Efficient allelic exchange and transposon mutagenesis in Mycobacterium avium by specialized transduction

Mycobacterium tuberculosis and Mycobacterium avium are pathogenic slow-growing mycobacteria that cause distinct human diseases. In contrast to recent advances in M. tuberculosis genetics and pathogenesis investigation, M. avium has remained genetically intractable and, consequently, its pathogenic strategies remain poorly understood. Here we report the successful development of efficient allelic exchange and transposon mutagenesis in an opaque clinical strain of M. avium by specialized transduction. Efforts to disrupt the leuD gene of M. avium by specialized transduction were successful but were complicated by inefficient isolation of recombinants secondary to high spontaneous antibiotic resistance. However, by using this leucine auxotroph as a genetic host and the Streptomyces coelicolor leuD gene as a selectable marker, we achieved efficient allelic exchange at the M. avium pcaA locus. A leuD-marked transposon delivered by specialized transduction mutagenized M. avium with efficiencies similar to M. tuberculosis. These results establish a system for random and directed mutagenesis of M. avium. In combination with the forthcoming M. avium genome sequence, these tools will allow the distinct physiologic and pathogenic properties of M. avium to be dissected in molecular detail.     

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.     

A crtB homolog essential for photochromogenicity in Mycobacterium marinum: isolation, characterization, and gene disruption via homologous recombination.

A gene essential for light-induced pigment production was isolated from the photochromogen Mycobacterium marinum by heterologous complementation of an M. marinum cosmid library in the nonchromogen Mycobacterium smegmatis. This gene is part of an operon and homologous to the Streptomyces griseus and Myxococcus xanthus crtB genes encoding phytoene synthase. Gene replacement at this locus was achieved via homologous recombination, demonstrating that its expression is essential for photochromogenicity. The ease of targeted gene disruption in this pathogenic Mycobacterium allows for the dissection of the molecular basis of mycobacterial pathogenesis.     

Detection and identification of mycobacteria by amplification of mycobacterial DNA

A 383bp segment of the gene coding for the 65kD mycobacterial antigens from Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, Mycobacterium paratuberculosis, and Mycobacterium fortuitum was amplified using Taq polymerase and synthetic oligonucleotide primers and the amplified DNAs from four of these species were compared by nucleotide sequencing. Although the gene segments from these species showed considerable similarity, oligonucleotide probes which could distinguish M. tuberculosis/M. bovis, M. avium/M. paratuberculosis and M. fortuitum could be identified. Samples containing 10(6) human cells and serial dilutions of a suspension of intact mycobacteria were prepared, DNA was extracted, the segment of the mycobacterial DNA sequence amplified, and the amplified DNA hybridized with oligonucleotide probes. In two independent experiments, this procedure permitted the detection and identification of less than 100 mycobacteria in the original sample. These results suggest that this approach may prove useful in the early diagnosis of mycobacterial infection.     

结核杆菌DnaA蛋白在耻垢分枝杆菌中的同源表达

目的：在耻垢分枝杆菌中表达重组结核杆菌DnaA蛋白并对表达产物进行鉴定。方法：用PCR的方法扩增结核杆菌dnaA基因并克隆至表达载体pMF406中,构建重组大肠杆菌-分枝杆菌穿梭质粒pMF-dnaA。经双酶切及测序鉴定后,用电转化的方法将重组质粒转至耻垢分枝杆菌mc2155中。用0.02%乙酰胺诱导重组耻垢分枝杆菌,对表达产物进行SDS-PAGE和Western blotting检测和鉴定。结果：重组耻垢分枝杆菌构建成功,SDS-PAGE及Western blotting结果显示该重组耻垢杆菌可以实现结核杆菌DnaA蛋白的同源高效表达。结论：结核杆菌DnaA蛋白的同源表达为结核杆菌DNA复制机制的研究奠定了基础。     

Gene replacement through homologous recombination in Mycobacterium intracellulare.

Mycobacterium intracellulare is a slow-growing pathogenic mycobacterium closely related to Mycobacterium avium. In contrast to Mycobacterium tuberculosis and Mycobacterium bovis BCG, M. intracellulare has received little attention as a model species for studies of mycobacterial molecular biology and genetics. This study shows that M. intracellulare 1403 (ATCC 35761) can be transformed by electroporation with high frequencies (up to 10(6) transformants per microgram of DNA), using plasmids pYT937 and pMH94 as replicative and integrative vectors, respectively. We also describe an experimental system that we used to study DNA recombination in M. intracellulare. First, an integrative plasmid was introduced into M. intracellulare 1403. A nonreplicative, nonintegrative plasmid having homology with the integrated plasmid was then introduced, and the resultant recombinants were analyzed to distinguish between events of homologous and illegitimate recombination. No illegitimate recombination occurred; in all recombinants, a single crossover between homologous regions of the two plasmids was noted. During subsequent growth of a recombinant clone, a spontaneous deletion occurred that resulted in a gene replacement on the chromosome of M. intracellulare 1403. The ability to construct site-specific mutations in M. intracellulare will provide novel insights into the biology of slow-growing mycobacteria.     

The Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth

The receptor-like protein kinase PknB from Mycobacterium tuberculosis is encoded by the distal gene in a highly conserved operon, present in all actinobacteria, that may control cell shape and cell division. Genes coding for a PknB-like protein kinase are also found in many more distantly related gram-positive bacteria. Here, we report that the pknB gene can be disrupted by allelic replacement in M. tuberculosis and the saprophyte Mycobacterium smegmatis only in the presence of a second functional copy of the gene. We also demonstrate that eukaryotic Ser/Thr protein kinase inhibitors, which inactivate PknB in vitro with a 50% inhibitory concentration in the submicromolar range, are able to kill M. tuberculosis H37Rv, M. smegmatis mc(2)155, and Mycobacterium aurum A+ with MICs in the micromolar range. Furthermore, significantly higher concentrations of these compounds are required to inhibit growth of M. smegmatis strains overexpressing PknB, suggesting that this protein kinase is the molecular target. These findings demonstrate that the Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth and support the development of protein kinase inhibitors as new potential antituberculosis drugs.     

Epitope-tagging vectors for the expression and detection of recombinant proteins in mycobacteria

New tools are required to study the growing number of uncharacterised genes derived from genome sequence projects that are specific to bacterial pathogens such as Mycobacterium tuberculosis. We have developed a series of vectors that permit the specific detection of recombinant proteins expressed in mycobacterial species. Gene expression in these vectors is driven by the strong hsp60 promoter of Mycobacterium bovis BCG and detection of expressed products is facilitated by C-terminal fusion of residues 409-419 of the human c-myc proto-oncogene. Using the M. tuberculosis Ag85B as a reporter of gene expression, we demonstrate that the vectors permit the specific detection of recombinant products expressed in the host species M. bovis BCG. BCG over-expressing Ag85B was a potent inducer of Ag85B-specific T cells in immunised mice, indicating that the C-terminal c-myc tag did not alter the characteristics of the recombinant protein. The versatility of the epitope-tagging vectors was demonstrated by the efficient secretion and detection of recombinant products in BCG. The vectors described in this study will facilitate the expression of foreign proteins in mycobacterial host systems.     

Rv1818c-encoded PE_PGRS protein of Mycobacterium tuberculosis is surface exposed and influences bacterial cell structure

Identification of the novel PE multigene family was an unexpected finding of the genomic sequencing of Mycobacterium tuberculosis. Presently, the biological role of the PE and PE_PGRS proteins encoded by this unique family of mycobacterial genes remains unknown. In this report, a representative PE_PGRS gene (Rv1818c/PE_PGRS33) was selected to investigate the role of these proteins. Cell fractionation studies and fluorescence analysis of recombinant strains of Mycobacterium smegmatis and M. tuberculosis expressing green fluorescent protein (GFP)-tagged proteins indicated that the Rv1818c gene product localized in the mycobacterial cell wall, mostly at the bacterial cell poles, where it is exposed to the extracellular milieu. Further analysis of this PE_PGRS protein showed that the PE domain is necessary for subcellular localization. In addition, the PGRS domain, but not PE, affects bacterial shape and colony morphology when Rv1818c is overexpressed in M. smegmatis and M. tuberculosis. Taken together, the results indicate that PE_PGRS and PE proteins can be associated with the mycobacterial cell wall and influence cellular structure as well as the formation of mycobacterial colonies. Regulated expression of PE genes could have implications for the survival and pathogenesis of mycobacteria within the human host and in other environmental niches.     

Genes for the protein antigens of the tuberculosis and leprosy bacilli

The lambda gt 11 expression vector permitted us to survey protein antigens of Mycobacterium leprae and Mycobacterium tuberculosis expressed in Escherichia coli. Using monoclonal antibodies, recombinant clones were detected producing three major antigens of M. tuberculosis and five major protein antigens of M. leprae. These recombinant antigens produced in E. coli should prove useful for diagnosis, epidemiology and possibly the development of recombinant mycobacterial vaccines.     

Mycobacterium smegmatis displays the Mycobacterium tuberculosis virulence-related neutral red character when expressing the Rv0577 gene

Neutral red staining is a cytochemical reaction that has been found to be related to Mycobacterium tuberculosis virulence and, therefore, the component involved in it is thought to be a virulence factor. To study the molecular basis of this reaction we constructed an M. tuberculosis cosmid library in Mycobacterium smegmatis and selected recombinant neutral red positive clones. Heterologous complementation identified Rv0577 as the gene responsible for this trait and we have also shown that it is expressed as a single polycistronic unit together with Rv0576 which could also be involved in the neutral red staining.     

Characterization of the functional replication origin of Mycobacterium tuberculosis.

The gene order in the 5kb Mycobacterium tuberculosis dnaA region is rnpA, rpmH, dnaA, dnaN and recF. We show that M. tuberculosis DNA fragment containing the dnaA-dnaN intergenic region functioned as oriC, i.e., allowed autonomous replication to otherwise nonreplicative plasmids, in M. tuberculosis H37Ra (H37Ra), avirulent strain of M. tuberculosis, and in Mycobacterium bovis BCG (BCG), a closely related, slowly growing mycobacterial strain. Removal of Escherichia coli plasmid replication origin (ColE1) from the M. tuberculosis oriC plasmids did not abolish their ability to function as oriC, confirming that the autonomous replication activity of these plasmids is due to the presence of the DNA fragment containing the dnaA-dnaN intergenic region. Deletion analyses revealed that the minimal oriC DNA fragment is 814bp. The copy number of M. tuberculosis oriC plasmids containing ColE1 ori relative to chromosomal oriC is one and the 5' flanking region of minimal oriC contains features that support stable autonomous replication. The M. tuberculosis oriC did not function in rapidly growing mycobacterial species such as M. smegmatis. M. smegmatis oriC functioned only in M. fortuitum, but not in any of the slowly growing mycobacterial species such as M. tuberculosis and BCG. Together these data suggest that the replication initiation mechanisms in the slowly growing Mycobacteria are similar and probably different from those in the rapidly growing Mycobacteria and vice versa.     

Molecular dissection of the role of two methyltransferases in the biosynthesis of phenolglycolipids and phthiocerol dimycoserosate in the Mycobacterium tuberculosis complex

A few mycobacterial species, most of which are pathogenic for humans, produce dimycocerosates of phthiocerol (DIM) and of glycosylated phenolphthiocerol, also called phenolglycolipid (PGL), two groups of molecules shown to be important virulence factors. The biosynthesis of these molecules is a very complex pathway that involves more than 15 enzymatic steps and has just begun to be elucidated. Most of the genes known to be involved in these pathways are clustered on the chromosome of M. tuberculosis. Based on their amino acid sequences, we hypothesized that the proteins encoded by Rv2952 and Rv2959c, two open reading frames of this locus, are involved in the transfer of methyl groups onto various hydroxyl functions during the biosynthesis of DIM, PGL, and related p-hydroxybenzoic acid derivatives (p-HBAD). Using allelic exchange and site-specific recombination, we produced three recombinant strains of Mycobacterium tuberculosis carrying insertions in Rv2952 or Rv2959c. Analysis of these mutants revealed that (i) the protein encoded by Rv2952 is a methyltransferase catalyzing the transfer of a methyl group onto the lipid moiety of phthiotriol and glycosylated phenolphthiotriol dimycocerosates to form DIM and PGL, respectively, (ii) Rv2959c is part of an operon including the newly characterized Rv2958c gene that encodes a glycosyltransferase also involved in PGL and p-HBAD biosynthesis, and (iii) the enzyme encoded by Rv2959c catalyzes the O-methylation of the hydroxyl group located on carbon 2 of the rhamnosyl residue linked to the phenolic group of PGL and p-HBAD produced by M. tuberculosis. These data further extend our understanding of the biosynthesis of important mycobacterial virulence factors and provide additional tools to decipher the molecular mechanisms of action of these molecules during the pathogenesis of tuberculosis.     

Cloning and characterization of the aroA gene from Mycobacterium tuberculosis.

The aroA gene from Mycobacterium tuberculosis has been cloned by complementation of an aroA mutant of Escherichia coli after lysogenization with a recombinant DNA library in the lambda gt11 vector. Detailed characterization of the M. tuberculosis aroA gene by nucleotide sequencing and by immunochemical analysis of the expressed product indicates that it encodes a 5-enolpyruvylshikimate-3-phosphate synthase that is structurally related to analogous enzymes from other bacterial, fungal, and plant sources. The potential use of the cloned gene in construction of genetically defined mutant strains of M. tuberculosis by gene replacement is proposed as a novel approach to the rational attenuation of mycobacterial pathogens and the possible development of new antimycobacterial vaccines.     

Studies on alpha(1-->5) linked octyl arabinofuranosyl disaccharides for mycobacterial arabinosyl transferase activity

The appearance multi-drug resistant Mycobacterium tuberculosis (MTB) throughout the world has prompted a search for new, safer and more active agents against tuberculosis. Based on studies of the biosynthesis of mycobacterial cell wall polysaccharides, octyl 5-O-(alpha-D-arabinofuranosyl)-alpha-D-arabinofuranoside analogues were synthesized and evaluated as inhibitors for M. tuberculosis and Mycobacterium avium. A cell free assay system has been used for the evaluation of these disaccharides as substrates for mycobacterial arabinosyltransferase activity.     

The Mycobacterium tuberculosis protein serine/threonine kinase PknG is linked to cellular glutamate/glutamine levels and is important for growth in vivo

The function of the Mycobacterium tuberculosis eukaryotic-like protein serine/threonine kinase PknG was investigated by gene knock-out and by expression and biochemical analysis. The pknG gene (Rv0410c), when cloned and expressed in Escherichia coli, encodes a functional kinase. An in vitro kinase assay of the recombinant protein demonstrated that PknG can autophosphorylate its kinase domain as well as its 30 kDa C-terminal portion, which contains a tetratricopeptide (TPR) structural signalling motif. Western analysis revealed that PknG is located in the cytosol as well as in mycobacterial membrane. The pknG gene was inactivated by allelic exchange in M. tuberculosis. The resulting mutant strain causes delayed mortality in SCID mice and displays decreased viability both in vitro and upon infection of BALB/c mice. The reduced growth of the mutant was more pronounced in the stationary phase of the mycobacterial growth cycle and when grown in nutrient-depleted media. The PknG-deficient mutant accumulates glutamate and glutamine. The cellular levels of these two amino acids reached approximately threefold of their parental strain levels. Higher cellular levels of the amine sugar-containing molecules, GlcN-Ins and mycothiol, which are derived from glutamate, were detected in the DeltapknG mutant. De novo glutamine synthesis was shown to be reduced by 50%. This is consistent with current knowledge suggesting that glutamine synthesis is regulated by glutamate and glutamine levels. These data support our hypothesis that PknG mediates the transfer of signals sensing nutritional stress in M. tuberculosis and translates them into metabolic adaptation.     

A protein secretion pathway critical for Mycobacterium tuberculosis virulence is conserved and functional in Mycobacterium smegmatis

The Snm protein secretion system is a critical determinant of Mycobacterium tuberculosis virulence. However, genes encoding components of this pathway are conserved among all mycobacteria, including the nonpathogenic saprophyte Mycobacterium smegmatis. We show that the Snm system is operational in M. smegmatis and that secretion of its homologous ESAT-6 and CFP-10 substrates is regulated by growth conditions. Importantly, we show that Snm secretion in M. smegmatis requires genes that are homologous to those required for secretion in M. tuberculosis. Using a gene knockout strategy in M. smegmatis, we have also discovered four new gene products that are essential for Snm secretion, including the serine protease mycosin 1. Despite the evolutionary distance between M. smegmatis and M. tuberculosis, the M. smegmatis Snm system can secrete the M. tuberculosis ESAT-6 and CFP-10 proteins, suggesting that substrate recognition is also conserved between the two species. M. smegmatis, therefore, represents a powerful system to study the multicomponent Snm secretory machine and to understand the role of this conserved system in mycobacterial biology.     

Comparative genomics of metabolic pathways in Mycobacterium species: gene duplication, gene decay and lateral gene transfer

The genus Mycobacterium comprises significant pathogenic species that infect both humans and animals. One species within this genus, Mycobacterium tuberculosis, is the primary killer of humans resulting from bacterial infections. Five mycobacterial genomes belonging to four different species (M. tuberculosis, Mycobacterium bovis, Mycobacterium leprae and Mycobacterium avium ssp. paratuberculosis) have been sequenced to date and another 14 mycobacterial genomes are at various stages of completion. A comparative analysis of the gene products of key metabolic pathways revealed that the major differences among these species are in the gene products constituting the cell wall and the gene families encoding the acidic glycine-rich (PE/PPE/PGRS) proteins. Mycobacterium leprae has evolved by retaining a minimal gene set for most of the gene families, whereas M. avium ssp. paratuberculosis has acquired some of the virulence factors by lateral gene transfer.     

Characterization of an exported monoglyceride lipase from Mycobacterium tuberculosis possibly involved in the metabolism of host cell membrane lipids

The Rv0183 gene of the Mycobacterium tuberculosis H37Rv strain, which has been implicated as a lysophospholipase, was cloned and expressed in Escherichia coli. The purified Rv0183 protein did not show any activity when lysophospholipid substrates were used, but preferentially hydrolysed monoacylglycerol substrates with a specific activity of 290 units x mg(-1) at 37 degrees C. Rv0183 hydrolyses both long chain di- and triacylglycerols, as determined using the monomolecular film technique, although the turnover was lower than with MAG (monoacyl-glycerol). The enzyme shows an optimum activity at pH values ranging from 7.5 to 9.0 using mono-olein as substrate and is inactivated by serine esterase inhibitors such as E600, PMSF and tetrahydrolipstatin. The catalytic triad is composed of Ser110, Asp226 and His256 residues, as confirmed by the results of site-directed mutagenesis. Rv0183 shows 35% sequence identity with the human and mouse monoglyceride lipases and well below 15% with the other bacterial lipases characterized so far. Homologues of Rv0183 can be identified in other mycobacterial genomes such as Mycobacterium bovis, Mycobacterium smegmatis, and even Mycobacterium leprae, which is known to contain a low number of genes involved in the replication process within the host cells. The results of immunolocalization studies performed with polyclonal antibodies raised against the purified recombinant Rv0183 suggested that the enzyme was present only in the cell wall and culture medium of M. tuberculosis. Our results identify Rv0183 as the first exported lipolytic enzyme to be characterized in M. tuberculosis and suggest that Rv0183 may be involved in the degradation of the host cell lipids.     

Molecular and biochemical characterisation of Mycobacterium smegmatis alcohol dehydrogenase C

The gene encoding of an alcohol dehydrogenase C (ADHC) from Mycobacterium smegmatis was cloned and sequenced. The protein encoded by this gene has 78% identity with Mycobacterium tuberculosis and Mycobacterium bovis BCG ADHC. The M. smegmatis ADHC was purified from M. smegmatis and the kinetic parameters of this enzyme showed that using NADPH as electron donor it has a strong preference for aliphatic and aromatic aldehyde substrates. Like the M. bovis BCG ADHC, this enzyme is more likely to act as an aldehyde reductase than as an alcohol dehydrogenase. The discovery of such an ADHC in a fast-growing, and easily engineered mycobacterial species opens the way to the utilisation of this M. smegmatis enzyme as a convenient model for the study of the physiological role of this alcohol dehydrogenase in mycobacteria.