Biochemical characterization of the 49 kDa penicillin-binding protein of Mycobacterium smegmatis.
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ABSTRACT:
SUBMITTER: Mukherjee T
PROVIDER: S-EPMC1217917 | biostudies-other | 1996 Nov
REPOSITORIES: biostudies-other
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Project description:The ponA gene of Mycobacterium smegmatis encodes a 95-kDa penicillin binding protein, PBP1, that is similar to PBP1s of Mycobacterium tuberculosis and Mycobacterium leprae. Transposon disruption of ponA in M. smegmatis resulted in a PBP1-deficient mutant that was sensitive to beta-lactam antibiotics, was more permeable to glycine, and grew slowly in liquid culture.
| S-EPMC89605 | biostudies-literature
Project description:Antibiotic resistance has been one of the biggest threats to global health. Despite the available prevention and control strategies and efforts in developing new antibiotics, the need remains for effective approaches against antibiotic resistance. Efficient strategies to cope with antimicrobial resistance require a quantitative and deeper understanding of microbial behavior, which can be obtained using different techniques to provide the missing pieces of the current antibiotic-resistance puzzle. Microfluidic-microscopy techniques are among the most promising methods that contribute modernization of traditional assays in microbiology. They provide monitoring and manipulation of cells at micro-scale volumes. Here, we combined population-level, culture-based assays with single-cell resolution, microfluidic-microscopy systems to investigate isoniazid response of Mycobacterium smegmatis penicillin-binding protein (PBP) mutant. This mutant exhibited normal growth in plain medium and sensitivity to stress responses when treated with thermal stress (45 °C), detergent stress (0.1% sodium dodecyl sulfate), acid stress (pH 4.5), and nutrient starvation (1XPBS). The impact of msm0031 transposon insertion on drug-mediated killing was determined for isoniazid (INH, 50 µg/mL), rifampicin (RIF, 200 µg/mL), ethionamide (ETH, 200 µg/mL), and ethambutol (EMB, 5 µg/mL). The PBP mutant demonstrated remarkable isoniazid-killing phenotype in batch culture. Therefore, we hypothesized that single-cell analysis will show increased lysis kinetics and fewer intact cells after drug treatment. However, the single-cell analysis data showed that upon isoniazid exposure, the percentage of the intact PBP mutant cells was 24%, while the percentage of the intact wild-type cells was 4.6%. The PBP mutant cells exhibited decreased cell-lysis profile. Therefore, the traditional culture-based assays were not sufficient to provide insights about the subpopulation of viable but non-culture cells. Consequently, we need more adequate tools to be able to comprehend and fight the antibiotic resistance of bacteria.
| S-EPMC6266593 | biostudies-literature
Project description:Class A penicillin-binding proteins (PBPs) are large, bifunctional proteins that are responsible for glycan chain assembly and peptide cross-linking of bacterial peptidoglycan. Bacteria in the genus Mycobacterium have been reported to have only two class A PBPs, PonA1 and PonA2, that are encoded in their genomes. We report here that the genomes of Mycobacterium smegmatis and other soil mycobacteria contain an additional gene encoding a third class A penicillin-binding protein, PonA3, which is a paralog of PonA2. Both the PonA2 and PonA3 proteins contain a penicillin-binding protein and serine/threonine protein kinase-associated (PASTA) domain that we propose may be involved in sensing the cell cycle and a C-terminal proline-rich region (PRR) that may have a role in protein-protein or protein-carbohydrate interactions. We show here that an M. smegmatis Delta ponA2 mutant has an unusual antibiotic susceptibility profile, exhibits a spherical morphology and an altered cell surface in stationary phase, and is defective for stationary-phase survival and recovery from anaerobic culture. In contrast, a Delta ponA3 mutant has no discernible phenotype under laboratory conditions. We demonstrate that PonA2 and PonA3 can bind penicillin and that PonA3 can partially substitute for PonA2 when ponA3 is expressed from a constitutive promoter on a multicopy plasmid. Our studies suggest that PonA2 is involved in adaptation to periods of nonreplication in response to starvation or anaerobiosis and that PonA3 may have a similar role. However, the regulation of PonA3 is likely different, suggesting that its importance could be related to stresses encountered in the environmental niches occupied by M. smegmatis and other soil-dwelling mycobacteria.
| S-EPMC2901687 | biostudies-literature
Project description:Glycosyltransferases (GTs) are widely present in several organisms. These enzymes specifically transfer sugar moieties to a range of substrates. The processes of bacterial glycosylation of the cell wall and their relations with host-pathogen interactions have been studied extensively, yet the majority of mycobacterial GTs involved in the cell wall synthesis remain poorly characterized. Glycopeptidolipids (GPLs) are major class of glycolipids present on the cell wall of various mycobacterial species. They play an important role in drug resistance and host-pathogen interaction virulence. Gtf3 enzyme performs a key step in the biosynthesis of triglycosylated GPLs. Here, we describe a general procedure to achieve expression, purification, and crystallization of recombinant protein Gtf3 from Mycobacterium smegmatis using an E. coli expression system. We reported also a combined bioinformatics and biochemical methods to predict aggregation propensity and improve protein solubilization of recombinant Gtf3. NVoy, a carbohydrate-based polymer reagent, was added to prevent protein aggregation by binding to hydrophobic protein surfaces of Gtf3. Using intrinsic tryptophan fluorescence quenching experiments, we also demonstrated that Gtf3-NVoy enzyme interacted with TDP and UDP nucleotide ligands. This case report proposes useful tools for the study of other glycosyltransferases which are rather difficult to characterize and crystallize.
| S-EPMC7494702 | biostudies-literature
Project description:BACKGROUND: Obg is a highly conserved GTP-binding protein that has homologues in bacteria, archaea and eukaryotes. In bacteria, Obg proteins are essential for growth, and they participate in spore formation, stress adaptation, ribosome assembly and chromosomal partitioning. This study was undertaken to investigate the biochemical and physiological characteristics of Obg in Mycobacterium tuberculosis, which causes tuberculosis in humans. RESULTS: We overexpressed M. tuberculosis Obg in Escherichia coli and then purified the protein. This protein binds to, hydrolyzes and is phosphorylated with GTP. An anti-Obg antiserum, raised against the purified Obg, detects a 55 kDa protein in immunoblots of M. tuberculosis extracts. Immunoblotting also discloses that cultured M. tuberculosis cells contain increased amounts of Obg in the late log phase and in the stationary phase. Obg is also associated with ribosomes in M. tuberculosis, and it is distributed to all three ribosomal fractions (30 S, 50 S and 70 S). Finally, yeast two-hybrid analysis reveals that Obg interacts with the stress protein UsfX, indicating that M. tuberculosis Obg, like other bacterial Obgs, is a stress related protein. CONCLUSIONS: Although its GTP-hydrolyzing and phosphorylating activities resemble those of other bacterial Obg homologues, M. tuberculosis Obg differs from them in these respects: (a) preferential association with the bacterial membrane; (b) association with all three ribosomal subunits, and (c) binding to the stress protein UsfX, rather than to RelA. Generation of mutant alleles of Obg of M. tuberculosis, and their characterization in vivo, may provide additional insights regarding its role in this important human pathogen.
| S-EPMC3056739 | biostudies-literature
Project description:AAA proteins (ATPases associated with various cellular activities) use the energy of ATP hydrolysis to drive conformational changes in diverse macromolecular targets. Here, we report the biochemical characterization and 2.5-Å crystal structure of a Mycobacterium smegmatis AAA protein Msm0858, the ortholog of Mycobacterium tuberculosis Rv0435c. Msm0858 is a magnesium-dependent ATPase and is active with all nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) as substrates. The Msm0858 structure comprises (i) an N-terminal domain (amino acids [aa] 17 to 201) composed of two ?-barrel modules and (ii) two AAA domains, D1 (aa 212 to 473) and D2 (aa 476 to 744), each of which has ADP in the active site. Msm0858-ADP is a monomer in solution and in crystallized form. Msm0858 domains are structurally homologous to the corresponding modules of mammalian p97. However, the position of the N-domain modules relative to the AAA domains in the Msm0858-ADP tertiary structure is different and would impede the formation of a p97-like hexameric quaternary structure. Mutational analysis of the A-box and B-box motifs indicated that the D1 and D2 AAA domains are both capable of ATP hydrolysis. Simultaneous mutations of the D1 and D2 active-site motifs were required to abolish ATPase activity. ATPase activity was effaced by mutation of the putative D2 arginine finger, suggesting that Msm0858 might oligomerize during the ATPase reaction cycle. A truncated variant Msm0858 (aa 212 to 745) that lacks the N domain was characterized as a catalytically active homodimer.Recent studies have underscored the importance of AAA proteins (ATPases associated with various cellular activities) in the physiology of mycobacteria. This study reports the ATPase activity and crystal structure of a previously uncharacterized mycobacterial AAA protein, Msm0858. Msm0858 consists of an N-terminal ?-barrel domain and two AAA domains, each with ADP bound in the active site. Msm0858 is a structural homolog of mammalian p97, with respect to the linear order and tertiary structures of their domains.
| S-EPMC4859601 | biostudies-literature
Project description:The pyrazinamidase from Mycobacterium smegmatis was purified to homogeneity to yield a product of approximately 50 kDa. The deduced amino-terminal amino acid sequence of this polypeptide was used to design an oligonucleotide probe for screening a DNA library of M. smegmatis. An open reading frame, designated pzaA, which encodes a polypeptide of 49.3 kDa containing motifs conserved in several amidases was identified. Targeted knockout of the pzaA gene by homologous recombination yielded a mutant, pzaA::aph, with a more-than-threefold-reduced level of pyrazinamidase activity, suggesting that this gene encodes the major pyrazinamidase of M. smegmatis. Recombinant forms of the M. smegmatis PzaA and the Mycobacterium tuberculosis pyrazinamidase/nicotinamidase (PncA) were produced in Escherichia coli and were partially purified and compared in terms of their kinetics of nicotinamidase and pyrazinamidase activity. The comparable Km values obtained from this study suggested that the unique specificity of pyrazinamide (PZA) for M. tuberculosis was not based on an unusually high PZA-specific activity of the PncA protein. Overexpression of pzaA conferred PZA susceptibility on M. smegmatis by reducing the MIC of this drug to 150 micrograms/ml.
| S-EPMC107651 | biostudies-literature
Project description:Mycobacterium smegmatis SigF is a group III sigma factor. Its ortholog in M. tuberculosis is reported to have role in regulation and function of cell wall components. In present study we have created an M. smegmatis ΔsigF mutant by allele exchange method. M. smegmatis sigF mutant shows non pigmented phenotype and is more sensitive to hydrogen peroxide generated oxidative stress. DNA microarray analysis of M. smegmatis wild type and ΔsigF mutant suggests that SigF in this species controls the expression of several energy and central intermediary metabolism genes along with regulation of carotenoid biosynthesis.
2010-08-20 | GSE19774 | GEO
Project description:Mycobacterium smegmatis represents one model for studying the biology of its pathogenic relative Mycobacterium tuberculosis. The structural characterization of a M. tuberculosis ortholog protein can serve as a valid tool for the development of molecules active against the M. tuberculosis target. In this context, we report the biochemical and structural characterization of M. smegmatis phosphoribosylpyrophosphate synthetase (PrsA), the ortholog of M. tuberculosis PrsA, the unique enzyme responsible for the synthesis of phosphoribosylpyrophosphate (PRPP). PRPP is a key metabolite involved in several biosynthetic pathways including those for histidine, tryptophan, nucleotides and decaprenylphosphoryl-arabinose, an essential precursor for the mycobacterial cell wall biosynthesis. Since M. tuberculosis PrsA has been validated as a drug target for the development of antitubercular agents, the data presented here will add to the knowledge of the mycobacterial enzyme and could contribute to the development of M. tuberculosis PrsA inhibitors of potential pharmacological interest.
| S-EPMC5395218 | biostudies-literature
Project description:The product of the gene ponA present in cosmid MTCY21D4, one of the collection of clones representing the genome of Mycobacterium tuberculosis, has been named penicillin-binding protein 1* (PBP1*), by analogy to the previously characterized PBP1* of M. leprae. This gene has been overexpressed in Escherichia coli. His(6)-tagged PBP1* localizes to the membranes of induced E. coli cells. Its susceptibility to degradation upon proteinase K digestion of spheroplasts from E. coli expressing the protein supports the view that the majority of the protein translocates to the periplasmic side of the membrane. Recombinant PBP1* binds benzylpenicillin and several other beta-lactams, notably cefotaxime, with high affinity. Truncation of the N-terminal 64 amino acid residues results in an expressed protein present exclusively in inclusion bodies and unable to associate with the membrane. The C-terminal module encompassing amino acids 272-663 can be extracted from inclusion bodies under denaturing conditions using guanidine/HCl and refolded to give a protein fully competent in penicillin-binding. Deletion of Gly(95)-Gln(143) results in the expression of a protein, which is localized in the cytosol. The soluble derivative of PBP1* binds benzylpenicillin with the same efficiency as the full-length protein. This is the first report of a soluble derivative of a class A high-molecular-mass PBP.
| S-EPMC1222347 | biostudies-other