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At least 19 recordsLinked to original sources
PubMed1999

Comparison of the construction of unmarked deletion mutations in Mycobacterium smegmatis, Mycobacterium bovis bacillus Calmette-Guérin, and Mycobacterium tuberculosis H37Rv by allelic exchange.

Until recently, genetic analysis of Mycobacterium tuberculosis, the causative agent of tuberculosis, was hindered by a lack of methods for gene disruptions and allelic exchange. Several groups have described different methods for disrupting genes marked with antibiotic resistance determinants in the slow-growing organisms Mycobacterium bovis bacillus Calmette-Guérin (BCG) and M. tuberculosis. In this study, we described the first report of using a mycobacterial suicidal plasmid bearing the counterselectable marker sacB for the allelic exchange of unmarked deletion mutations in the chromosomes of two substrains of M. bovis BCG and M. tuberculosis H37Rv. In addition, our comparison of the recombination frequencies in these two slow-growing species and that of the fast-growing organism Mycobacterium smegmatis suggests that the homologous recombination machinery of the three species is equally efficient. The mutants constructed here have deletions in the lysA gene, encoding meso-diaminopimelate decarboxylase, an enzyme catalyzing the last step in lysine biosynthesis. We observed striking differences in the lysine auxotrophic phenotypes of these three species of mycobacteria. The M. smegmatis mutant can grow on lysine-supplemented defined medium or complex rich medium, while the BCG mutants grow only on lysine-supplemented defined medium and are unable to form colonies on complex rich medium. The M. tuberculosis lysine auxotroph requires 25-fold more lysine on defined medium than do the other mutants and is dependent upon the detergent Tween 80. The mutants described in this work are potential vaccine candidates and can also be used for studies of cell wall biosynthesis and amino acid metabolism.

Alleles↗
PubMed1996

Organization of the origins of replication of the chromosomes of Mycobacterium smegmatis, Mycobacterium leprae and Mycobacterium tuberculosis and isolation of a functional origin from M. smegmatis.

The genus Mycobacterium is composed of species with widely differing growth rates ranging from approximately three hours in Mycobacterium smegmatis to two weeks in Mycobacterium leprae. As DNA replication is coupled to cell duplication, it may be regulated by common mechanisms. The chromosomal regions surrounding the origins of DNA replication from M. smegmatis, M. tuberculosis, and M. leprae have been sequenced, and show very few differences. The gene order, rnpA-rpmH-dnaA-dnaN-recF-orf-gyrB-gyrA, is the same as in other Gram-positive organisms. Although the general organization in M. smegmatis is very similar to that of Streptomyces spp., a closely related genus, M. tuberculosis and M. leprae differ as they lack an open reading frame, between dnaN and recF, which is similar to the gnd gene of Escherichia coli. Within the three mycobacterial species, there is extensive sequence conservation in the intergenic regions flanking dnaA, but more variation from the consensus DnaA box sequence was seen than in other bacteria. By means of subcloning experiments, the putative chromosomal origin of replication of M. smegmatis, containing the dnaA-dnaN region, was shown to promote autonomous replication in M. smegmatis, unlike the corresponding regions from M. tuberculosis or M. leprae.

Bacterial Proteins↗
PubMed1991

Site-specific integration of mycobacteriophage L5: integration-proficient vectors for Mycobacterium smegmatis, Mycobacterium tuberculosis, and bacille Calmette-Guérin.

Mycobacteriophage L5, a temperate phage of mycobacteria, integrates site-specifically into the Mycobacterium smegmatis chromosome. We have identified the int gene and attP site of L5, characterized the chromosomal attachment site (attB), and constructed plasmid vectors that efficiently transform M. smegmatis through stable site-specific integration of the plasmid into the bacterial genome. These integration-proficient plasmids also efficiently transform slow-growing mycobacteria such as the pathogen Mycobacterium tuberculosis and the vaccine strain bacille Calmette-Guérin (BCG). The ability to easily generate stable recombinants in these slow-growing mycobacteria without the requirement for continual selection is of particular importance for the construction of recombinant BCG vaccines and for the isolation and characterization of mycobacterial pathogenic determinants in animal model systems. Integration vectors of this type should be of general use in a number of additional bacterial systems where temperate phages have been identified.

Amino Acid Sequence↗
PubMed2006

Identification of Nudix hydrolase family members with an antimutator role in Mycobacterium tuberculosis and Mycobacterium smegmatis.

Mycobacterium tuberculosis and Mycobacterium smegmatis MutT1, MutT2, MutT3, and Rv3908 (MutT4) enzymes were screened for an antimutator role. Results indicate that both MutT1, in M. tuberculosis and M. smegmatis, and MutT4, in M. smegmatis, have that role. Furthermore, an 8-oxo-guanosine triphosphatase function for MutT1 and MutT2 is suggested.

Gene Deletion↗
PubMed2000

Polyprenyl phosphate biosynthesis in Mycobacterium tuberculosis and Mycobacterium smegmatis.

Mycobacterium smegmatis has been shown to contain two forms of polyprenyl phosphate (Pol-P), while Mycobacterium tuberculosis contains only one. Utilizing subcellular fractions from M. smegmatis and M. tuberculosis, we show that Pol-P synthesis is different in these species. The specific activities of the prenyl diphosphate synthases in M. tuberculosis are 10- to 100-fold lower than those in M. smegmatis. In M. smegmatis decaprenyl diphosphate and heptaprenyl diphosphate were the main products synthesized in vitro, whereas in M. tuberculosis only decaprenyl diphosphate was synthesized. The data from both organisms suggest that geranyl diphosphate is the allylic substrate for two distinct prenyl diphosphate synthases, one located in the cell membrane that synthesizes omega,E,Z-farnesyl diphosphate and the other present in the cytosol that synthesizes omega,E,E,E-geranylgeranyl diphosphate. In M. smegmatis, the omega,E, Z-farnesyl diphosphate is utilized by a membrane-associated prenyl diphosphate synthase activity to generate decaprenyl diphosphate, and the omega,E,E,E-geranylgeranyl diphosphate is utilized by a membrane-associated activity for the synthesis of the heptaprenyl diphosphate. In M. tuberculosis, however, omega,E,E,E-geranylgeranyl diphosphate is not utilized for the synthesis of heptaprenyl diphosphate. Thus, the difference in the compositions of the Pol-P of M. smegmatis and M. tuberculosis can be attributed to distinct enzymatic differences between these two organisms.

Cell Membrane↗
PubMed1999

Identification and characterization of a new intermediate in the ribosylative inactivation pathway of rifampin by Mycobacterium smegmatis.

Mycobacterium smegmatis DSM 43756 inactivates rifampin by ribosylation. To study this process of rifampicin, all possible inactivated forms of the antibiotic were extracted and purified. Structural studies showed the presence of a new inactivation product, designated RIP-TAp(23-phosphoribosyl-rifampin). Formation of 23-(O-ADP-ribosyl)rifampin (RIP-TAs) is the first step, followed by removal of AMP to give rise to the newly identified compound. Lastly, dephosphorylation leads to formation of 23-ribosyl-rifampin (RIP-Mb). Feeding experiments with the ADP-ribosylated antibiotic obtained from the cell homogenates of an Escherichia coli strain carrying the cloned M. smegmatis gene confirmed this rifampin inactivation process.

Antibiotics, Antitubercular↗
PubMed1998

Effect of IS900 gene of Mycobacterium paratuberculosis on Mycobacterium smegmatis.

Mycobacterium paratuberculosis is mycobactin dependent and contains multiple copies of the IS900 gene that encodes for p43 (46.5K protein). The correlation between the two characteristics has been investigated. A 3.2-kb BamHI fragment from M. paratuberculosis containing the 1.451 kb IS900 gene was cloned in Escherichia coli and Mycobacterium smegmatis with pcDNA II and pNEZ6.3 plasmids, respectively. Surprisingly, the recombinant M. smegmatis grew poorly and slower in 7H9 broth supplemented with OADC (12 day) compared with M. smegmatis wild type or to M. smegmatis transformed with pNEZ6.3 (2 day). The growth rate of the recombinant M. smegmatis was restored by the addition of 2.4 microM ferric mycobactin J to the media. There was no effect on the growth rate of E. coli recombinants. Western blot analysis with p43-specific anti-peptide antibodies resulted in the expression of 46.5K and a cleaved form of 33.5K protein bands in the recombinant E. coli. There was no expression in the recombinant M. smegmatis. A lower expression of 33. 5K protein band was detected in the native M. paratuberculosis protein. The nucleotide sequence of the 3.2-kb fragment confirmed the presence of p43-encoded ORF. There was no additional encoding sequence in the fragment. This suggests that the IS900 gene and/or its encoding products are involved in mycobactin dependency and possibly the slow growth rate of M. paratuberculosis.

Bacterial Proteins↗
PubMed2006

Distinct DNA repair pathways involving RecA and nonhomologous end joining in Mycobacterium smegmatis.

Mycobacterium smegmatis was used to study the relationship between DNA repair processes involving RecA and nonhomologous end joining (NHEJ). The effect of gene deletions in recA and/or in two genes involved in NHEJ (ku and ligD) was tested on the ability of bacteria to join breaks in plasmids transformed into them and in their response to chemicals that damage DNA. The results provide in vivo evidence that only NHEJ is required for the repair of noncompatible DNA ends. By contrast, the response of mycobacteria to mitomycin C preferentially involved a RecA-dependent pathway.

DNA Damage↗
PubMed1999

Cloning and characterization of arylamine N-acetyltransferase genes from Mycobacterium smegmatis and Mycobacterium tuberculosis: increased expression results in isoniazid resistance.

Arylamine N-acetyltransferases (NATs) are found in many eukaryotic organisms, including humans, and have previously been identified in the prokaryote Salmonella typhimurium. NATs from many sources acetylate the antitubercular drug isoniazid and so inactivate it. nat genes were cloned from Mycobacterium smegmatis and Mycobacterium tuberculosis, and expressed in Escherichia coli and M. smegmatis. The induced M. smegmatis NAT catalyzes the acetylation of isoniazid. A monospecific antiserum raised against pure NAT from S. typhimurium recognizes NAT from M. smegmatis and cross-reacts with recombinant NAT from M. tuberculosis. Overexpression of mycobacterial nat genes in E. coli results in predominantly insoluble recombinant protein; however, with M. smegmatis as the host using the vector pACE-1, NAT proteins from M. tuberculosis and M. smegmatis are soluble. M. smegmatis transformants induced to express the M. tuberculosis nat gene in culture demonstrated a threefold higher resistance to isoniazid. We propose that NAT in mycobacteria could have a role in acetylating, and hence inactivating, isoniazid.

Amino Acid Sequence↗
PubMed1988

Human disease due to Mycobacterium smegmatis.

Mycobacterium smegmatis is a rapidly growing environmental species not considered a human pathogen. We identified 22 human isolates of M. smegmatis from Australia and the southern United States: 19 were from skin or soft-tissue infections, and none were from urine or the male genital tract. These isolates closely resembled Mycobacterium fortuitum, except for a negative three-day arylsulfatase test; growth at 43-45 C; a low semiquantitative catalase test; and, in 50% of isolates, a late-developing, yellow-to-orange pigment. The isolates were biochemically identical to four reference strains and the type strain of M. smegmatis. Isolates were resistant to isoniazid and rifampin but susceptible to ethambutol, doxycycline, sulfamethoxazole, ciprofloxacin, imipenem, and amikacin. Eleven patients treated on the basis of in vitro susceptibility tests responded well to therapy. The similarity of M. smegmatis to M. fortuitum and the failure to recognize that the former is an environmental species may have contributed to previous failures to recognize it as a human pathogen.

Arylsulfatases↗
PubMed1980

Immunotherapy of experimental cancer by intralesional injection of emulsified nonliving mycobacteria: comparison of Mycobacterium bovis (BCG), Mycobacterium phlei, and Mycobacterium smegmatis.

Mycobacterium bovis (BCG), Mycobacterium phlei, and Mycobacterium smegmatis were each tested in emulsified form for their potency to cause regression of transplants of a syngeneic murine fibrosarcoma and of a syngeneic guinea pig hepatoma. On a weight basis, M. phlei and M. smegmatis were as effective as BCG in causing tumor regression. M. phlei and M. smegmatis were comparable to BCG in provoking delayed cutaneous hypersensitivity reactions in guinea pigs sensitized to M. phlei or M. smegmatis. In BCG-sensitized guinea pigs, M. phlei and M. smegmatis provoked weaker delayed cutaneous hypersensitivity reactions than did BCG. Purified protein derivative of M. tuberculosis was more active in eliciting delayed cutaneous hypersensitivity in BCG-sensitized guinea pigs than in animals sensitized with M. phlei or M. smegmatis.

Animals↗
PubMed1988

Methanol production by Mycobacterium smegmatis.

Mycobacterium smegmatis cells produce [3H]methanol when incubated with [methyl-3H]methionine. The methanol is derived from S-adenosylmethionine rather than methyltetrahydrofolate. M. smegmatis cells carboxymethylate several proteins, and some of the methanol probably results from their demethylation, but most of the methanol may come from an unidentified component with a high gel mobility. Although methanol in the medium reached 19 microM, it was not incorporated into the methylated mannose polysaccharide, a lipid carrier in this organism.

Mannosides↗
PubMed1976

Metabolism of triacylglycerol in Mycobacterium smegmatis.

Mycobacterium smegmatis cells incorporated [1-14C]oleic acid into triacylglycerols (TG) from the medium more rapidly than shorter chain fatty acids, caprilic and butyric acids. This incorporation was inhibited more strongly by 10(-3) M N-ethylmaleimide than by 10(-3) M KCN. [14C]TG in the bacterial cells was utilized when the cells were in poor nutritional conditions, such as phosphate buffer (pH 7.0) containing oleic acid. Accumulation of TG was observed in the cells at late stages of growth. Diglyceride acyltransferase [EC 2.3.1.20] activity was detected in a cell-free extract from this bacterium. The pH optimum of this enzyme was between pH 7 and 9. F- and Tween 20 showed remarkable enhancing and inhibitory effects, respectively.

Acyltransferases↗
PubMed1989

Metabolism of aspartate in Mycobacterium smegmatis.

Mycobacterium smegmatis grows best on L-asparagine as a sole nitrogen source; this was confirmed. [14C]Aspartate was taken up rapidly (46 nmol.mg dry cells-1.h-1 from 1 mM L-asparagine) and metabolised to CO2 as well as to amino acids synthesised through the aspartate pathway. Proportionately more radioactivity appeared in the amino acids in bacteria grown in medium containing low nitrogen. Activities of aspartokinase and homoserine dehydrogenase, the initial enzymes of the aspartate pathway, were carried by separate proteins. Aspartokinase was purified as three isoenzymes and represented up to 8% of the soluble protein of M. smegmatis. All three isoenzymes contained molecular mass subunits of 50 kDa and 11 kDa which showed no activity individually; full enzyme activity was recovered on pooling the subunits. Km values for aspartate were: aspartokinases I and III, 2.4 mM; aspartokinase II, 6.4 mM. Aspartokinase I was inhibited by threonine and homoserine and aspartokinase III by lysine, but aspartokinase II was not inhibited by any amino acids. Aspartokinase activity was repressed by methionine and lysine with a small residue of activity attributable to unrepressed aspartokinase I. Homoserine dehydrogenase activity was 96% inhibited by 2 mM threonine; isoleucine, cysteine and valine had lesser effects and in combination gave additive inhibition. Homoserine dehydrogenase was repressed by threonine and leucine. Only amino acids synthesised through the aspartate pathway were tested for inhibition and repression. Of these, only one, meso-diaminopimilate, had no discernable effect on either enzyme activity.

Aspartate Kinase↗
PubMed2004

Methylation of GPLs in Mycobacterium smegmatis and Mycobacterium avium.

Several species of mycobacteria express abundant glycopeptidolipids (GPLs) on the surfaces of their cells. The GPLs are glycolipids that contain modified sugars including acetylated 6-deoxy-talose and methylated rhamnose. Four methyltransferases have been implicated in the synthesis of the GPLs of Mycobacterium smegmatis and Mycobacterium avium. A rhamnosyl 3-O-methytransferase and a fatty acid methyltransferase of M. smegmatis have been previously characterized. In this paper, we characterize the methyltransferases that are responsible for modifying the hydroxyl groups at positions 2 and 4 of rhamnose and propose the biosynthetic sequence of GPL trimethylrhamnose formation. The analysis of M. avium genes through the creation of specific mutants is technically difficult; therefore, an alternative approach to determine the function of putative methyltransferases of M. avium was undertaken. Complementation of M. smegmatis methyltransferase mutants with M. avium genes revealed that MtfC and MtfB of the latter species have 4-O-methyltransferase activity and that MtfD is a 3-O-methyltransferase which can modify rhamnose of GPLs in M. smegmatis.

Amino Acid Sequence↗