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.     

Characterization of protein acyltransferase function of recombinant purified GlnA1 from Mycobacterium tuberculosis: a moon lighting property

The protein acetyltransferase (MTAase) function of glutamine synthetase of Mycobacterium smegmatis was established earlier. In this paper, studies were undertaken to examine MTAase function of recombinant glutamine synthetase (rGlnA1) of Mycobacterium tuberculosis, which showed >80% similarity with M. smegmatis GlnA. The specificity of MTAase to several acyl derivative of coumarins was examined. The results clearly indicated that MTAase exhibited differential specificities to several acyloxycoumarins. Further, MTAase was also found capable of transferring propionyl and butyryl groups from propoxy and butoxy derivatives of 4-methylcoumarin. These observations characterized MTAase in general as a protein acyltransferase. MTAase catalyzed acetylation of GST by 7,8-diacetoxy-4-methylcoumarin (DAMC), a model acetoxy coumarin was confirmed by MALDI-TOF-MS as well as western blot analysis using acetylated lysine polyclonal antibody. In order to validate the active site of rGlnA1 for TAase activity, effect of DAMC and L-methionine-S-sulfoximine (MSO) on GS and TAase activity of rGlnA1 were studied. The results indicated that the active sites of GS and TAase were found different. Acetyl CoA, a universal biological acetyl group donor, was also found to be a substrate for MTAase. These results appropriately characterize glutamine synthetase of Mtb exhibiting transacylase action as a moonlighting protein.     

Biochemical and molecular characterization of the biosynthesis of glutamine and glutamate, two major compatible solutes in the moderately halophilic bacterium Halobacillus halophilus

The moderately halophilic, chloride-dependent bacterium Halobacillus halophilus produces glutamate and glutamine as main compatible solutes at external salinities of 1.0 to 1.5 M NaCl. The routes for the biosynthesis of these solutes and their regulation were examined. The genome contains two genes potentially encoding glutamate dehydrogenases and two genes for the small subunit of a glutamate synthase, but only one gene for the large subunit. However, the expression of these genes was not salt dependent, nor were the corresponding enzymatic activities detectable in cell extracts of cells grown at different salinities. In contrast, glutamine synthetase activity was readily detectable in H. halophilus. Induction of glutamine synthetase activity was strictly salt dependent and reached a maximum at 3.0 M NaCl; chloride stimulated the production of active enzyme by about 300%. Two potential genes encoding a glutamine synthetase, glnA1 and glnA2, were identified. The expression of glnA2 but not of glnA1 was increased up to fourfold in cells adapted to high salt, indicating that GlnA2 is the glutamine synthetase involved in the synthesis of the solutes glutamate and glutamine. Furthermore, expression of glnA2 was stimulated twofold by the presence of chloride ions. Chloride exerted an even more pronounced effect on the enzymatic activity of preformed enzyme: in the absence of chloride in the assay buffer, glutamine synthetase activity was decreased by as much as 90%. These data demonstrate for the first time a regulatory role of a component of common salt, chloride, in the biosynthesis of compatible solutes.     

Growth of mycobacteria on carbon monoxide and methanol

Several mycobacterial strains, such as Mycobacterium flavescens, Mycobacterium gastri, Mycobacterium neoaurum, Mycobacterium parafortuitum, Mycobacterium peregrinum, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium tuberculosis, and Mycobacterium vaccae, were found to grow on carbon monoxide (CO) as the sole source of carbon and energy. These bacteria, except for M. tuberculosis, also utilized methanol as the sole carbon and energy source. A CO dehydrogenase (CO-DH) assay, staining by activity of CO-DH, and Western blot analysis using an antibody raised against CO-DH of Mycobacterium sp. strain JC1 (formerly Acinetobacter sp. strain JC1 [J. W. Cho, H. S. Yim, and Y. M. Kim, Kor. J. Microbiol. 23:1-8, 1985]) revealed that CO-DH is present in extracts of the bacteria prepared from cells grown on CO. Ribulose bisphosphate carboxylase/oxygenase (RubisCO) activity was also detected in extracts prepared from all cells, except M. tuberculosis, grown on CO. The mycobacteria grown on methanol, except for M. gastri, which showed hexulose phosphate synthase activity, did not exhibit activities of classic methanol dehydrogenase, hydroxypyruvate reductase, or hexulose phosphate synthase but exhibited N,N-dimethyl-4-nitrosoaniline-dependent methanol dehydrogenase and RuBisCO activities. Cells grown on methanol were also found to have dihydroxyacetone synthase. Double immunodiffusion revealed that the antigenic sites of CO-DHs, RuBisCOs, and dihydroxyacetone synthases in all mycobacteria tested are identical with those of the Mycobacterium sp. strain JC1 enzymes.     

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.     

Functional complementation of the essential gene fabG1 of Mycobacterium tuberculosis by Mycobacterium smegmatis fabG but not Escherichia coli fabG

Mycolic acids are a key component of the mycobacterial cell wall, providing structure and forming a major permeability barrier. In Mycobacterium tuberculosis mycolic acids are synthesized by type I and type II fatty acid synthases. One of the enzymes of the type II system is encoded by fabG1. We demonstrate here that this gene can be deleted from the M. tuberculosis chromosome only when another functional copy is provided elsewhere, showing that under normal culture conditions fabG1 is essential. FabG1 activity can be replaced by the corresponding enzyme from the closely related species Mycobacterium smegmatis but not by the enzyme from Escherichia coli. M. tuberculosis carrying FabG from M. smegmatis showed no phenotypic changes, and both the mycolic acids and cell wall permeability were unchanged. Thus, M. tuberculosis and M. smegmatis enzymes are interchangeable and do not control the lengths and types of mycolic acids synthesized.     

Pyruvate carboxylase from Mycobacterium smegmatis: stabilization, rapid purification, molecular and biochemical characterization and regulation of the cellular level

This is the first report on the purification and characterization of an anaplerotic enzyme from a Mycobacterium. The anaplerotic reactions play important roles in the biochemical differentiation of mycobacteria into non-replicating stages. We have purified and characterized a pyruvate carboxylase (PYC) from Mycobacterium smegmatis and cloned and sequenced its gene. We have developed a very rapid and efficient purification protocol that provided PYC with very high specific activities (up to 150 U/mg) that remained essentially unchanged over a month. The enzyme was found to be a homomultimer of 121 kDa subunits, mildly thermophilic, absolutely dependent on acyl-CoAs for activity and inhibited by ADP, by excess Mg(2+), Co(2+), and Mn(2+), by aspartate, but not by glutamate and alpha-ketoglutarate. Supplementation of minimal growth medium with aspartate did not lower the cellular PYC level, rather doubled it; with glutamate the level remained unchanged. These observations would not fit the idea that the M. smegmatis enzyme fulfills a straightforward anaplerotic function; in a closely related organism, Corynebacterium glutamicum, PYC is the major anaplerotic enzyme. Growth on glucose provided 2-fold higher cellular PYC level than that observed with glycerol. The PYCs of M. smegmatis and Mycobacterium tuberculosis were highly homologous to each other. In M. smegmatis, M. tuberculosis and M. lepra, pyc was flanked by a putative methylase and a putative integral membrane protein genes in an identical operon-like arrangement. Thus, M. smegmatis could serve as a model for studying PYC-related physiological aspects of mycobacteria. Also, the ease of purification and the extraordinary stability could make the M. smegmatis enzyme a model for studying the structure-function relationships of PYCs in general. It should be noted that no crystal structure is available for this enzyme of paramount importance in all three domains of life, archaea, bacteria, and eukarya.     

Large Mg(2+)-dependent currents are associated with the increased expression of ALR1 in Saccharomyces cerevisiae

Mycobacteria adapt to a decrease in oxygen tension by entry into a non-replicative persistent phase. It was shown earlier that the two-component system, DevR-DevS, was induced in Mycobacterium tuberculosis and Mycobacterium bovis BCG cultures during hypoxia, suggesting that it may play a regulatory role in their adaptation to oxygen limitation. The presence of a homologous genetic system in Mycobacterium smegmatis was predicted by scanning its unfinished genome sequence with devR and devS genes of M. tuberculosis. Rv3134c, which is cotranscribed with devR-devS in M. tuberculosis, was also present in M. smegmatis at a similar location upstream from devR. The expression of all three genes was induced at the RNA and protein levels in M. smegmatis cultures grown under microaerobic and anaerobic conditions. The M. smegmatis genome also contained the hspX gene, encoding chaperone alpha-crystallin, Acr, that was induced during hypoxia. The similarity in sequences and hypoxia-responsive behaviour of devR-devS, Rv3134c and hspX genes in M. smegmatis and M. tuberculosis suggests that the molecular mechanisms involved in the dormancy response are likely conserved in these two species. M. smegmatis could therefore serve as a useful model for the delineation of the hypoxia response in general and DevR-DevS regulated pathways in particular.     

A genomic view of sugar transport in Mycobacterium smegmatis and Mycobacterium tuberculosis

We present a comprehensive analysis of carbohydrate uptake systems of the soil bacterium Mycobacterium smegmatis and the human pathogen Mycobacterium tuberculosis. Our results show that M. smegmatis has 28 putative carbohydrate transporters. The majority of sugar transport systems (19/28) in M. smegmatis belong to the ATP-binding cassette (ABC) transporter family. In contrast to previous reports, we identified genes encoding all components of the phosphotransferase system (PTS), including permeases for fructose, glucose, and dihydroxyacetone, in M. smegmatis. It is anticipated that the PTS of M. smegmatis plays an important role in the global control of carbon metabolism similar to those of other bacteria. M. smegmatis further possesses one putative glycerol facilitator of the major intrinsic protein family, four sugar permeases of the major facilitator superfamily, one of which was assigned as a glucose transporter, and one galactose permease of the sodium solute superfamily. Our predictions were validated by gene expression, growth, and sugar transport analyses. Strikingly, we detected only five sugar permeases in the slow-growing species M. tuberculosis, two of which occur in M. smegmatis. Genes for a PTS are missing in M. tuberculosis. Our analysis thus brings the diversity of carbohydrate uptake systems of fast- and a slow-growing mycobacteria to light, which reflects the lifestyles of M. smegmatis and M. tuberculosis in their natural habitats, the soil and the human body, respectively.     

Mycobacterium tuberculosis mammalian cell entry operon (mce) homologs in Mycobacterium other than tuberculosis (MOTT)

The cloned mammalian cell entry gene mce1a from Mycobacterium tuberculosis confers to non-pathogenic Escherichia coli the ability to invade and survive inside macrophages and HeLa cells. The aim of this work was to search for and characterize homologs of the four M. tuberculosis mammalian cell entry operons (mce1, mce2, mce3 and mce4) in mycobacteria other than tuberculosis (MOTT). The dot-blot and polymerase chain reaction (PCR) experiments performed on 24 clinical isolates representing 20 different mycobacterial species indicated that the mce operons were widely distributed throughout the genus Mycobacterium. BLAST search results showed the presence of mce1, mce2 and mce4 homologs in Mycobacterium bovis, Mycobacterium avium and Mycobacterium smegmatis. A homologous region for the mce3 operon was also found in M. avium and M. smegmatis. DNA and protein alignments were done to compare the M. tuberculosis mce operons and the deduced M. bovis, M. avium, and M. smegmatis homologs. The deduced proteins of M. bovis mce1, mce2 and mce4 operons had 99.6-100% homology with the respective M. tuberculosis mce proteins (MTmce). The similarity between M. avium mce proteins and the individual M. tuberculosis homologs ranged from 56.2 to 85.5%. The alignment results between M. smegmatis mce proteins and the respective MTmce proteins ranged from 58.5% to 68.5%. Primer sets were designed from the M. tuberculosis mce4a gene for amplification of 379-bp fragments. Amplification was successful in 14 strains representing 11 different mycobacterial species. The PCR fragments were sequenced from 10 strains representing eight species. Alignment of the sequenced PCR products showed that mce4a homologs are highly conserved in the genus Mycobacterium. In conclusions, the four mce operons in different mycobacterial species are generally organized in the same manner. The phylogenetic tree comparing the different mce operons showed that the mce1 operon was closely related to the mce2 operon and mce3 diverged from the other operons. The wide distribution of the mce operons in pathogenic and non-pathogenic mycobacteria implicates that the presence of these putative virulence genes is not an indicator for the pathogenicity of the bacilli. Instead, the pathogenicity of these factors might be determined by their expression.     

Isolation, characterization, and complementation of Rhizobium meliloti 104A14 mutants that lack glutamine synthetase II activity.

The glutamine synthetase (GS)-glutamate synthase pathway is the primary route used by members of the family Rhizobiaceae to assimilate ammonia. Two forms of glutamine synthetase, GSI and GSII, are found in Rhizobium and Bradyrhizobium species. These are encoded by the glnA and glnII genes, respectively. Starting with a Rhizobium meliloti glnA mutant as the parent strain, we isolated mutants unable to grow on minimal medium with ammonia as the sole nitrogen source. For two auxotrophs that lacked any detectable GS activity, R. meliloti DNA of the mutated region was cloned and partially characterized. Lack of cross-hybridization indicated that the cloned regions were not closely linked to each other or to glnA; they therefore contain two independent genes needed for GSII synthesis or activity. One of the cloned regions was identified as glnII. An R. meliloti glnII mutant and an R. meliloti glnA glnII double mutant were constructed. Both formed effective nodules on alfalfa. This is unlike the B. japonicum-soybean symbiosis, in which at least one of these GS enzymes must be present for nitrogen-fixing nodules to develop. However, the R. meliloti double mutant was not a strict glutamine auxotroph, since it could grow on media that contained glutamate and ammonia, an observation that suggests that a third GS may be active in this species.     

Mapping essential domains of Mycobacterium smegmatis WhmD: insights into WhiB structure and function

A growing body of evidence suggests that the WhiB-like proteins exclusive to the GC-rich actinomycete genera play significant roles in pathogenesis and cell division. Each of these proteins contains four invariant cysteine residues and a conserved helix-turn-helix motif. whmD, the Mycobacterium smegmatis homologue of Streptomyces coelicolor whiB, is essential in M. smegmatis, and the conditionally complemented mutant M. smegmatis 628-53 undergoes filamentation under nonpermissive conditions. To identify residues critical to WhmD function, we developed a cotransformation-based assay to screen for alleles that complement the filamentation phenotype of M. smegmatis 628-53 following inducer withdrawal. Mycobacterium tuberculosis whiB2 and S. coelicolor whiB complemented the defect in M. smegmatis 628-53, indicating that these genes are true functional orthologues of whmD. Deletion analysis suggested that the N-terminal 67 and C-terminal 12 amino acid residues are dispensable for activity. Site-directed mutagenesis indicated that three of the four conserved cysteine residues (C90, C93, and C99) and a conserved aspartate (D71) are essential. Mutations in a predicted loop glycine (G111) and an unstructured leucine (L116) were poorly tolerated. The region essential for WhmD activity encompasses 6 of the 10 residues conserved in all seven M. tuberculosis WhiBs, as well as in most members of the WhiB family identified thus far. WhmD structure was found to be sensitive to the presence of a reducing agent, suggesting that the cysteine residues are involved in coordinating a metal ion. Iron-specific staining strongly suggested that WhmD contains a bound iron atom. With this information, we have now begun to comprehend the functional significance of the conserved sequence and structural elements in this novel family of proteins.     

Investigation of the functional properties and regulation of three glutamine synthetase-like genes in Streptomyces coelicolor A3(2)

Streptomyces coelicolor A3(2) has three additional glnA-type genes besides the glutamine synthetase genes glnA (encoding GSI) and glnII (encoding GSII). The aim of this work was to characterize their functional properties and regulation. Sequence analyses revealed that GlnA2, GlnA3, and GlnA4 are dissimilar to S. coelicolor GSI and lack highly conserved amino acid residues involved in catalysis. In heterologous expression experiments, glnA2, glnA3, and glnA4, in contrast to glnA and glnII, were not capable of complementing the l-glutamine auxotrophy of an Escherichia coli glnA mutant. The lack of a conserved sequence motif reflecting adenylylation control of enzyme activity suggests that GlnA2, GlnA3, and GlnA4 are not regulated via adenylyltransferase-mediated modification. In DNA-binding assays, the OmpR-like regulator of nitrogen metabolism GlnRII, which interacts with the glnA and glnII promoters, did not bind to the upstream regions of glnA2, glnA3, and glnA4. These findings support the conclusion that glnA2, glnA3, and glnA4 are not directly involved in l-glutamine synthesis and nitrogen assimilation and are not subject to nitrogen control in S. coelicolor. The glnA3 gene product is similar to FluG, which is required for asexual sporulation in Aspergillus nidulans. However, inactivation of glnA3 does not block morphological differentiation in S. coelicolor.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

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.     

A fragment of 21 ORFs around the direct repeat (DR) region of Mycobacterium tuberculosis is absent from the other sequenced mycobacterial genomes: implications for the evolution of the DR region

The direct repeat (DR) region is a singular locus of the Mycobacterium tuberculosis complex genome. This region consists of 36 bp repetitive sequences separated by non-repetitive unique spacer sequences. Around this region there are several genes coding for proteins of unknown function. To determine whether the M. smegmatis, M. avium, M. marinum and M. leprae genomes contain sequences and ORFs similar to those of the DR locus of the M. tuberculosis complex, we analysed the corresponding regions in these species. As a first step, some conserved genes that flank the DR genes [Rv2785c (rpsO), Rv2786c (ribF), Rv2790c (ltp1 ), Rv2793c (truB), Rv2800, Rv2825, Rv2828, Rv2831 (echA16 ), Rv2838 (rbfA) and Rv2845 (proS )] were used as markers to locate the corresponding orthologues in M. smegmatis, M. avium, M. marinum and M. leprae in silico. Most of these M. tuberculosis marker genes have highly similar orthologues located in the same order and orientation in the other mycobacteria. In contrast, no orthologues were found for ORFs Rv2801-Rv2824, suggesting that these genes are unique to M. tuberculosis within the genus Mycobacterium.We observed that in M. smegmatis and M. avium, Rv2800 and Rv2825 are adjacent. This observation was experimentally confirmed by PCR. In conclusion, as the DR locus and the ORFs around it are absent in M. smegmatis and M. avium and, as it is possible that these species are older than M. tuberculosis, we postulated that the DR locus was acquired by the M. tuberculosis complex species or by an ancestor bacterium.