Effects of clofazimine on potassium uptake by a Trk-deletion mutant of Mycobacterium tuberculosis.

OBJECTIVES: This study was designed to investigate the effects of the membrane-active, anti-mycobacterial agent, clofazimine, on potassium (K+)-uptake by a mutant of Mycobacterium tuberculosis (MTB), in which the Trk system, the major K+ transporter of this microbial pathogen, had been selectively inactivated. METHODS: The ceoB and ceoC genes of MTB, which encode the TrkA proteins, CeoB and CeoC, were deleted by homologous recombination, and the double-knockout mutant and wild-type strains compared with respect to K+ uptake and growth in the presence and absence of clofazimine (0.015-2.5 mg/L) using radioassay procedures. RESULTS: Surprisingly, the magnitudes of K+ uptake and rate of growth of the ceoBC-knockout mutant were significantly (P < 0.05) greater than those of the wild-type strain, due, presumably, to induction of a back-up transporter. Exposure of both the wild-type strain and ceoBC-knockout mutant of MTB to clofazimine was accompanied by dose-related decreases in K+ uptake, as well as growth, which were of comparable magnitude for both strains. CONCLUSIONS: These observations demonstrate that the major K+ transporter of MTB, Trk, as well as an uncharacterized inducible back-up system, is equally sensitive to the inhibitory actions of clofazimine.

Global expression profiling of strains harbouring null mutations reveals that the five rpf-like genes of Mycobacterium tuberculosis show functional redundancy.

SETTING: Aged, dormant cultures of Mycobacterium tuberculosis can be resuscitated by a secreted, proteinaceous growth factor from Micrococcus luteus, known as resuscitation-promoting factor (Rpf). M. tuberculosis contains five rpf-like genes, rpfA (Rv0867c), rpfB (Rv1009), rpfC (Rv1884c), rpfD (Rv2389c) and rpfE (Rv2450c), that bear significant similarity to Mi. luteus rpf, suggesting that these too may play a role in growth and/or reactivation from a quiescent state. OBJECTIVE AND DESIGN: Unmarked deletion mutants of each of the five rpf-like genes of M. tuberculosis H37Rv were constructed and their phenotypes and global gene expression profiles were assessed. RESULTS AND CONCLUSIONS: Deletion of any one of the rpf-like genes did not affect growth or survival of M. tuberculosis in liquid culture, but some alterations in colony-forming ability and colonial morphology were observed. Global gene expression profiling suggested that loss of rpfC affected the expression of the largest number of genes and there was a significant overlap in the differential gene expression profile of the rpfC mutant with those of the rpfB, rpfD and rpfE mutants. The expression profile of the rpfA mutant was notably less similar, but inverse associations with genes affected in the other mutants were observed. These results, together with those obtained by real-time, quantitative RT-PCR, suggest that the rpf-like genes serve wholly or partially overlapping functions in M. tuberculosis.

Characterization of the cydAB-encoded cytochrome bd oxidase from Mycobacterium smegmatis.

The cydAB genes from Mycobacterium smegmatis have been cloned and characterized. The cydA and cydB genes encode the two subunits of a cytochrome bd oxidase belonging to the widely distributed family of quinol oxidases found in prokaryotes. The cydD and cydC genes located immediately downstream of cydB encode a putative ATP-binding cassette-type transporter. At room temperature, reduced minus oxidized difference spectra of membranes purified from wild-type M. smegmatis displayed spectral features that are characteristic of the gamma-proteobacterial type cytochrome bd oxidase. Inactivation of cydA or cydB by insertion of a kanamycin resistance marker resulted in loss of d-heme absorbance at 631 nm. The d-heme could be restored by transformation of the M. smegmatis cyd mutants with a replicating plasmid carrying the highly homologous cydABDC gene cluster from Mycobacterium tuberculosis. Inactivation of cydA had no effect on the ability of M. smegmatis to exit from stationary phase at 37 or 42 degrees C. The growth rate of the cydA mutant was tested under oxystatic conditions. Although no discernible growth defect was observed under moderately aerobic conditions (9.2 to 37.5 x 10(2) Pa of pO(2) or 5 to 21% air saturation), the mutant displayed a significant growth disadvantage when cocultured with the wild type under extreme microaerophilia (0.8 to 1.7 x 10(2) Pa of pO(2) or 0.5 to 1% air saturation). These observations were in accordance with the two- to threefold increase in cydAB gene expression observed upon reduction of the pO(2) of the growth medium from 21 to 0.5% air saturation and with the concomitant increase in d-heme absorbance in spectra of membranes isolated from wild-type M. smegmatis cultured at 1% air saturation. Finally, the cydA mutant displayed a competitive growth disadvantage in the presence of the terminal oxidase inhibitor, cyanide, when cocultured with wild type at 21% air saturation in an oxystat. In conjunction with these findings, our results suggest that cytochrome bd is an important terminal oxidase in M. smegmatis.

DNA metabolism in mycobacterium tuberculosis: implications for drug resistance and strain variability.

In this paper, we review the evidence supporting the notion that the genome of Mycobacterium tuberculosis sustains considerable damage as a result of exposure to nitrosative and oxidative stress. On these grounds, we propose a model in which stress-induced DNA damage in M. tuberculosis plays a role in the evolution of chromosomally encoded drug resistance mutations by altering the global mutation rate by mechanisms akin to SOS mutagenesis. Finally we review some of the factors determining the evolution of PE/PPE and MIRU (There are many abbreviations in this paper which are not defined, e.g. SOS, PE/PPE and MIRU. Please indicate whether these are well known and will be understood by readers or whether they should be defined at first mention) loci whose sequence characteristics are suggestive of their classification as heritable local mutators.

Expression of Mycobacterium smegmatis pyrazinamidase in Mycobacterium tuberculosis confers hypersensitivity to pyrazinamide and related amides.

A pyrazinamidase (PZase)-deficient pncA mutant of Mycobacterium tuberculosis, constructed by allelic exchange, was used to investigate the effects of heterologous amidase gene expression on the susceptibility of this organism to pyrazinamide (PZA) and related amides. The mutant was highly resistant to PZA (MIC, >2,000 microg/ml), in accordance with the well-established role of pncA in the PZA susceptibility of M. tuberculosis (A. Scorpio and Y. Zhang, Nat. Med. 2:662-667, 1996). Integration of the pzaA gene encoding the major PZase/nicotinamidase from Mycobacterium smegmatis (H. I. M. Boshoff and V. Mizrahi, J. Bacteriol. 180:5809-5814, 1998) or the M. tuberculosis pncA gene into the pncA mutant complemented its PZase/nicotinamidase defect. In both pzaA- and pncA-complemented mutant strains, the PZase activity was detected exclusively in the cytoplasm, suggesting an intracellular localization for PzaA and PncA. The pzaA-complemented strain was hypersensitive to PZA (MIC, /=20 microg/ml) and was also sensitive to benzamide (MIC, 20 microg/ml), unlike the wild-type and pncA-complemented mutant strains, which were highly resistant to this amide (MIC, >500 microg/ml). This finding was consistent with the observation that benzamide is hydrolyzed by PzaA but not by PncA. Overexpression of PzaA also conferred sensitivity to PZA, nicotinamide, and benzamide on M. smegmatis (MIC, 150 microg/ml in all cases) and rendered Escherichia coli hypersensitive for growth at low pH.

Construction and application of mycobacterial reporter transposons.

The transposon Tn5367, which is a derivative of the mycobacterial insertion sequence IS1096, was modified by introducing novel genes to produce reporter transposons which can be used to generate transposon insertion libraries containing mycobacterial gene or operon fusions. A plasmid that is temperature-sensitive for replication in mycobacteria was used to deliver promoterless lacZ or aph reporter genes to Mycobacterium smegmatis as transcriptional (lacZ), or translational ('aph) fusions. Mutants containing lacZ produced varying intensities of blue colour on indicator media. This reporter activity could be used as a quantitative measure of promoter strength. Mutants displaying varying levels of resistance to kanamycin were obtained by transpositional insertion of the 'aph reporter lacking a promoter, ribosome binding site and start codon to form functionally active translational fusions. Finally, inclusion of the R6Kgamma origin within Tn5367 allowed transposon insertions to be rescued in an Escherichia coli host strain permissive for the replication of this origin. This study demonstrates that transcriptional and translational reporter derivatives of Tn5367 are functional, and they supplement the growing range of molecular tools available for the study of mycobacteria.

The stringent response of Mycobacterium tuberculosis is required for long-term survival.

The stringent response utilizes hyperphosphorylated guanine [(p)ppGpp] as a signaling molecule to control bacterial gene expression involved in long-term survival under starvation conditions. In gram-negative bacteria, (p)ppGpp is produced by the activity of the related RelA and SpoT proteins. Mycobacterium tuberculosis contains a single homolog of these proteins (Rel(Mtb)) and responds to nutrient starvation by producing (p)ppGpp. A rel(Mtb) knockout strain was constructed in a virulent strain of M. tuberculosis, H37Rv, by allelic replacement. The rel(Mtb) mutant displayed a significantly slower aerobic growth rate than the wild type in synthetic liquid media, whether rich or minimal. The growth rate of the wild type was equivalent to that of the mutant when citrate or phospholipid was employed as the sole carbon source. These two organisms also showed identical growth rates within a human macrophage-like cell line. These results suggest that the in vivo carbon source does not represent a stressful condition for the bacilli, since it appears to be utilized in a similar Rel(Mtb)-independent manner. In vitro growth in liquid media represents a condition that benefits from Rel(Mtb)-mediated adaptation. Long-term survival of the rel(Mtb) mutant during in vitro starvation or nutrient run out in normal media was significantly impaired compared to that in the wild type. In addition, the mutant was significantly less able to survive extended anaerobic incubation than the wild-type virulent organism. Thus, the Rel(Mtb) protein is required for long-term survival of pathogenic mycobacteria under starvation conditions.

Staphylococcus aureus nuclease is a useful secretion reporter for mycobacteria.

A secretion reporter system based on Staphylococcus aureus nuclease (nuc) was developed for use in mycobacteria. Fusion of secretion signals to the reporter cloned in a shuttle vector, pBPnuc1, resulted in halo formation around colonies of Mycobacterium smegmatis and Mycobacterium tuberculosis grown on DNase agar plates containing Methyl Green indicator dye. This in-situ detection system was used to identify secreted proteins by screening a pBPnuc1::H37Rv nuc gene fusion library in M. smegmatis. The clones identified in this screen all formed colony halos when present in M. tuberculosis grown on indicator media. The proteins corresponded to DesA2, a stearoyl-acyl carrier protein desaturase, PepA, a putative serine protease and the Apa antigen, which is the ATP-binding subunit of an ABC transport system. Of these proteins, only PepA and Apa contained recognizable leader peptides.

Purification, gene cloning, targeted knockout, overexpression, and biochemical characterization of the major pyrazinamidase from Mycobacterium smegmatis.

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.

DNA repair in Mycobacterium tuberculosis. What have we learnt from the genome sequence?

The genome sequence of Mycobacterium tuberculosis was analysed by searching for homologues of genes known to be involved in the reversal or repair of DNA damage in Escherichia coli and related organisms. Genes necessary to perform nucleotide excision repair (NER), base excision repair (BER), recombination, and SOS repair and mutagenesis were identified. In particular, all of the genes known to be directly involved in the repair of oxidative and alkylative damage are present in M. tuberculosis. In contrast, we failed to identify homologues of genes involved in mismatch repair. This finding has potentially significant implications with respect to genome stability, strain variability at repeat loci and the emergence of chromosomally encoded drug resistance mutations.

Single-frequency 1559-nm erbium-doped fiber laser pumped by a 650-nm semiconductor laser.

A single-frequency laser with a 2-cm-length erbium-doped fiber and fiber-grating coupler mirrors was operated successfully with a 650-nm semiconductor pump laser. Laser pump threshold was 0.91-mW and 34- ?W output power at 1559 nm was obtained for 6-mW pump power.

The RLEP-flanked polA gene from Mycobacterium leprae is not transcribed in Mycobacterium smegmatis.

A polA mutant of Mycobacterium smegmatis (Ms) lacking DNA polymerase activity could not support the replication of a pAL5000-derived plasmid or a derivative harbouring the RLEP-flanked polA gene from M. leprae (Mlep). In contrast, the plasmid containing the M. tuberculosis polA gene transformed the mutant with high efficiency and complemented its damage-sensitive phenotype suggesting that the replication of the pAL5000 origin is dependent on host PolI function and that the RLEP-flanked Mlep polA gene is not expressed in Ms.

SOS induction in mycobacteria: analysis of the DNA-binding activity of a LexA-like repressor and its role in DNA damage induction of the recA gene from Mycobacterium smegmatis.

The protein encoded by the lexA gene from Mycobacterium leprae was overproduced in Escherichia coli. The recombinant protein bound to the promoter regions of the M. leprae lexA, M. leprae recA and M. smegmatis recA genes at sites with the sequences 5'-GAACACATGTTT and 5'-GAACAGGTGTTC, which belong to the 'Cheo box' family of binding sites recognized by the SOS repressor from Bacillus subtilis. Gel mobility shift assays were used to confirm that proteins with the same site specificity of DNA binding are also present in Mycobacterium tuberculosis and M. smegmatis. Complex formation was impaired by mutagenic disruption of the dyad symmetry of the M. smegmatis recA Cheo box. LexA binding was also inhibited by preincubation of the M. smegmatis and M. tuberculosis extracts with anti-M. leprae LexA antibodies, suggesting that the mycobacterial LexA proteins are functionally conserved at the level of DNA binding. Finally, exposure of M. smegmatis to DNA-damaging agents resulted in induction of the M. smegmatis recA promoter with concomitant loss of DNA binding of LexA to its Cheo box, confirming that this organism possesses the key regulatory elements of a functional SOS induction system.

Deoxy- and dideoxynucleotide discrimination and identification of critical 5' nuclease domain residues of the DNA polymerase I from Mycobacterium tuberculosis.

The DNA polymerase I (PolI) from Mycobacterium tuberculosis (Mtb) was overproduced in Escherichia coli as an enzymatically active, recombinant protein with or without an N-terminal His-tag. The proteins catalysed both the DNA polymerisation of homo- and heteropolymer template-primers and the 5'-3' exonucleolytic hydrolysis of gapped and nicked substrates but lacked an associated proofreading activity. In accordance with recent predictions [Tabor, S. and Richardson, C.C. (1995) Proc. Natl. Acad. Sci. USA, 92, 6339-6343], both recombinant forms of the M. tuberculosis enzyme were unable to discriminate against dideoxynucleotide 5'-triphosphates and were thus efficiently inhibited by these chain-terminating nucleotide analogues during DNA synthesis. This unusual property might be potentially exploitable in terms of novel anti-mycobacterial drug design. A mutational analysis of 5' nuclease domain residues allowed the roles of nine invariant acidic residues to be evaluated. Acidic side chain neutralisation resulted in a > or = 20-fold reduction in activity, with the most profound reduction (> or = 10(4)-fold) being caused by neutralisation of the Asp125, Asp148 and Asp150 residues.

Construction by homologous recombination and phenotypic characterization of a DNA polymerase domain polA mutant of Mycobacterium smegmatis.

Gene replacement was achieved by homologous recombination in Mycobacterium smegmatis (Ms) using a cloned segment of the polA gene (encoding the DNA polymerase I) disrupted within the region encoding the C-terminal DNA polymerase domain by a kanamycin-resistance marker. The Ms polA755:aph mutant thus generated displayed a phenotype of hypersensitivity to DNA damage induced by UV irradiation and by hydrogen peroxide challenge.

Cloning, sequence analysis, overproduction in Escherichia coli and enzymatic characterization of the RNase HI from Mycobacterium smegmatis.

Activity gel analysis of cell extracts from slow- and fast-growing mycobacteria confirmed the presence of several RNase H activities in both classes of organism. The rnhA gene from Mycobacterium smegmatis (Ms) was subsequently cloned using an internal gene segment probe [Mizrahi et al., Gene 136 (1993) 287-290]. The gene encodes a polypeptide of 159 amino acids that shares 50% identity with the RNase HI from Escherichia coli (Ec). However, unlike its counterparts from Gram- bacteria, Ms rnhA does not form an overlapping divergent transcriptional unit with dnaQ (encoding the epsilon (proofreading) subunit of DNA polymerase III). Ms RNase HI was overproduced in Ec as an enzymatically active maltose-binding protein (MBP) fusion protein which cleaved the RNA strand of an RNA.DNA hybrid with a similar site selectivity to that of its Ec homologue.