Reactive Chlorine Species Reversibly Inhibit DnaB Protein Splicing in Mycobacteria.

Intervening proteins, or inteins, are mobile genetic elements that are translated within host polypeptides and removed at the protein level by splicing. In protein splicing, a self-mediated reaction removes the intein, leaving a peptide bond in place. While protein splicing can proceed in the absence of external cofactors, several examples of conditional protein splicing (CPS) have emerged. In CPS, the rate and accuracy of splicing are highly dependent on environmental conditions. Because the activity of the intein-containing host protein is compromised prior to splicing and inteins are highly abundant in the microbial world, CPS represents an emerging form of posttranslational regulation that is potentially widespread in microbes. Reactive chlorine species (RCS) are highly potent oxidants encountered by bacteria in a variety of natural environments, including within cells of the mammalian innate immune system. Here, we demonstrate that two naturally occurring RCS, namely, hypochlorous acid (the active compound in bleach) and N-chlorotaurine, can reversibly block splicing of DnaB inteins from Mycobacterium leprae and Mycobacterium smegmatis in vitro. Further, using a reporter that monitors DnaB intein activity within M. smegmatis, we show that DnaB protein splicing is inhibited by RCS in the native host. DnaB, an essential replicative helicase, is the most common intein-housing protein in bacteria. These results add to the growing list of environmental conditions that are relevant to the survival of the intein-containing host and influence protein splicing, as well as suggesting a novel mycobacterial response to RCS. We propose a model in which DnaB splicing, and therefore replication, is paused when these mycobacteria encounter RCS. IMPORTANCE Inteins are both widespread and abundant in microbes, including within several bacterial and fungal pathogens. Inteins are domains translated within host proteins and removed at the protein level by splicing. Traditionally considered molecular parasites, some inteins have emerged in recent years as adaptive posttranslational regulatory elements. Several studies have demonstrated CPS, in which the rate and accuracy of protein splicing, and thus host protein functions, are responsive to environmental conditions relevant to the intein-containing organism. In this work, we demonstrate that two naturally occurring RCS, including the active compound in household bleach, reversibly inhibit protein splicing of Mycobacterium leprae and Mycobacterium smegmatis DnaB inteins. In addition to describing a new physiologically relevant condition that can temporarily inhibit protein splicing, this study suggests a novel stress response in Mycobacterium, a bacterial genus of tremendous importance to humans.

Features of the biochemistry of Mycobacterium smegmatis, as a possible model for Mycobacterium tuberculosis.

An alternate host for mycobacteria is Mycobacterium smegmatis which is used frequently. It is a directly budding eco-friendly organism not emulated as human infection. It is mainly useful for the investigation of various microorganisms in the sort of Mycobacteria in cell culture laboratories. Some Mycobacterium species groups that is normal, unsafe ailments, likely to Mycobacterium leprae, Mycobacterium tuberculosis and Mycobacterium bovis. At present, various laboratories are clean and culture this type of species to make an opinion that fascinating route of harmful Mycobacteria. This publication provides aggregate data on cell shape, genome studies, ecology, pathology and utilization of M. smegmatis.

Evaluation of the intracellular accumulation of fluoroquinolones in mycobacteria by fluorometric assays.

Background: Fluoroquinolones (FQs) are being used as second-line agents in the treatment of tuberculosis caused by multidrug-resistant strains. Ofloxacin (OFX) is being tried as a part of modified multidrug therapy regimens for leprosy. A preliminary study was carried out to evaluate the accumulation of FQs - OFX, levofloxacin (LFX), norfloxacin (NFX), and ciprofloxacin (CIF) in Mycobacterium smegmatis. Methods: M. smegmatis were grown in Sauton's medium till log phase, harvested and resuspended in phosphate buffer (0.1 M, pH 7.2, Optical Density (OD) of 0.4-0.5) The suspensions were incubated with OFX, LFX, NFX, and CIF (10 µg/ml) at 37°C. The drugs were estimated in the supernatants using spectrofluorimeteric methods. The experiments were also conducted with the addition of carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a proton motive force inhibitor, at 100 µM, 10 min before and/or immediately after the addition of the drugs. Results: The time taken to achieve a Steady State Concentration (SSC) of OFX in M. smegmatis was 3 min and the level of accumulation was 102 ng/mg dry weight of the bacilli; with LFX the time for SSC was 5 min and the level of accumulation was 90 ng/mg; in case of NFX the accumulation to SSC was 87 ng/mg in 3 min. CIF accumulation attained a steady state (SSC level of 79 ng/mg) in 4 min. The accumulation kinetics for NFX in M. smegmatis using the spectrofluorimetric method is comparable with radioactive assays. Dose-related accumulation was observed with 10 µg/ml exposure concentrations. The addition of CCCP failed to influence the accumulation of each of these quinolones. Conclusion: The findings of dose-related accumulation of OFX, LFX NFX, and CIF suggest simple diffusion as the possible mechanism of transport of these drugs.

Impact of the epoxide hydrolase EphD on the metabolism of mycolic acids in mycobacteria.

Mycolic acids are the hallmark of the cell envelope in mycobacteria, which include the important human pathogens Mycobacterium tuberculosis and Mycobacterium leprae Mycolic acids are very long C60-C90 α-alkyl ß-hydroxy fatty acids having a variety of functional groups on their hydrocarbon chain that define several mycolate types. Mycobacteria also produce an unusually large number of putative epoxide hydrolases, but the physiological functions of these enzymes are still unclear. Here, we report that the mycobacterial epoxide hydrolase EphD is involved in mycolic acid metabolism. We found that orthologs of EphD from M. tuberculosis and M. smegmatis are functional epoxide hydrolases, cleaving a lipophilic substrate, 9,10-cis-epoxystearic acid, in vitro and forming a vicinal diol. The results of EphD overproduction in M. smegmatis and M. bovis BCG Δhma strains producing epoxymycolic acids indicated that EphD is involved in the metabolism of these forms of mycolates in both fast- and slow-growing mycobacteria. Moreover, using MALDI-TOF-MS and 1H NMR spectroscopy of mycolic acids and lipids isolated from EphD-overproducing M. smegmatis, we identified new oxygenated mycolic acid species that accumulated during epoxymycolate depletion. Disruption of the ephD gene in M. tuberculosis specifically impaired the synthesis of ketomycolates and caused accumulation of their precursor, hydroxymycolate, indicating either direct or indirect involvement of EphD in ketomycolate biosynthesis. Our results clearly indicate that EphD plays a role in metabolism of oxygenated mycolic acids in mycobacteria.

Mycobacterial DNA-binding protein 1 is critical for long term survival of Mycobacterium smegmatis and simultaneously coordinates cellular functions.

Bacteria can proliferate perpetually without ageing, but they also face conditions where they must persist. Mycobacteria can survive for a long period. This state appears during mycobacterial diseases such as tuberculosis and leprosy, which are chronic and develop after long-term persistent infections. However, the fundamental mechanisms of the long-term living of mycobacteria are unknown. Every Mycobacterium species expresses Mycobacterial DNA-binding protein 1 (MDP1), a histone-like nucleoid associated protein. Mycobacterium smegmatis is a saprophytic fast grower and used as a model of mycobacterial persistence, since it shares the characteristics of the long-term survival observed in pathogenic mycobacteria. Here we show that MDP1-deficient M. smegmatis dies more rapidly than the parental strain after entering stationary phase. Proteomic analyses revealed 21 upregulated proteins with more than 3-fold in MDP1-deficient strain, including DnaA, a replication initiator, NDH, a NADH dehydrogenase that catalyzes downhill electron transfer, Fas1, a critical fatty acid synthase, and antioxidants such as AhpC and KatG. Biochemical analyses showed elevated levels of DNA and ATP syntheses, a decreased NADH/NAD+ ratio, and a loss of resistance to oxidative stress in the MDP1-knockout strain. This study suggests the importance of MDP1-dependent simultaneous control of the cellular functions in the long-term survival of mycobacteria.

Division of labor among Mycobacterium smegmatis RNase H enzymes: RNase H1 activity of RnhA or RnhC is essential for growth whereas RnhB and RnhA guard against killing by hydrogen peroxide in stationary phase.

RNase H enzymes sense the presence of ribonucleotides in the genome and initiate their removal by incising the ribonucleotide-containing strand of an RNA:DNA hybrid. Mycobacterium smegmatis encodes four RNase H enzymes: RnhA, RnhB, RnhC and RnhD. Here, we interrogate the biochemical activity and nucleic acid substrate specificity of RnhA. We report that RnhA (like RnhC characterized previously) is an RNase H1-type magnesium-dependent endonuclease with stringent specificity for RNA:DNA hybrid duplexes. Whereas RnhA does not incise an embedded mono-ribonucleotide, it can efficiently cleave within tracts of four or more ribonucleotides in duplex DNA. We gained genetic insights to the division of labor among mycobacterial RNases H by deleting the rnhA, rnhB, rnhC and rnhD genes, individually and in various combinations. The salient conclusions are that: (i) RNase H1 activity is essential for mycobacterial growth and can be provided by either RnhC or RnhA; (ii) the RNase H2 enzymes RnhB and RnhD are dispensable for growth and (iii) RnhB and RnhA collaborate to protect M. smegmatis against oxidative damage in stationary phase. Our findings highlight RnhC, the sole RNase H1 in pathogenic mycobacteria, as a candidate drug discovery target for tuberculosis and leprosy.

Mycobacterium smegmatis HelY Is an RNA-Activated ATPase/dATPase and 3'-to-5' Helicase That Unwinds 3'-Tailed RNA Duplexes and RNA:DNA Hybrids.

UNLABELLED: Mycobacteria have a large and distinctive ensemble of DNA helicases that function in DNA replication, repair, and recombination. Little is known about the roster of RNA helicases in mycobacteria or their roles in RNA transactions. The 912-amino-acid Mycobacterium smegmatis HelY (MSMEG_3885) protein is a bacterial homolog of the Mtr4 and Ski2 helicases that regulate RNA 3' processing and turnover by the eukaryal exosome. Here we characterize HelY as an RNA-stimulated ATPase/dATPase and an ATP/dATP-dependent 3'-to-5' helicase. HelY requires a 3' single-strand RNA tail (a loading RNA strand) to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The findings that HelY ATPase is unresponsive to a DNA polynucleotide cofactor and that HelY is unable to unwind a 3'-tailed duplex in which the loading strand is DNA distinguish HelY from other mycobacterial nucleoside triphosphatases/helicases characterized previously. The biochemical properties of HelY, which resemble those of Mtr4/Ski2, hint at a role for HelY in mycobacterial RNA catabolism. IMPORTANCE: RNA helicases play crucial roles in transcription, RNA processing, and translation by virtue of their ability to alter RNA secondary structure or remodel RNA-protein interactions. In eukarya, the RNA helicases Mtr4 and Ski2 regulate RNA 3' resection by the exosome. Mycobacterium smegmatis HelY, a bacterial homolog of Mtr4/Ski2, is characterized here as a unidirectional helicase, powered by RNA-dependent ATP/dATP hydrolysis, that tracks 3' to 5' along a loading RNA strand to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The biochemical properties of HelY suggest a role in bacterial RNA transactions. HelY homologs are present in pathogenic mycobacteria (e.g., M. tuberculosis and M. leprae) and are widely prevalent in Actinobacteria and Cyanobacteria but occur sporadically elsewhere in the bacterial domain.

Production of recombinant proteins in Mycobacterium smegmatis for structural and functional studies.

Protein production using recombinant DNA technology has a fundamental impact on our understanding of biology through providing proteins for structural and functional studies. Escherichia coli (E. coli) has been traditionally used as the default expression host to over-express and purify proteins from many different organisms. E. coli does, however, have known shortcomings for obtaining soluble, properly folded proteins suitable for downstream studies. These shortcomings are even more pronounced for the mycobacterial pathogen Mycobacterium tuberculosis, the bacterium that causes tuberculosis, with typically only one third of proteins expressed in E. coli produced as soluble proteins. Mycobacterium smegmatis (M. smegmatis) is a closely related and non-pathogenic species that has been successfully used as an expression host for production of proteins from various mycobacterial species. In this review, we describe the early attempts to produce mycobacterial proteins in alternative expression hosts and then focus on available expression systems in M. smegmatis. The advantages of using M. smegmatis as an expression host, its application in structural biology and some practical aspects of protein production are also discussed. M. smegmatis provides an effective expression platform for enhanced understanding of mycobacterial biology and pathogenesis and for developing novel and better therapeutics and diagnostics.

Noncanonical SMC protein in Mycobacterium smegmatis restricts maintenance of Mycobacterium fortuitum plasmids.

Research on tuberculosis and leprosy was revolutionized by the development of a plasmid transformation system in the fast-growing surrogate, Mycobacterium smegmatis. This transformation system was made possible by the successful isolation of a M. smegmatis mutant strain mc(2)155, whose efficient plasmid transformation (ept) phenotype supported the replication of Mycobacterium fortuitum pAL5000 plasmids. In this report, we identified the EptC gene, the loss of which confers the ept phenotype. EptC shares significant amino acid sequence homology and domain structure with the MukB protein of Escherichia coli, a structural maintenance of chromosomes (SMC) protein. Surprisingly, M. smegmatis has three paralogs of SMC proteins: EptC and MSMEG_0370 both share homology with Gram-negative bacterial MukB; and MSMEG_2423 shares homology with Gram-positive bacterial SMCs, including the single SMC protein predicted for Mycobacterium tuberculosis and Mycobacterium leprae. Purified EptC was shown to bind ssDNA and stabilize negative supercoils in plasmid DNA. Moreover, an EptC-mCherry fusion protein was constructed and shown to bind to DNA in live mycobacteria, and to prevent segregation of plasmid DNA to daughter cells. To our knowledge, this is the first report of impaired plasmid maintenance caused by a SMC homolog, which has been canonically known to assist the segregation of genetic materials.

Dielectrophoresis-based purification of antibiotic-treated bacterial subpopulations.

Persistence of bacteria during antibiotic therapy is a widespread phenomenon, of particular importance in refractory mycobacterial infections such as leprosy and tuberculosis. Persistence is characterized by the phenotypic tolerance of a subpopulation of bacterial cells to antibiotics. Characterization of these "persister" cells is often difficult due to the transient, non-heritable nature of the phenotype and due to the presence of contaminating material from non-persisting cells, which usually comprise the larger fraction. In this study, we use 3D carbon-electrode arrays for dielectrophoresis-based separation of intact cells from damaged cells, revealed by differential staining with propidium iodide, and we use this procedure to purify intact cells from cultures of Mycobacterium smegmatis treated with isoniazid, a frontline anti-tuberculosis drug. The method presented in this study could be used for rapid label-free enrichment of intact persister cells from antibiotic-treated cultures while preserving the metastable persister phenotype. This approach would facilitate the downstream analysis of low-frequency subpopulations of cells using conventional omics techniques, such as transcriptomic and proteomic analysis.

Protective role of Mycobacterium leprae small heat-shock protein in heterologous hosts, Escherichia coli and Mycobacterium smegmatis, grown under stress.

The aim of this study is to examine the in vivo role of a small heat-shock protein (sHsp18) from Mycobacterium leprae in the survival of heterologous recombinant hosts carrying the gene encoding this protein under different environmental conditions that are normally encountered by M. leprae during its infection of the human host. Using an Escherichia coli system where shsp18 expression is controlled by its native promoter, we show that expression of shsp18 is induced under low oxygen tension, nutrient depletion and oxidative stress, all of which reflect the natural internal environment of the granulomas where the pathogen resides for long periods. We demonstrate the in vivo chaperone activity of sHsp18 through its ability to confer survival advantage to recombinant E. coli at heat-shock temperatures. Additional evidence for the protective role of sHsp18 was obtained when Mycobacterium smegmatis harbouring a copy of shsp18 was found to multiply better in human macrophages. Furthermore, the autokinase activity of sHsp18 protein demonstrated for what is believed to be the first time in this study implies that some of the functions of sHsp18 might be controlled by the phosphorylation state of this protein. Results from this study suggest that shsp18 might be one of the factors that facilitate the survival and persistence of M. leprae under stress and autophosphorylation of sHsp18 protein could be a mechanism used by this protein to sense changes in the external environment.

Characterization of the MSMEG_2631 gene (mmp) encoding a multidrug and toxic compound extrusion (MATE) family protein in Mycobacterium smegmatis and exploration of its polyspecific nature using biolog phenotype microarray.

In Mycobacterium, multidrug efflux pumps can be associated with intrinsic drug resistance. Comparison of putative mycobacterial transport genes revealed a single annotated open reading frame (ORF) for a multidrug and toxic compound extrusion (MATE) family efflux pump in all sequenced mycobacteria except Mycobacterium leprae. Since MATE efflux pumps function as multidrug efflux pumps by conferring resistance to structurally diverse antibiotics and DNA-damaging chemicals, we studied this gene (MSMEG_2631) in M. smegmatis mc(2)155 and determined that it encodes a MATE efflux system that contributes to intrinsic resistance of Mycobacterium. We propose that the MSMEG_2631 gene be named mmp, for mycobacterial MATE protein. Biolog Phenotype MicroArray data indicated that mmp deletion increased susceptibility for phleomycin, bleomycin, capreomycin, amikacin, kanamycin, cetylpyridinium chloride, and several sulfa drugs. MSMEG_2619 (efpA) and MSMEG_3563 mask the effect of mmp deletion due to overlapping efflux capabilities. We present evidence that mmp is a part of an MSMEG_2626-2628-2629-2630-2631 operon regulated by a strong constitutive promoter, initiated from a single transcription start site. All together, our results show that M. smegmatis constitutively encodes an Na(+)-dependent MATE multidrug efflux pump from mmp in an operon with putative genes encoding proteins for apparently unrelated functions.

Stoichiometric protein complex formation and over-expression using the prokaryotic native operon structure.

In prokaryotes, operon encoded proteins often form protein-protein complexes. Here, we show that the native structure of operons can be used to efficiently overexpress protein complexes. This study focuses on operons from mycobacteria and the use of Mycobacterium smegmatis as an expression host. We demonstrate robust and correct stoichiometric expression of dimers to higher oligomers. The expression efficacy was found to be largely independent of the intergenic distances. The strategy was successfully extended to express mycobacterial protein complexes in Escherichia coli, showing that the operon structure of gram-positive bacteria is also functional in gram-negative bacteria. The presented strategy could become a general tool for the expression of large quantities of pure prokaryotic protein complexes for biochemical and structural studies.

Pharmacokinetic of ALA and h-ALA induced porphyrins in the models Mycobacterium phlei and Mycobacterium smegmatis.

Photodynamic inactivation (PDI) of bacterial strains presents an attractive potential alternative to antibiotic therapies. Success is dependent on the effective accumulation in bacterial cells of photochemical substances called photosensitizers, which are usually porphyrins. It is also important to know the distribution of the photosensitizer in bacteria at the microscopic level. The present results examine the accumulation of photosensitizers by Mycobacterium phlei and Mycobacterium smegmatis, which serve as models for the important pathogens Mycobacterium tuberculosis, Mycobacterium leprae and Mycobacterium bovis. The kinetics of porphyrin synthesis after treatment with the precursors ALA and h-ALA were studied. The goal was to describe the biosynthesis and the pharmacokinetics of sensitizers in both bacterial strains using fluorescence microscopy and spectroscopy. We could show that both Mycobacterium strains enrich porphyrins after ALA and h-ALA administration detected by fluorescence peaks at about 620nm. By HPLC analyses the major porphyrin could be identified as coproporphyrin. In the future we will apply the new knowledge in in vitro and in vivo experiments to strains of M. tuberculosis, M. leprae and M. bovis and examine cell destruction by PDI.

Identification of two proteins that interact with the Erp virulence factor from Mycobacterium tuberculosis by using the bacterial two-hybrid system.

BACKGROUND: The exported repetitive protein (erp) gene encodes a secreted 36-kDa protein with a central domain containing several proline-glycine-leucine-threonine-serine (PGLTS) repeats. It has been demonstrated that erp is a virulence-associated factor since the disruption of this gene impairs the growth of Mycobacterium bovis and Mycobacterium tuberculosis in mice. RESULTS: In order to elucidate the function of Erp we searched for Erp-binding proteins from M. tuberculosis by using a bacterial two-hybrid system. Our results indicate that Erp interacts specifically with two putative membrane proteins, Rv1417 and Rv2617c. Further analysis revealed that the latter two interact with each other, indicating that Rv1417, Rv2617c and Erp are connected through multiple interactions. While Rv1417 is disseminated in several Actinomycetales genera, orthologues of Rv2617c are exclusively present in members of the M. tuberculosis complex (MTC). The central and amino-terminal regions of Erp were determined to be involved in the interaction with Rv1417 and Rv2627c. Erp forms from Mycobacterium smegmatis and Mycobacterium leprae were not able to interact with Rv2617c in two-hybrid assays. Immunolocalization experiments showed that Rv1417 and Rv2617c are found on the cell membrane and Erp on the bacterial cell wall. Finally, comparative genomics and expression studies revealed a possible role of Rv1417 in riboflavin metabolism. CONCLUSION: We identified interactive partners of Erp, an M. tuberculosis protein involved in virulence, which will be the focus of future investigation to decipher the function of the Erp family protein.

Identification, characterization, and azole-binding properties of Mycobacterium smegmatis CYP164A2, a homolog of ML2088, the sole cytochrome P450 gene of Mycobacterium leprae.

The genome sequence of Mycobacterium leprae revealed a single open reading frame, ML2088 (CYP164A1), encoding a putative full-length cytochrome P450 monooxygenase and 12 pseudogenes. We have identified a homolog of ML2088 in Mycobacterium smegmatis and report here the cloning, expression, purification, and azole-binding characteristics of this cytochrome P450 (CYP164A2). CYP164A2 is 1,245 bp long and encodes a protein of 414 amino acids and molecular mass of 45 kDa. CYP164A2 has 60% identity with Mycobacterium leprae CYP161A1 and 66 to 69% identity with eight other mycobacterial CYP164A1 homologs, with three identified highly conserved motifs. Recombinant CYP164A2 has the typical spectral characteristics of a cytochrome P450 monooxygenase, predominantly in the ferric low-spin state. Unusually, the spin state was readily modulated by increasing ionic strength at pH 7.5, with 50% high-spin occupancy achieved with 0.14 M NaCl. CYP164A2 bound clotrimazole, econazole, and miconazole strongly (K(d), 1.2 to 2.5 muM); however, strong binding with itraconazole, ketoconazole, and voriconazole was only observed in the presence of 0.5 M NaCl. Fluconazole did not bind to CYP164A2 at pH 7.5 and no discernible type II binding spectrum was observed.

Cyclic AMP in mycobacteria: characterization and functional role of the Rv1647 ortholog in Mycobacterium smegmatis.

Mycobacterial genomes are endowed with many eukaryote-like nucleotide cyclase genes encoding proteins that can synthesize 3',5'-cyclic AMP (cAMP). However, the roles of cAMP and the need for such redundancy in terms of adenylyl cyclase genes remain unknown. We measured cAMP levels in Mycobacterium smegmatis during growth and under various stress conditions and report the first biochemical and functional characterization of the MSMEG_3780 adenylyl cyclase, whose orthologs in Mycobacterium tuberculosis (Rv1647) and Mycobacterium leprae (ML1399) have been recently characterized in vitro. MSMEG_3780 was important for producing cAMP levels in the logarithmic phase of growth, since the DeltaMSMEG_3780 strain showed lower intracellular cAMP levels at this stage of growth. cAMP levels decreased in wild-type M. smegmatis under conditions of acid stress but not in the DeltaMSMEG_3780 strain. This was correlated with a reduction in MSMEG_3780 promoter activity, indicating that the effect of the reduction in cAMP levels on acid stress was caused by a decrease in the transcription of MSMEG_3780. Complementation of the DeltaMSMEG_3780 strain with the genomic integration of MSMEG_3780 or the Rv1647 gene could restore cAMP levels during logarithmic growth. The Rv1647 promoter was also acid sensitive, emphasizing the biochemical and functional similarities in these two adenylyl cyclases. This study therefore represents the first detailed biochemical and functional analysis of an adenylyl cyclase that is important for maintaining cAMP levels in mycobacteria and underscores the subtle roles that these genes may play in the physiology of the organism.

The Rv0805 gene from Mycobacterium tuberculosis encodes a 3',5'-cyclic nucleotide phosphodiesterase: biochemical and mutational analysis.

Mycobacterium tuberculosis is an important human pathogen and has developed sophisticated mechanisms to evade the host immune system. These could involve the use of cyclic nucleotide-dependent signaling systems, since the M. tuberculosis genome encodes a large number of functional adenylyl cyclases. Using bioinformatic approaches, we identify, clone, and biochemically characterize the Rv0805 gene product, the first cyclic nucleotide phosphodiesterase identified in M. tuberculosis and a homologue of the cAMP phosphodiesterase present in Escherichia coli (cpdA). The Rv0805 gene product, a class III phosphodiesterase, is a member of the metallophosphoesterase family, and computational modeling and mutational analyses indicate that the protein possesses interesting properties not reported earlier in this class of enzymes. Mutational analysis of critical histidine and aspartate residues predicted to be essential for metal coordination reduced catalytic activity by 90-50%, and several mutant proteins showed sigmoidal kinetics with respect to Mn in contrast to the wild-type enzyme. Mutation of an asparagine residue in the GNHD motif that is conserved throughout the metallophosphoesterase enzymes almost completely abolished catalytic activity, and these studies therefore represent the first mutational analysis of this class of phosphodiesterases. The Rv0805 protein hydrolyzes cAMP and cGMP in vitro, and overexpression in Mycobacterium smegmatis and E. coli reduces intracellular cAMP levels. The presence of an orthologue of Rv0805 in Mycobacterium leprae suggests that the Rv0805 protein could have an important role to play in regulating cAMP levels in these bacteria and adds an additional level of complexity to cyclic nucleotide signaling in this organism.

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

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

Identification of Mycobacterium avium genes up-regulated in cultured macrophages and in mice.

To investigate Mycobacterium avium gene expression upon infection of macrophages, we created a M. avium-promoter library upstream of a promoter-less gene encoding the green fluorescent protein (GFP) in Mycobacterium smegmatis. Clones were evaluated for increased expression of GFP after infection of U937 macrophages. A number of M. avium genes were up-regulated more than 3-fold after 24 and 48 h following macrophage infection. M. avium genes expressed by M. smegmatis during growth in macrophages include genes encoding transport/binding proteins, synthesis, modification and degradation of macromolecules, and a great majority of genes for which no function is currently known. For some of the unknown genes, homologues were identified in bacteria such as Mycobacterium leprae, Salmonella typhimurium and Agrobacterium tumefaciens. In order to investigate if these genes were also expressed in M. avium during macrophage infection in vitro and in vivo, transcripts of selected genes were quantified using real time RT-PCR. Evaluation of most expressed genes in M. smegmatis confirmed their up-regulation in M. avium after 24 h infection of macrophages in vitro and mice.