; Protein Subunits; Steroid Isomerases
PubMed: 33992958
DOI: 10.1016/j.bbrc.2021.05.007 -
The Journal of Antibiotics Dec 2023Mycobacterium tuberculosis is exposed to diverse stresses inside the host during dormancy. Meanwhile, many metabolic and transcriptional regulatory changes occur,...
Mycobacterium tuberculosis is exposed to diverse stresses inside the host during dormancy. Meanwhile, many metabolic and transcriptional regulatory changes occur, resulting in physiological modifications that help M. tuberculosis to adapt to these stresses. The same physiological changes also cause antibiotic tolerance in dormant M. tuberculosis. However, the transcriptional regulatory mechanism of antibiotic tolerance during dormancy remains unclear. Here, we showed that the expression of Rv1255c, an uncharacterised member of the tetracycline repressor family of transcriptional regulators, is upregulated during different stresses and hypoxia-induced dormancy. Antibiotic tolerance and efflux activities of Mycobacterium smegmatis constitutively expressing Rv1255c were analysed, and interestingly, it showed increased isoniazid tolerance and efflux activity. The intrabacterial isoniazid concentrations were found to be low in M. smegmatis expressing Rv1255c. Moreover, orthologs of the M. tuberculosis katG, gene of the enzyme which activates the first-line prodrug isoniazid, are overexpressed in this strain. Structural analysis of isoforms of KatG enzymes in M. smegmatis identified major amino acid substitutions associated with isoniazid resistance. Thus, we showed that Rv1255c helps M. smegmatis tolerate isoniazid by orchestrating drug efflux machinery. In addition, we showed that Rv1255c also causes overexpression of katG isoform in M. smegmatis which has amino acid substitutions as found in isoniazid-resistant katG in M. tuberculosis.
Topics: Humans; Anti-Bacterial Agents; Antitubercular Agents; Bacterial Proteins; Catalase; Isoniazid; Mycobacterium smegmatis; Mycobacterium tuberculosis; Tuberculosis
PubMed: 37821540
DOI: 10.1038/s41429-023-00661-8 -
The Journal of Biological Chemistry May 2022Oxidation of malate to oxaloacetate, catalyzed by either malate dehydrogenase (Mdh) or malate quinone oxidoreductase (Mqo), is a critical step of the tricarboxylic acid...
Oxidation of malate to oxaloacetate, catalyzed by either malate dehydrogenase (Mdh) or malate quinone oxidoreductase (Mqo), is a critical step of the tricarboxylic acid cycle. Both Mqo and Mdh are found in most bacterial genomes, but the level of functional redundancy between these enzymes remains unclear. A bioinformatic survey revealed that Mqo was not as widespread as Mdh in bacteria but that it was highly conserved in mycobacteria. We therefore used mycobacteria as a model genera to study the functional role(s) of Mqo and its redundancy with Mdh. We deleted mqo from the environmental saprophyte Mycobacterium smegmatis, which lacks Mdh, and found that Mqo was essential for growth on nonfermentable carbon sources. On fermentable carbon sources, the Δmqo mutant exhibited delayed growth and lowered oxygen consumption and secreted malate and fumarate as terminal end products. Furthermore, heterologous expression of Mdh from the pathogenic species Mycobacterium tuberculosis shortened the delayed growth on fermentable carbon sources and restored growth on nonfermentable carbon sources at a reduced growth rate. In M. tuberculosis, CRISPR interference of either mdh or mqo expression resulted in a slower growth rate compared to controls, which was further inhibited when both genes were knocked down simultaneously. These data reveal that exergonic Mqo activity powers mycobacterial growth under nonenergy limiting conditions and that endergonic Mdh activity complements Mqo activity, but at an energetic cost for mycobacterial growth. We propose Mdh is maintained in slow-growing mycobacterial pathogens for use under conditions such as hypoxia that require reductive tricarboxylic acid cycle activity.
Topics: Bacterial Proteins; Carbon; Citric Acid Cycle; Malate Dehydrogenase; Malates; Mycobacterium smegmatis; Oxaloacetic Acid; Oxidoreductases
PubMed: 35337802
DOI: 10.1016/j.jbc.2022.101859 -
The FEBS Journal Oct 2020Whereas intracellular proteolysis is essential for proper cellular function, it is a destructive process, which must be tightly regulated. In some bacteria, a...
Whereas intracellular proteolysis is essential for proper cellular function, it is a destructive process, which must be tightly regulated. In some bacteria, a Pup-proteasome system tags target proteins for degradation by a bacterial proteasome. Pup, a small modifier protein, is attached to target proteins by PafA, the sole Pup ligase, in a process termed pupylation. In mycobacteria, including Mycobacterium smegmatis and Mycobacterium tuberculosis, Pup undergoes a deamidation step by the enzyme Dop prior to its PafA-mediated attachment to a target. The catalytic mechanism of Pup deamidation is also used by Dop to perform depupylation, namely the removal of Pup from already tagged proteins. Hence, Dop appears to play contradictory roles: On the one hand, deamidation of Pup promotes pupylation, while on the other hand, depupylation reduces tagged protein levels. To avoid futile pupylation-depupylation cycles, Dop activity must be regulated. An intramolecular regulatory mechanism directs Dop to catalyze deamidation more effectively than depupylation. A complementary intermolecular mechanism results in Dop depletion under conditions where protein pupylation and degradation are favorable. In this work, we studied these regulatory mechanisms and identified a flexible loop in Dop, previously termed the Dop-loop, that acts as an intramolecular regulatory element that allosterically controls substrate preference. To investigate regulation at the intermolecular level, we used the CRISPR interference system to knock down the expression of M. smegmatis ATP-dependent intracellular proteases and found that the ClpCP protease is responsible for Dop depletion under starvation conditions. These findings clarify previous observations and introduce a new level for the regulation of Dop activity. DATABASE: Structural data are available in the PDB database under the accession numbers 4BJR and 4B0S.
Topics: Amidohydrolases; Bacterial Proteins; Mycobacterium smegmatis
PubMed: 32037686
DOI: 10.1111/febs.15245 -
Journal of Bacteriology Apr 2023As a master nitrogen regulator in most actinomycetes, GlnR both governs central nitrogen metabolism and regulates many carbon, phosphate, and secondary metabolic...
As a master nitrogen regulator in most actinomycetes, GlnR both governs central nitrogen metabolism and regulates many carbon, phosphate, and secondary metabolic pathways. To date, most studies have been focused on the GlnR regulon, while little is known about the transcriptional regulator for itself. It has been observed that transcription can be upregulated in Mycobacterium smegmatis under nitrogen-limited growth conditions; however, the detailed regulatory mechanism is still unclear. Here, we demonstrate that the gene in M. smegmatis is transcriptionally activated by its product GlnR in response to nitrogen limitation. The precise GlnR binding site was successfully characterized in its promoter region using the electrophoretic mobility shift assay and the DNase I footprinting assay. Site mutagenesis and genetic analyses confirmed that the binding site was essential for self-activation of transcription. Moreover, based on bioinformatic analyses, we discovered that most of the mycobacterial promoter regions (144 out of 147) contain potential GlnR binding sites, and we subsequently proved that the purified M. smegmatis GlnR protein could specifically bind 16 promoter regions that represent 119 mycobacterial species, including Mycobacterium tuberculosis. Together, our findings not only elucidate the transcriptional self-regulation mechanism of transcription in M. smegmatis but also indicate the ubiquity of the mechanism in other mycobacterial species. In actinomycetes, the nitrogen metabolism not only is essential for the construction of life macromolecules but also affects the biosynthesis of secondary metabolites and even virulence (e.g., Mycobacterium tuberculosis). The transcriptional regulation of genes involved in nitrogen metabolism has been thoroughly studied and involves the master nitrogen regulator GlnR. However, the transcriptional regulation of itself remains elusive. Here, we demonstrated that GlnR functions as a transcriptional self-activator in response to nitrogen starvation in the fast-growing model Mycobacterium species Mycobacterium smegmatis. We further showed that this self-regulation mechanism could be widespread in other mycobacteria, which might be beneficial for those slow-growing mycobacteria to adapt to the nitrogen-starvation environments such as within human macrophages for M. tuberculosis.
Topics: Humans; Nitrogen; Gene Expression Regulation, Bacterial; Transcription Factors; Mycobacterium smegmatis; Mycobacterium tuberculosis; Self-Control; Bacterial Proteins
PubMed: 36943048
DOI: 10.1128/jb.00479-22 -
The International Journal of... Jul 2020Mycobacterium smegmatis MSMEG_0129 and Rv0164, its homologue in Mycobacterium tuberculosis, are single START-domain proteins essential for bacterial growth and survival,...
Mycobacterium smegmatis MSMEG_0129 and Rv0164, its homologue in Mycobacterium tuberculosis, are single START-domain proteins essential for bacterial growth and survival, but their biochemical activities and biological roles remain undetermined. Here, we probed the possible functions of MSMEG_0129 and its underlying mechanisms by determining its cellular location, searching for its interaction partners and monitoring its transcription profile. MSMEG_0129, and Rv0164 by extension, were found to be cytosolic proteins rather than secreted components as previously understood. Increases in MSMEG_0129 expression at physiological levels accelerated bacterial growth in a proportional manner, but additional growth acceleration was not observed when MSMEG_0129 was overexpressed up to 20 fold. MSMEG_0129 is a short-lived protein, unstable at both the mRNA and protein levels. Co-IP and GST pull-down assays showed that MSMEG_0129 interacts with the ClpP2 protease and a global transcription factor, CarD, their expression being correlated with that of MSMEG_0129. Nutrient deficiency led to the downregulation of MSMEG_0129 but upregulation of CarD. However, in the context of constitutive MSMEG_0129 overexpression under nutrient-rich or starvation conditions, the mRNA level of CarD was reduced 3 fold. Conversely, expression of ClpP2 decreased with MSMEG_0129 downregulation under starvation conditions, but increased 4-8 fold when MSMEG_0129 was overexpressed. Our data suggest that MSMEG_0129, and Rv0164 by analogy, are likely to be nutrition sensing factors that regulate mycobacterial growth and may be involved in signal transfer under nutrient deficiency, possibly via physical and regulatory interactions with CarD and ClpP2.
Topics: Bacterial Proteins; Cytoplasm; Immunoprecipitation; Mass Spectrometry; Mycobacterium smegmatis; Mycobacterium tuberculosis; Nutrients; Protein Binding; Protein Domains; Protein Stability; Serine Endopeptidases; Transcription Factors
PubMed: 32389745
DOI: 10.1016/j.biocel.2020.105763 -
Future Microbiology Apr 2020To identify and characterize new mycobacterium pyrazinamide (PZA) resistance genes in addition to , and . To screen a Tn7 mc155 transposon library using 50 μM PZA...
To identify and characterize new mycobacterium pyrazinamide (PZA) resistance genes in addition to , and . To screen a Tn7 mc155 transposon library using 50 μM PZA and a PZA hypersensitive mutant (M492) was obtained. MIC was further used to confirm the hypersensitivity of M492 mutant by culturing the mutant in Middlebrook 7H9 liquid medium at 37°C. is the gene underlying the hypersensitive phenotype of mutant M492. The observed resistance to PZA and fluoroquinolones involved the alteration of cell wall permeability and the dissipation of the proton motive force. NAD/NADH dysregulation and attenuated glyoxylate shunt might underlie the declined scavenging capacity of reactive oxygen species in the -deficient mutants. is a novel gene involved in pyrazinamide resistance and might be a new candidate for drugs target.
Topics: 2,2'-Dipyridyl; Anti-Bacterial Agents; Antitubercular Agents; Cell Membrane Permeability; Drug Resistance, Bacterial; Fluoroquinolones; Genes, Bacterial; Glyoxylates; Hydrogen Peroxide; Membrane Potentials; Membrane Proteins; Microbial Sensitivity Tests; Mutation; Mycobacterium smegmatis; Mycobacterium tuberculosis; NAD; Pyrazinamide; Reactive Oxygen Species; Thiourea; Transcription Factors
PubMed: 32250176
DOI: 10.2217/fmb-2019-0071