Defective mycolic acid biosynthesis in a mutant of Mycobacterium smegmatis.

A mutant of Mycobacterium smegmatis defective in mycolic acid biosynthesis was isolated following chemical mutagenesis. Fatty acids were extracted from the mutant and subjected to structural analysis by thin-layer chromatography and high-performance liquid chromatography (HPLC) of both methyl and p-bromophenacyl ester derivatives. Thin-layer chromatography did not show the presence of any fatty acid of RF comparable to that of standard methyl mycolate. The HPLC profile revealed a broad peak in the standard mycolic acid ester region. No characteristic peaks of mycolic acid esters comparable to the wild-type could be resolved. Mass spectral analysis of the HPLC-purified peak demonstrated the presence of shorter-chain fatty acids in the mutant. These data support the idea that the mutant accumulates precursors of mycolic acids and is incapable of carrying out the final conversion to mycolic acids of 60-90 carbon atoms.     

Efficient transposition in mycobacteria: construction of Mycobacterium smegmatis insertional mutant libraries.

The Tn611 transposon was inserted into pCG63, a temperature-sensitive plasmid isolated from an Escherichia coli-mycobacterial shuttle vector which contains the pAL5000 and pUC18 replicons. The resulting plasmid, pCG79, was used to generate a large number of insertional mutations in Mycobacterium smegmatis. These are the first mycobacterial insertional mutant libraries to be constructed by transposition directly into a mycobacterium. No highly preferential insertion sites were detected by Southern blot analysis of the chromosomal DNAs isolated from the insertion mutants. Auxotrophic mutants with various phenotypes were isolated at a frequency ranging from 0.1 to 0.4%, suggesting that the libraries are representative. The pCG79 system thus seems to be a useful tool for the study of M. smegmatis genetics and may be applicable to other mycobacteria, such as the M. tuberculosis complex.     

Identification and characterization of a diamide sensitive mutant of Mycobacterium smegmatis

A mutant, T7, highly sensitive to oxidative stress as caused by diamide was isolated from a Mycobacterium smegmatis mc(2)155 transposon mutant library. While wild-type M. smegmatis is able to grow well on solid media supplemented with 10 mM diamide, T7 is only able to grow on solid media containing up to 1 mM diamide. This mutant is also sensitive to other thiol modifying agents such as iodoacetamide and chlorodinitrobenzene. By sequencing the genomic DNA flanking the transposon, T7 was found to be mutated in the region upstream of the homolog of M. tuberculosis Rv0274 open reading frame. Sequence analysis revealed that Rv0274 is a member of a superfamily of metalloenzymes comprising enzymes such as extradiol dioxygenases, glyoxalases, and fosfomycin resistant glutathione transferases. Cloning and epichromosomal expression of M. tuberculosis Rv0274 in the mutant resulted in complementation of the sensitivity to diamide.     

Genetic analysis of sliding motility in Mycobacterium smegmatis

A screen for nonsliding mutants of Mycobacterium smegmatis yielded 20 mutants with transposon insertions in the mps gene, which is involved in glycopeptidolipid biosynthesis. One mutant had an insertion in a gene predicted to encode a membrane transport protein. All mutants lacked glycopeptidolipids and were unable to form biofilms on polyvinyl chloride.     

Fluorescent probes for investigating peptidoglycan biosynthesis in mycobacteria

Tuberculosis killed 1.5 million people in 2018. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the most deadly infectious bacteria in the world. A strength of mycobacterial pathogens — their formidable cell wall — could also be one of their greatest molecular vulnerabilities. As in other bacteria, peptidoglycan (PG) maintenance and integrity is essential to mycobacterial survival. But Mtb PG is unique, and a better understanding of its biosynthetic machinery could lead to new drugs or more effective treatment regimens. Such investigations are being accelerated by the application of fluorescent probes, including those based on vancomycin, β-lactams, PG stem mimics, d-amino acids, and reactive glycans. This review will describe how fluorescent probes are being used to uncover new information on the regulation and drug susceptibility of two classes of enzymes that fortify the Mtb PG: the penicillin-binding proteins and the L,D-transpeptidases.     

Cell wall structure of a mutant of Mycobacterium smegmatis defective in the biosynthesis of mycolic acids

A mutant strain of Mycobacterium smegmatis defective in the biosynthesis of mycolic acids was recently isolated (Liu, J., and Nikaido, H. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4011-4016). This mutant failed to synthesize full-length mycolic acids and accumulated a series of long chain beta-hydroxymeromycolates. In this work, we provide a detailed characterization of the localization of meromycolates and of the cell wall structure of the mutant. Thin layer chromatography showed that the insoluble cell wall matrix remaining after extraction with chloroform/methanol and SDS still contained a large portion of the total meromycolates. Matrix-assisted laser desorption/ionization and electrospray ionization mass spectroscopy analysis of fragments arising from Smith degradation of the insoluble cell wall matrix revealed that the meromycolates were covalently attached to arabinogalactan at the 5-OH positions of the terminal arabinofuranosyl residues. The arabinogalactan appeared to be normal in the mutant strain, as analyzed by NMR. Analysis of organic phase lipids showed that the mutant cell wall contained some of the extractable lipids but lacked glycopeptidolipids and lipooligosaccharides. Differential scanning calorimetry of the mutant cell wall failed to show the large cooperative thermal transitions typical of intact mycobacterial cell walls. Transmission electron microscopy showed that the mutant cell wall had an abnormal ultrastructure (without the electron-transparent zone associated with the asymmetric mycolate lipid layer). Taken together, these results demonstrate the importance of mycolic acids for the structural and functional integrity of the mycobacterial cell wall. The lack of highly organized lipid domains in the mutant cell wall explains the drug-sensitive and temperature-sensitive phenotypes of the mutant.     

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.     

Neutral lipid biosynthesis in Mycobacterium smegmatis.

The biosynthesis of neutral lipids in Mycobacterium smegmatis was studied using cell free extracts. Maximum neutral lipid production was obtained when the reaction mixture (400 microliter) consisted of 0.25 M potassium phosphate buffer (pH 7.5), 0.125 mM oleoyl-CoA, 3.75 mM sn-glycerol-3-P, 10 mM MgCl2 and 1.85 mg bovine serum albumin. No magnesium dependency for the acylation of sn-glycerol-3-P was observed. A slight stabilizing effect seemed to occur due to this ion. The enzyme phosphatidate phosphohydrolase, on the other hand, was shown to be magnesium dependent. The activity of this enzyme also appeared to be stimulated by high concentration (0.75 to 1.25 mM) of ATP which enhanced lipid formation at all concentrations tested (0.25 to 3.75 mM). A heat-stable protective factor having a molecular weight less than 16 000 which caused a stimulatory effect on sn-glycerol 3-phosphate acyltransferase activity was found in the cell-free extracts. Preliminary experiments suggest that the factor might be polysaccharide in nature.     

Comparative analysis of mutants in the mycothiol biosynthesis pathway in Mycobacterium smegmatis

The role of mycothiol in mycobacteria was examined by comparative analysis of mutants disrupted in the four known genes encoding the protein machinery needed for mycothiol biosynthesis. These mutants were sensitive to acid stress, antibiotic stress, alkylating stress, and oxidative stress indicating that mycothiol and mycothiol-dependent enzymes protect the mycobacterial cell against attack from various different types of stresses and toxic agents.     

An unusual mutation results in the replacement of diaminopimelate with lanthionine in the peptidoglycan of a mutant strain of Mycobacterium smegmatis

Mycobacterial peptidoglycan contains L-alanyl-D-iso-glutaminyl-meso-diaminopimelyl-D-alanyl-D-alanine peptides, with the exception of the peptidoglycan of Mycobacterium leprae, in which glycine replaces the L-alanyl residue. The third-position amino acid of the peptides is where peptidoglycan cross-linking occurs, either between the meso-diaminopimelate (DAP) moiety of one peptide and the penultimate D-alanine of another peptide or between two DAP residues. We previously described a collection of spontaneous mutants of DAP-auxotrophic strains of Mycobacterium smegmatis that can grow in the absence of DAP. The mutants are grouped into seven classes, depending on how well they grow without DAP and whether they are sensitive to DAP, temperature, or detergent. Furthermore, the mutants are hypersusceptible to beta-lactam antibiotics when grown in the absence of DAP, suggesting that these mutants assemble an abnormal peptidoglycan. In this study, we show that one of these mutants, M. smegmatis strain PM440, utilizes lanthionine, an unusual bacterial metabolite, in place of DAP. We also demonstrate that the abilities of PM440 to grow without DAP and use lanthionine for peptidoglycan biosynthesis result from an unusual mutation in the putative ribosome binding site of the cbs gene, encoding cystathionine beta-synthase, an enzyme that is a part of the cysteine biosynthetic pathway.     

Studies on cardiolipin biosynthesis in Mycobacterium smegmatis.

Supplementation of a growth medium with 5% glucose has been found to stimulate the formation of cardiolipin and phosphatidylethanolamine five- and threefold, respectively, in Mycobacterium smegmatis. The presence of both cytidine diphosphate diglyceride and phosphatidylglycerol pathways of biosynthesis of cardiolipin in cell-free extracts has been demonstrated. The enzymes were localized in the fractions which contained membranes. Isonicotinic acid hydrazide and streptomycin sulfate inhibited the formation of cardiolipin.     

Genetic antagonism and hypermutability in Mycobacterium smegmatis

Multidrug-resistant strains of Mycobacterium tuberculosis are a serious and continuing human health problem. Such strains may contain as many as four or five different mutations, and M. tuberculosis strains that are resistant to both streptomycin and rifampin contain mutations in the rpsL and rpoB genes, respectively. Coexisting mutations of this kind in Escherichia coli have been shown to interact negatively (S. L. Chakrabarti and L. Gorini, Proc. Natl. Acad. Sci. USA 72:2084-2087, 1975; S. L. Chakrabarti and L. Gorini, Proc. Natl. Acad. Sci. USA 74:1157-1161, 1977). We investigated this possibility in Mycobacterium smegmatis by analyzing the frequency and nature of spontaneous mutants that are resistant to either streptomycin or rifampin or to both antibiotics. Mutants resistant to streptomycin were isolated from characterized rifampin-resistant mutants of M. smegmatis under selection either for one or for both antibiotics. Similarly, mutants resistant to rifampin were isolated from streptomycin-resistant strains. The second antibiotic resistance mutation occurred at a lower frequency in both cases. Surprisingly, in both cases a very high rate of reversion of the initial antibiotic resistance allele was detected when single antibiotic selection was used; the majority of strains resistant to only one antibiotic were isolated by this process. Determinations of rates of mutation to antibiotic resistance in M. smegmatis showed that the frequencies were enhanced up to 10(4)-fold during stationary phase. If such behavior is also typical of slow-growing pathogenic mycobacteria, these studies suggest that the generation of multiply drug-resistant strains by successive mutations may be a more complex genetic phenomenon than suspected.     

Functional analysis of molybdopterin biosynthesis in mycobacteria identifies a fused molybdopterin synthase in Mycobacterium tuberculosis

Most mycobacterial species possess a full complement of genes for the biosynthesis of molybdenum cofactor (MoCo). However, a distinguishing feature of members of the Mycobacterium tuberculosis complex is their possession of multiple homologs associated with the first two steps of the MoCo biosynthetic pathway. A mutant of M. tuberculosis lacking the moaA1-moaD1 gene cluster and a derivative in which moaD2 was also deleted were significantly impaired for growth in media containing nitrate as a sole nitrogen source, indicating a reduced availability of MoCo to support the assimilatory function of the MoCo-dependent nitrate reductase, NarGHI. However, the double mutant displayed residual respiratory nitrate reductase activity, suggesting that it retains the capacity to produce MoCo. The M. tuberculosis moaD and moaE homologs were further analyzed by expressing these genes in mutant strains of M. smegmatis that lacked one or both of the sole molybdopterin (MPT) synthase-encoding genes, moaD2 and moaE2, and were unable to grow on nitrate, presumably as a result of the loss of MoCo-dependent nitrate assimilatory activity. Expression of M. tuberculosis moaD2 in the M. smegmatis moaD2 mutant and of M. tuberculosis moaE1 or moaE2 in the M. smegmatis moaE2 mutant restored nitrate assimilation, confirming the functionality of these genes in MPT synthesis. Expression of M. tuberculosis moaX also restored MoCo biosynthesis in M. smegmatis mutants lacking moaD2, moaE2, or both, thus identifying MoaX as a fused MPT synthase. By implicating multiple synthase-encoding homologs in MoCo biosynthesis, these results suggest that important cellular functions may be served by their expansion in M. tuberculosis.     

Purification and characterization of a deoxyriboendonuclease from Mycobacterium smegmatis

A deoxyriboendonuclease has been purified to near homogeneity from a fast growing mycobacterium species, M. smegmatis and characterized to some extent. The size of enzyme is about 43 kDa as determined by a denaturing gel analysis. It shows optimum activity at 32 degrees C in Tris-HCl buffer (pH 7.2) containing 2.5 mM of MgCl2. Both EDTA and K+ but not Na+ inhibit its activity. Evidences show that the enzyme is not a restriction endonuclease but catalyzes the endonucleolytic cleavage of both the double- as well as the single-strand DNA non-specifically. It has been shown that the cleavage by this enzyme generates DNA fragments carrying phosphate groups at 5' ends and hydroxyl group at the 3' ends, respectively. Analysis reveals that no endonuclease having size and property identical to our deoxyriboendonuclease had been purified from M. smegmatis before. The property of our enzymes closely matches with the deoxyriboendonucleases purified from diverse sources including bacteria.     

Polymethylpolysaccharide synthesis in an ethionine-resistant mutant of Mycobacterium smegmatis.

Mutants of Mycobacterium smegmatis were selected for resistance to ethionine in an effort to obtain methylation-defective strains that were altered in their ability to make methylmannose polysaccharides (MMP) or methylglucose lipopolysaccharides. Two methods were developed for the detection of MMP in cell extracts to aid in the screening for potential mutants, one a qualitative procedure based on iodine binding by the sample after paper chromatography and the other a quantitative procedure based on fluorimetric titration of the MMP with parinaric acid. An ethionine-resistant mutant was obtained that contained only about 25% of the normal level of S-adenosylmethionine and 10% of the normal level of methionine adenosyltransferase (adenosine 5'-triphosphate:L-methionine S-adenosyltransferase, EC 2.5.1.6) activity. When grown in the presence of 0.1% ethionine, the mutant cells contained about 50% of the wild-type levels of methylglucose lipopolysaccharides but only about 7% of the normal level of MMP (wild-type cells contain about 0.14 mM MMP and the mutant contains about 0.01 mM MMP). The amount of fatty acid synthesis in the ethionine-resistant mutant grown in the presence of ethionine was not dramatically altered although the mutant accumulated more short-chain and less long-chain unsaturated fatty acids than the wild-type cells.     

MmpS4 promotes glycopeptidolipids biosynthesis and export in Mycobacterium smegmatis

The MmpS family (mycobacterial membrane protein small) includes over 100 small membrane proteins specific to the genus Mycobacterium that have not yet been studied experimentally. The genes encoding MmpS proteins are often associated with mmpL genes, which are homologous to the RND (resistance nodulation cell division) genes of Gram-negative bacteria that encode proteins functioning as multidrug efflux system. We showed by molecular genetics and biochemical analysis that MmpS4 in Mycobacterium smegmatis is required for the production and export of large amounts of cell surface glycolipids, but is dispensable for biosynthesis per se. A new specific and sensitive method utilizing single-chain antibodies against the surface-exposed glycolipids was developed to confirm that MmpS4 was dispensable for transport to the surface. Orthologous complementation demonstrated that the MmpS4 proteins are exchangeable, thus not specific to a defined lipid species. MmpS4 function requires the formation of a protein complex at the pole of the bacillus, which requires the extracytosolic C-terminal domain of MmpS4. We suggest that MmpS proteins facilitate lipid biosynthesis by acting as a scaffold for coupled biosynthesis and transport machinery.