Novel substrates of Mycobacterium tuberculosis PknH Ser/Thr kinase.

PknH Ser/Thr protein kinase of Mycobacterium tuberculosis controls the expression of a variety of cell wall related enzymes and regulates the in vivo growth in mice. Therefore, we predicted that the PknH kinase could phosphorylate several substrates controlling different metabolic and physiological pathways. Using a bioinformatic approach, we identified 40 potential substrates. Two substrates were shown to be phosphorylated by recombinant PknH kinase in vitro. Point mutation studies verified that substrates are phosphorylated at the in silico-predicted sites. Kinetic studies revealed a similar relative-phosphorylation rate (V(max)) of PknH towards two new substrates and the only previously known substrate, EmbR. Unlike the EmbR protein, the Rv0681 and DacB1 proteins do not contain an FHA domain and are possible participants of new signaling pathways mediated by the PknH kinase in M. tuberculosis.

Deletion of the Mycobacterium tuberculosis pknH gene confers a higher bacillary load during the chronic phase of infection in BALB/c mice.

The role of the serine/threonine kinase PknH in the physiology and virulence of Mycobacterium tuberculosis was assessed by the construction of a pknH deletion mutant. Deletion of the pknH gene did not affect sensitivity to the antimycobacterial drug ethambutol, although it was previously thought to be involved in regulating expression of emb genes encoding arabinosyl transferases, the targets of ethambutol. Nevertheless, transcription analyses revealed that genes associated with mycobacterial cell wall component synthesis, such as emb and ini operons, are downstream substrates of the PknH signaling cascade. In vitro survival studies revealed that a mutant with a deletion of the pknH gene displayed increased resistance to acidified nitrite stress, suggesting that nitric oxide is one of the potential environmental triggers for PknH activation. The effect of pknH deletion on mycobacterial virulence was investigated in BALB/c mice. In this model, the DeltapknH mutant was found to survive and replicate to a higher bacillary load in mouse organs than its parental strain and the pknH-complemented strain. In contrast, another closely related kinase mutant, the DeltapknE mutant, obtained from the same parental strain, was not affected in its virulence phenotype. Infection of THP-1 cells or in vitro growth studies in 7H9 medium did not reveal a significant in vitro growth advantage phenotype for the DeltapknH mutant. In conclusion, we propose that the serine/threonine kinase PknH plays a role in regulating bacillary load in mouse organs to facilitate adaptation to the host environment, possibly by enabling a regulated chronic infection by M. tuberculosis.

The mechanism of upstream activation in the rrnB operon of Mycobacterium smegmatis is different from the Escherichia coli paradigm.

Mycobacteria are slow-growing bacteria with a generation time of from 2-3 h up to several weeks. Consistent with the low growth rate, mycobacterial species have a maximum of two rRNA operons, rrnA and rrnB. The rrnA operon is present in all mycobacteria and has between two and five promoters, depending on species, whereas the rrnB operon, with a single promoter, is only found in some of the faster-growing species. The promoter region of the rrnB operon of a typical fast grower, Mycobacterium smegmatis, was investigated. By using lacZ reporter gene fusions it was demonstrated that the rrnB operon contains a highly activating region upstream of the core promoter, comparable to other bacterial rrn operons. However, the results suggest that, unlike the situation in, for example, Escherichia coli, the activating mechanism is solely factor dependent, and that no UP element is involved.

The AraC family transcriptional regulator Rv1931c plays a role in the virulence of Mycobacterium tuberculosis.

A Mycobacterium tuberculosis strain disrupted in the AraC homologue Rv1931c was isolated. The mutant strain exhibited reduced survival both in macrophages and in a mouse infection model, with survival being restored on complementation with the Rv1931c gene. These results suggest that Rv1931c regulates genes important for virulence of M. tuberculosis.

Autophosphorylation of the 16 kDa and 70 kDa antigens (Hsp 16.3 and Hsp 70) of Mycobacterium tuberculosis.

Several antigens of Mycobacterium tuberculosis, identified by monoclonal antibodies, have been previously cloned and are being exploited in the development of improved vaccines and diagnostic reagents. In this study, the molecular characteristics of two of these antigens, the immunodominant proteins Hsp 16.3 and Hsp 70, were analysed in further detail by assessing their capacity to undergo protein phosphorylation, a chemical modification frequently used by organisms to adjust to environmental variations. Hsp 16.3 was overproduced in an Escherichia coli expression system and purified to homogeneity. Upon incubation in the presence of radioactive ATP, it was shown to possess autophosphorylation activity. Two-dimensional analysis of its phosphoamino acid content revealed that it was modified exclusively at serine residues. In addition, cross-linking experiments demonstrated that it could tightly bind to ATP. Purified Hsp 70 was also shown to autophosphorylate but phosphorylation occurred exclusively at threonine residues. This reaction was found to be strongly stimulated by calcium ions. These data indicate that both structural and functional similarities exist between Hsp 16.3 (Acr) and alpha-crystallin, a eukaryotic protein which plays an important role in maintaining the transparency of the vertebrate eye, and that the functional properties of Hsp 70 from M. tuberculosis are similar to those of other bacterial members of the Hsp 70 family, particularly the E. coli homologue DnaK.

DNA alkylation damage as a sensor of nitrosative stress in Mycobacterium tuberculosis.

One of the cellular consequences of nitrosative stress is alkylation damage to DNA. To assess whether nitrosative stress is registered on the genome of Mycobacterium tuberculosis, mutants lacking an alkylation damage repair and reversal operon were constructed. Although hypersensitive to the genotoxic effects of N-methyl-N'-nitro-N-nitrosoguanidine in vitro, the mutants displayed no phenotype in vivo, suggesting that permeation of nitrosative stress to the level of cytotoxic DNA damage is restricted.

DNA damage induction of recA in Mycobacterium tuberculosis independently of RecA and LexA.

The ubiquitous and highly conserved RecA protein is generally expressed from a single promoter, which is regulated by LexA in conjunction with RecA. We show here using transcriptional fusions to a reporter gene that the Mycobacterium tuberculosis recA gene is expressed from two promoters. Although one promoter is clearly regulated in the classical way, the other remains DNA damage inducible in the absence of RecA or when LexA binding is prevented. These observations demonstrate convincingly for the first time that there is a novel mechanism of DNA damage induction in M. tuberculosis that is independent of LexA and RecA.

The functions of OmpATb, a pore-forming protein of Mycobacterium tuberculosis.

The functions of OmpATb, the product of the ompATb gene of Mycobacterium tuberculosis and a putative porin, were investigated by studying a mutant with a targeted deletion of the gene, and by observing expression of the gene in wild-type M. tuberculosis H37Rv by real-time polymerase chain reaction (PCR) and immunoblotting. The loss of ompATb had no effect on growth under normal conditions, but caused a major reduction in ability to grow at reduced pH. The gene was substantially upregulated in wild-type bacteria exposed to these conditions. The mutant was impaired in its ability to grow in macrophages and in normal mice, although it was as virulent as the wild type in mice that lack T cells. Deletion of the ompATb gene reduced permeability to several small water-soluble substances. This was particularly evident at pH 5.5; at this pH, uptake of serine was minimal, suggesting that, at this pH, OmpATb might be the only functioning porin. These data indicate that OmpATb has two functions: as a pore-forming protein with properties of a porin, and in enabling M. tuberculosis to respond to reduced environmental pH. It is not known whether this second function is related to the porin-like activity at low pH or involves a completely separate role for OmpATB. The involvement with pH is likely to contribute to the ability of M. tuberculosis to overcome host defence mechanisms and grow in a mammalian host.

Slow induction of RecA by DNA damage in Mycobacterium tuberculosis.

In mycobacteria, as in most bacterial species, the expression of RecA is induced by DNA damage. However, the authors show here that the kinetics of recA induction in Mycobacterium smegmatis and in Mycobacterium tuberculosis are quite different: whilst maximum expression in M. smegmatis occurred 3-6 h after addition of a DNA-damaging agent, incubation for 18-36 h was required to reach peak levels in M. tuberculosis. This is despite the fact that the M. tuberculosis promoter can be activated more rapidly when transferred to M. smegmatis. In addition, it is demonstrated that in both species the DNA is sufficiently damaged to give maximum induction within the first hour of incubation with mitomycin C. The difference in the induction kinetics of recA between the two species was mirrored by a difference in the levels of DNA-binding-competent LexA following DNA damage. A decrease in the ability of LexA to bind to the SOS box was readily detected by 2 h in M. smegmatis, whilst a decrease was not apparent until 18-24 h in M. tuberculosis and then only a very small decrease was observed.

Crystal structure of the transcription elongation/anti-termination factor NusA from Mycobacterium tuberculosis at 1.7 A resolution.

Mycobacterium tuberculosis is the cause of tuberculosis in humans, a disease that affects over a one-third of the world's population. This slow-growing pathogen has only one ribosomal RNA operon, thus making its transcriptional apparatus a fundamentally interesting target for drug discovery. NusA binds to RNA polymerase and modulates several of the ribosomal RNA transcriptional processes. Here, we report the crystal structure of NusA, and reveal that the molecule consists of four domains. They are organised as two distinct entities. The N-terminal domain (residues 1 to 99) that resembles the B chain of the Rad50cd ATP binding cassette-ATPase (ABC-ATPase) and a C-terminal module (residues 108 to 329) consisting of a ribosomal S1 protein domain followed by two K homology domains. The S1 and KH domains are tightly integrated together to form an extensive RNA-binding structure, but are flexibly tethered to the N-terminal domain. The molecule's surfaces and architecture provide insights into RNA and polymerase interactions and the mechanism of pause site discrimination. They also allow us to rationalize certain termination-defective and cold shock-sensitive mutations in the nusA gene that have been studied in Escherichia coli.

Silencing of oxidative stress response in Mycobacterium tuberculosis: expression patterns of ahpC in virulent and avirulent strains and effect of ahpC inactivation.

Intracellular pathogens such as Mycobacterium tuberculosis are able to survive in the face of antimicrobial products generated by the host cell in response to infection. The product of the alkyl hydroperoxide reductase gene (ahpC) of M. tuberculosis is thought to be involved in protecting the organism against both oxidative and nitrosative stress encountered within the infected macrophage. Here we report that, contrary to expectations, ahpC expression in virulent strains of M. tuberculosis and Mycobacterium bovis grown in vitro is repressed, often below the level of detection, whereas expression in the avirulent vaccine strain M. bovis BCG is constitutively high. The repression of the ahpC gene of the virulent strains is independent of the naturally occurring lesions of central regulator oxyR. Using a green fluorescence protein vector (gfp)-ahpC reporter construct we present data showing that repression of ahpC of virulent M. tuberculosis also occurred during growth inside macrophages, whereas derepression in BCG was again seen under identical conditions. Inactivation of ahpC on the chromosome of M. tuberculosis by homologous recombination had no effect on its growth during acute infection in mice and did not affect in vitro sensitivity to H2O2. However, consistent with AhpC function in detoxifying organic peroxides, sensitivity to cumene hydroperoxide exposure was increased in the ahpC::Km(r) mutant strain. The preservation of a functional ahpC gene in M. tuberculosis in spite of its repression under normal growth conditions suggests that, while AhpC does not play a significant role in establishing infection, it is likely to be important under certain, as yet undefined conditions. This is supported by the observation that repression of ahpC expression in vitro was lifted under conditions of static growth.

Crystallization and preliminary X-ray diffraction studies on the N-utilizing substance A (NusA) from Mycobacterium tuberculosis.

N-utilizing substance A (NusA) is a protein which performs several roles as a cofactor of DNA-dependent RNA polymerase. Its acts as an elongation factor and facilitates pausing, termination and the formation of a complex assembly that mediates transcription antitermination in eubacteria. Biochemical and biophysical data in the literature suggest that this protein performs these functions by binding to the core RNA polymerase, other protein factors and certain RNA fragments having specific signal sequences. The NusA of Mycobacterium tuberculosis has been cloned and overexpressed in Escherichia coli and crystallized using the hanging-drop vapour-diffusion method. The space group is P3(1)21, with unit-cell parameters a = b = 78.1, c = 180.3 A. A native data set complete to 1.7 A resolution has been collected from a single crystal.

Mycobacterium bovis BCG recA deletion mutant shows increased susceptibility to DNA-damaging agents but wild-type survival in a mouse infection model.

Pathogenic microorganisms possess antioxidant defense mechanisms for protection from reactive oxygen metabolites which are generated during the respiratory burst of phagocytic cells. These defense mechanisms include enzymes such as catalase, which detoxifies reactive oxygen species, and DNA repair systems, which repair damage resulting from oxidative stress. To (i) determine the relative importance of the DNA repair system when oxidative stress is encountered by the Mycobacterium tuberculosis complex during infection of the host and to (ii) provide improved mycobacterial hosts as live carriers to express foreign antigens, the recA locus was inactivated by allelic exchange in Mycobacterium bovis BCG. The recA mutants are sensitive to DNA-damaging agents and show increased susceptibility to metronidazole, the first lead compound active against the dormant M. tuberculosis complex. Surprisingly, the recA genotype does not affect the in vitro dormancy response, nor does the defect in the DNA repair system lead to attenuation as determined in a mouse infection model. The recA mutants will be a valuable tool for further development of BCG as an antigen delivery system to express foreign antigens and as a source of a genetically stable vaccine against tuberculosis.

Spectroscopic and thermodynamic characterization of the transcription antitermination factor NusE and its interaction with NusB from Mycobacterium tuberculosis.

N-utilizing proteins (Nus) form a complex involved in the regulation of rRNA biosynthesis in enteric bacteria by modulating the efficiency of transcriptional termination [Nodwell, J. R., and Greenblatt, J. (1993) Cell 72, 261-268]. The protein NusE (identical to the protein S10 of the small ribosomal subunit) from the pathogenic mycobacterium M. tuberculosis has been cloned and overexpressed in Escherichia coli. The pure protein has been characterized by circular dichroism, ultracentrifugation, NMR, and binding to NusB. The near-ultraviolet circular dichroism spectrum of this protein suggests that it has a moderate (ca. 12-16%) alpha-helical content at 30 degrees C. The protein undergoes cold denaturation, with a temperature of maximum stability near 40 degrees C, implying a substantial heat capacity difference between the folded and unfolded states. The sedimentation equilibrium and velocity data indicate that the protein is monomeric and expanded in solution. NMR spectroscopy shows that there is no significant tertiary structure, and confirms the low secondary structure content at low temperatures. Furthermore, there was evidence for more structure at 30 degrees C than at 10 degrees C. Well-defined shifts in peaks in the HSQC spectrum of (15)N labeled NusE/NusB when the unlabeled counterpart was added at approximately stoichiometric concentrations showed the formation of a NusE-NusB complex in the absence of RNA. The far-UV CD and ultracentrifuge experiments, however, indicated relatively weak binding. Isothermal titration calorimetry showed the binding was weak and endothermic at 15 degrees C, with a total DeltaH of > or =10 kcal/mol. This weak binding is consistent with a small interaction interface and lack of large conformational rearrangements in the predominantly unfolded NusE protein. The conformational flexibility of NusE may be important for its roles in both the ribosome and antitermination complexes.

A new single-copy mycobacterial plasmid, pMF1, from Mycobacterium fortuitum which is compatible with the pAL5000 replicon.

A 9.2 kb cryptic Mycobacterium fortuitum plasmid, pMF1, was isolated from strain 110 and its restriction map constructed. A 4.2 kb HindIII fragment of pMF1 was found to support replication in mycobacteria and this fragment was cloned and sequenced to characterize the replication elements of the plasmid. Computer analysis identified a putative Rep protein (362 amino acids) with high homology to the putative Rep protein of the Mycobacterium celatum plasmid pCLP and limited homology, mostly in the N-terminal region, to the Rep proteins of Mycobacterium avium pLR7, M. fortuitum pJAZ38 and Mycobacterium scrofulaceum pMSC262. A region containing a putative ori site was located upstream of the rep gene; this region displayed high homology at the nucleotide level with the predicted ori of pCLP and pJAZ38. A plasmid carrying the 4.2 kb HindIII fragment and a kanamycin resistance marker, designated pBP4, was maintained as a single-copy plasmid in Mycobacterium smegmatis and was stably inherited in the absence of antibiotic selection. Plasmid pBP4 was incompatible with the pJAZ38 replicon but was compatible with the widely used pAL5000 replicon, indicating that among the mycobacterial vectors now available there are two incompatibility groups. Significantly, the plasmid was able to replicate in the pathogen Mycobacterium tuberculosis, making it a useful tool for gene expression studies. To provide a choice of restriction sites and easy manipulation, a 2.1 kb fragment containing the minimal replication region was cloned to make the mycobacterial shuttle vector pBP10, which showed similar stability to pBP4.

Construction and complementation of a recA deletion mutant of Mycobacterium smegmatis reveals that the intein in Mycobacterium tuberculosis recA does not affect RecA function.

A recA deletion mutant of Mycobacterium smegmatis has been isolated by homologous recombination using a sacB counterselection strategy. Deletion of the recA gene from the chromosome was demonstrated by Southern hybridizations and by polymerase chain reaction (PCR). Western analysis using anti-RecA antibodies confirmed that the RecA protein was not made by the mutant strain. The recA deletion strain exhibited enhanced sensitivity to UV irradiation and failed to undergo homologous recombination. The results obtained from the recombination assays suggest that in wild-type M. smegmatis the majority of colonies arise from single cross-over homologous recombination events with only a very minor contribution from random integrations. The deficiencies in UV survival and recombination were complemented by introduction of the cloned M. smegmatis recA gene. Overexpression of RecA was found to be toxic in the absence of recX, which is found downstream of and co-transcribed with recA and is thus also affected by the deletion of recA. The M. smegmatis recA deletion strain was also complemented by the M. tuberculosis recA gene with or without its intein; most importantly, the frequency of double cross-over homologous recombination events was identical regardless of whether the M. tuberculosis recA gene contained or lacked the intein. Thus, the low frequency of homologous recombination observed in M. tuberculosis is not due to the presence of an intein-coding sequence in its recA gene per se.

Expression of a gene for a porin-like protein of the OmpA family from Mycobacterium tuberculosis H37Rv.

An open reading frame in the genomic database of Mycobacterium tuberculosis H37Rv was identified as having homology with an outer membrane protein. We found that the gene specified a protein belonging to the OmpA family, which includes some porins of gram-negative organisms. The gene was amplified by PCR and cloned into Escherichia coli. Overexpression of the gene was toxic to the host, but limited amounts could be purified from cells before growth ceased. A truncated gene devoid of the code for a presumed signal sequence was well expressed, but the protein had no pore-forming activity in the liposome swelling assay. However, the intact protein, OmpATb, behaved as a porin of low specific activity, with a pore diameter of 1.4 to 1.8 nm, and was also active in planar lipid bilayers, showing a single-channel conductance of 700 pS. The protein had a molecular mass of about 38 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polyclonal rabbit antiserum raised to the truncated protein recognized a protein of similar molecular mass in detergent extracts of broken M. tuberculosis cells. Reverse transcription-PCR confirmed that the gene for OmpATb was expressed in M. tuberculosis cells growing in culture. Comparison of the purified protein with that in the detergent-extracted preparation using liposomes and planar lipid bilayers showed that the two materials had similar pore-forming properties. OmpATb is different from either of the mycobacterial porins described so far. This is the first report of a porin-like molecule from M. tuberculosis; the porin is likely to be important in controlling the access of hydrophilic molecules to the bacterial cell.

Mycobacterial recA is cotranscribed with a potential regulatory gene called recX.

The recA gene of Mycobacterium smegmatis has been cloned and sequenced. The amino acid sequence of the RecA protein is highly homologous to other RecA proteins. Three other potential open reading frames were identified. One of these showed extensive homology to a protein, HypB, involved in the incorporation of nickel into hydrogenases. Another, found downstream of and overlapping recA, was similar to a gene, recX, which has been proposed to play a regulatory role related to recA function. The homology between the M. smegmatis sequence and that of Mycobacterium tuberculosis extended upstream of the recA coding region for 140 bp including a motif identical to the Cheo-box consensus sequence which has been shown to bind LexA. In addition, the transcriptional start sites were found to be identical to those identified previously for M. tuberculosis. Transcriptional fusions to the reporter gene chloramphenicol acetyltransferase (CAT) revealed that recA was DNA-damage inducible and that expression required sequences at some distance from the mapped transcriptional start sites. Although a motif with only one mismatch to the Cheo box was found in the intergenic region between orf1 and orf2 these open reading frames were not DNA-damage inducible, nor was this motif required for regulation of recA expression. Gel retardation assays revealed that the reason for this was that LexA did not bind to this sequence containing a mismatch. Reverse transcription/polymerase chain reaction analysis of M. smegmatis RNA demonstrated that recA and orf3 (recX) are within the same transcriptional unit.

Pisatin resistance in Dictyostelium discoideum and Neurospora crassa: comparison of mutant phenotypes.

The pea phytoalexin pisatin, at its inhibitory concentration, was shown to have two distinct inhibitory effects on amoebae of the cellular slime mould Dictyostelium discoideum. One effect was cytolytic and was demonstrable even in non-growing cells whereas the second effect was observed only under conditions favourable to growth. Pretreatment with a sublethal concentration of pisatin induced the amoebae to acquire resistance to both these effects. Mutations in nysC that alter membrane sterols and confer resistance to the polyene antibiotics nystatin and pimaricin blocked resistance to the growth-associated inhibitory effect but did not affect acquisition of resistance to the cytolytic effect. The nysB sunD double mutant HK412 displayed a partially constitutive resistance to the cytolytic effect but, like the nysC mutants, was blocked in the acquisition of resistance to the growth-associated inhibitory effect. Pistatin-treated cells incubated in pisatin-free medium lost their ability to grow on pisatin-containing medium much more rapidly than they lost resistance to the cytolytic effect of pisatin. These results suggest that the induction of pisatin resistance may involve the turning-on of independent resistance mechanisms against the two inhibitory effects of pisatin. This could account for our inability to isolate pisatin-resistant mutants in a single step. The Neurospora crassa erg1 and erg3 mutants that have altered membrane sterols and are nystatin resistant displayed sensitivity to pisatin. The pisatin-sensitivity phenotype of the erg mutants was used in selections to identify complementing plasmids from an ordered Neurospora genomic library.(ABSTRACT TRUNCATED AT 250 WORDS)

An inducible, nondegradative phytoalexin resistance mechanism in Dictyostelium discoideum is suppressed by mutations that alter membrane sterol composition.

Pretreatment of Dictyostelium discoideum amoebae with a sublethal concentration of the pea phytoalexin pisatin was shown to induce nondegradative resistance to subsequent challenges with inhibitory concentrations. An alteration of membrane sterol composition either with the azasterol A25822B or by mutations in nysC that confer resistance to the polyene antibiotic nystatin suppressed the induction of pisatin resistance. Wild-type cells grown on pisatin medium acquired resistance to nystatin; however, after transfer to nystatin medium, they lost their pisatin resistance phenotype but remained nystatin resistant. To account for this asymmetry in the induction and maintenance of cross-resistance after growth on pisatin and nystatin media, we propose a model in which the two resistance phenotypes are governed by distinct mechanisms. This model presumes that growth on pisatin induces membrane alterations that predispose cells to acquire nystatin resistance but that the pisatin-induced membrane alterations are not maintained in the absence of pisatin.