Evaluation of the role of sigma B in Mycobacterium smegmatis

The alternate sigma factor, sigB, is known to play a crucial role in maintaining the stationary phase in mycobacteria. In this communication, we have studied the proteomics of Mycobacterium smegmatis mc(2)155 and its two derivatives, one of which has a disrupted sigB gene and the other, PMVSigB, which contains a multicopy plasmid containing sigB. We have identified by two-dimensional gel analyses, several proteins that are over-expressed in PMVSigB compared to mc(2)155. These proteins are either stress proteins or participate actively in different metabolic pathways of the organisms. On the other hand, when sigB deleted mycobacteria were grown until the stationary phase and its two-dimensional protein profile was compared to that of mc(2)155, few DNA binding proteins were found to be up-regulated. We have shown recently that upon over-expressing sigB, the cell surface glycopeptidolipids of M. smegmatis are hyperglycosylated, a situation similar to what was observed for nutritionally starved bacteria. Gene expression profile through quantitative PCR presented here identified a Rhamnosyltransferase responsible for this hyperglycosylation.     

Biosynthesis of d-arabinose in Mycobacterium smegmatis: specific labeling from d-glucose

d-Arabinose is a major sugar in the cell wall polysaccharides of Mycobacterium tuberculosis and other mycobacterial species. The reactions involved in the biosynthesis and activation of d-arabinose represent excellent potential sites for drug intervention since d-arabinose is not found in mammalian cells, and the cell wall arabinomannan and/or arabinogalactan appear to be essential for cell survival. Since the pathway involved in conversion of d-glucose to d-arabinose is unknown, we incubated cells of Mycobacterium smegmatis individually with [1-(14)C]glucose, [3,4-(14)C]glucose, and [6-(14)C]glucose and compared the specific activities of the cell wall-bound arabinose. Although the specific activity of the arabinose was about 25% lower with [6-(14)C]glucose than with other labels, there did not appear to be selective loss of either carbon 1 or carbon 6, suggesting that arabinose was not formed by loss of carbon 1 of glucose via the oxidative step of the pentose phosphate pathway, or by loss of carbon 6 in the uronic acid pathway. Similar labeling patterns were observed with ribose isolated from the nucleic acid fraction. Since these results suggested an unusual pathway of pentose formation, labeling studies were also done with [1-(13)C]glucose, [2-(13)C]glucose, and [6-(13)C]glucose and the cell wall arabinose was examined by NMR analysis. This method allows one to determine the relative (13)C content in each carbon of the arabinose. The labeling patterns suggested that the most likely pathway was condensation of carbons 1 and 2 of fructose 6-phosphate produced by the transaldolase reaction with carbons 4, 5, and 6 (i.e., glyceraldehyde 3-phosphate) formed by fructose-1,6 bisphosphate aldolase. Cell-free enzyme extracts of M. smegmatis were incubated with ribose 5-phosphate, xylulose 5-phosphate, and d-arabinose 5-phosphate under a variety of experimental conditions. Although the ribose 5-phosphate and xylulose 5-phosphate were converted to other pentoses and hexoses, no arabinose 5-phosphate (or free arabinose) was detected in any of these reactions. In addition, these enzyme extracts did not convert arabinose 5-phosphate to any other pentose or hexose. In addition, incubation of [(14)C]glucose 6-phosphate and various nucleoside triphosphates (ATP, CTP, GTP, TTP, and UTP) with cytosolic or membrane fractions from the mycobacterial cells did not result in formation of a nucleotide form of arabinose, although other radioactive sugars including rhamnose and galactose were found in the nucleotide fraction. Furthermore, no radioactive arabinose was found in the nucleotide fraction isolated from M. smegmatis cells grown in [(3)H]glucose, nor was arabinose detected in a large-scale extraction of the sugar nucleotide fraction from 300 g of cells. The logical conclusion from these studies is that d-arabinose is probably produced from d-ribose by epimerization of carbon 2 of the ribose moiety of polyprenylphosphate-ribose to form polyprenylphosphate-arabinose, which is then used as the precursor for formation of arabinosyl polymers.     

Identification of small RNAs in Mycobacterium smegmatis using heterologous Hfq

Gene regulation by small RNAs (sRNAs) has been extensively studied in various bacteria. However, the presence and roles of sRNAs in mycobacteria remain largely unclear. Immunoprecipitation of RNA chaperone Hfq to enrich for sRNAs is one of the effective methods to isolate sRNAs. However, the lack of an identified mycobacterial hfq restricts the feasibility of this approach. We developed a novel method that takes advantage of the conserved inherent sRNAs-binding capability of heterologous Hfq from Escherichia coli to enrich sRNAs from Mycobacterium smegmatis, a model organism for studying Mycobacterium tuberculosis. We validated 12 trans-encoded and 12 cis-encoded novel sRNAs in M. smegmatis. Many of these sRNAs are differentially expressed at exponential phase compared with stationary phase, suggesting that sRNAs are involved in the growth of mycobacteria. Intriguingly, five of the cis-encoded novel sRNAs target known transposases. Phylogenetic conservation analysis shows that these sRNAs are pathogenicity dependent. We believe that our findings will serve as an important reference for future analysis of sRNAs regulation in mycobacteria and will contribute significantly to the development of sRNAs prediction programs. Moreover, this novel method of using heterologous Hfq for sRNAs enrichment can be of general use for the discovery of bacterial sRNAs in which no endogenous Hfq is identified.     

Metabolism of cyclic AMP in non-pathogenic Mycobacterium smegmatis

The intracellular concentration of cyclic AMP reached a maximum in 3.5-day old cultures of Mycobacterium smegmatis grown in the presence of glycerol as the main source of carbon. Glucose-grown cells exhibited decreased cyclic AMP levels at all stages of growth. When M. smegmatis cells were incubated with various metabolites, pyruvate increased whereas glucose, citric acid, succinic acid and lactic acid decreased intracellular cyclic AMP levels. No cyclic AMP was detected in the incubation medium. The presence of a cyclic AMP-binding protein was demonstrated in cellfree extracts of M. smegmatis.     

Functional Analysis of a c-di-AMP-specific Phosphodiesterase MsPDE from Mycobacterium smegmatis

Cyclic di‑AMP (c-di-AMP) is a second signaling molecule involved in the regulation of bacterial physiological processes and interaction between pathogen and host. However, the regulatory network mediated by c-di-AMP in Mycobacterium remains obscure. In M. smegmatis, a diadenylate cyclase (DAC) was reported recently, but there is still no investigation on c-di-AMP phosphodiesterase (PDE). Here, we provide a systematic study on signaling mechanism of c-di-AMP PDE in M. smegmatis. Based on our enzymatic analysis, MsPDE (MSMEG_2630), which contained a DHH-DHHA1 domain, displayed a 200-fold higher hydrolytic efficiency (k_cat/K_m) to c-di-AMP than to c-di-GMP. MsPDE was capable of converting c-di-AMP to pApA and AMP, and hydrolyzing pApA to AMP. Site-directed mutations in DHH and DHHA1 revealed that DHH domain was critical for the phosphodiesterase activity. To explore the regulatory role of c-di-AMP in vivo, we constructed the mspde mutant (Δmspde) and found that deficiency of MsPDE significantly enhanced intracellular C₁₂-C₂₀ fatty acid accumulation. Deficiency of DAC in many bacteria results in cell death. However, we acquired the M. smegmatis strain with DAC gene disrupted (ΔmsdisA) by homologous recombination approach. Deletion of msdisA reduced bacterial C₁₂-C₂₀ fatty acids production but scarcely affected bacterial survival. We also provided evidences that superfluous c-di-AMP in M. smegmatis could lead to abnormal colonial morphology. Collectively, our results indicate that MsPDE is a functional c-di-AMP-specific phosphodiesterase both in vitro and in vivo. Our study also expands the regulatory network mediated by c-di-AMP in M. smegmatis.     

Snapshots of RecA protein involving movement of the C-domain and different conformations of the DNA-binding loops: crystallographic and comparative analysis of 11 structures of Mycobacterium smegmatis RecA

Mycobacterium smegmatis RecA and its nucleotide complexes crystallize in three different, but closely related, forms characterized by specific ranges of unit cell dimensions. The six crystals reported here and five reported earlier, all grown under the same or very similar conditions, belong to these three forms, all in space group P6(1). They include one obtained by reducing relative humidity around the crystal. In all crystals, RecA monomers form filaments around a 6(1) screw axis. Thus, the c-dimension of the crystal corresponds to the pitch of the RecA filament. As reported for Escherichia coli RecA, the variation in the pitch among the three forms correlates well with the motion of the C-terminal domain of the RecA monomers with respect to the main domain. The domain motion is compatible with formation of inactive as well as active RecA filaments involving monomers with a fully ordered C domain. It does not appear to influence the movement upon nucleotide-binding of the switch residue, which is believed to provide the trigger for transmitting the effect of nucleotide binding to the DNA-binding region. Interestingly, partial dehydration of the crystal results in the movement of the residue similar to that caused by nucleotide binding. The ordering of the DNA-binding loops, which present ensembles of conformations, is also unaffected by domain motion. The conformation of loop L2 appears to depend upon nucleotide binding, presumably on account of the movement of the switch residue that forms part of the loop. The conformations of loops L1 and L2 are correlated and have implications for intermolecular communications within the RecA filament. The structures resulting from different orientations of the C domain and different conformations of the DNA-binding loops appear to represent snapshots of the RecA at different phases of activity, and provide insights into the mechanism of action of RecA.     

An orphan gyrB in the Mycobacterium smegmatis genome uncovered by comparative genomics

DNA gyrase is an essential topoisomerase found in all bacteria. It is encoded bygyrB andgyrA genes. These genes are organized differently in different bacteria. Direct comparison ofMycobacterium tuberculosis andMycobacterium smegmatis genomes reveals presence of an additionalgyrB inM. smegmatis flanked by novel genes. Analysis of the amino acid sequence of GyrB from different organisms suggests that the orphan GyrB inM. smegmatis may have an important cellular role.     

Turnover of lipids in Mycobacterium smegmatis CDC 46 and Mycobacterium phlei ATCC 354

The rates of breakdown and renewal of individual lipids in cultures of Mycobacterium smegmatis CDC 46 and Mycobacterium phlei ATCC 354 were investigated by means of a pulse labelling technique using palmitate-1-¹⁴C. The results indicated that in growing cultures of both strains phospholipids were broken down, and cardiolipin had a very rapid turnover. In chase experiments, almost 45% and 40% of the radioactivity of this component were lost respectively from M. smegmatis and M. phlei during one generation time of the cell. The other two major components, phosphatidyl ethanolamine and phosphatidylinositol mannosides showed relatively low turnover. The loss of radioactivity from phosphatidylinositol mannosides was greater in M. phlei than in M. smegmatis but the loss of radioactivity from phosphatidyl ethanolamine was higher in M. smegmatis. The pattern of loss of radioactivity from lipids was almost the same in both strains, the difference being only in the extent of loss. The differences in the cellular localization of the phospholipids indicate their different roles within the cell. Results obtained with the glyceride fraction indicated a very rapid turnover of triglycerides in both strains.Abbreviations CL Cardiolipin - PE Phosphatidyl ethanolamine - PIMx phosphatidylinositol mannosides - PIM₂A phosphatidylinositol dimannoside tetra acylated - PIM₂B phosphatidylinositol dimannoside tri acylated - PIM₅ phosphatidylinositol pentamannoside tetra acylated     

Oxygen binding and NO scavenging properties of truncated hemoglobin, HbN, of Mycobacterium smegmatis

Unraveling of microbial genome data has indicated that two distantly related truncated hemoglobins (trHbs), HbN and HbO, might occur in many species of slow-growing pathogenic mycobacteria. Involvement of HbN in bacterial defense against NO toxicity and nitrosative stress has been proposed. A gene, encoding a putative HbN homolog with conserved features of typical trHbs, has been identified within the genome sequence of fast-growing mycobacterium, Mycobacterium smegmatis. Sequence analysis of M. smegmatis HbN indicated that it is relatively smaller in size and lacks N-terminal pre-A region, carrying 12-residue polar sequence motif that is present in HbN of M. tuberculosis. HbN encoding gene of M. smegmatis was expressed in E. coli as a 12.8kD homodimeric heme protein that binds oxygen reversibly with high affinity (P50 approximately 0.081 mm Hg) and autooxidizes faster than M. tuberculosis HbN. The circular dichroism spectra indicate that HbN of M. smegmatis and M. tuberculosis are structurally similar. Interestingly, an hmp mutant of E. coli, unable to metabolize nitric oxide, exhibited very low NO uptake activity in the presence of M. smegmatis HbN as compared to HbN of M. tuberculosis. On the basis of cellular heme content, specific nitric oxide dioxygenase (NOD) activity of M. smegmatis HbN was nearly one-third of that from M. tuberculosis. Additionally, the hmp mutant of E. coli, carrying M. smegmatis HbN, exhibited nearly 10-fold lower cell survival under nitrosative stress and nitrite derived reactive nitrogen species as compared to the isogenic strain harboring HbN of M. tuberculosis. Taken together, these results suggest that NO metabolizing activity and protection provided by M. smegmatis HbN against toxicity of NO and reactive nitrogen is significantly lower than HbN of M. tuberculosis. The lower efficiency of M. smegmatis HbN for NO detoxification as compared to M. tuberculosis HbN might be related to different level of NO exposure and nitrosative stress faced by these mycobacteria during their cellular metabolism.     

Presence of mycobacteriophage I3-like DNA sequences in the genome of its host Mycobacterium smegmatis

The genomes of phage I3 and its host Mycobacterium smegmatis have been compared. From thermal melting studies the GC contents of DNA from mycobacteriophage I3 and its host M. smegmatis were found to be 66%. A new method, based only on the initial rates of reassociation, has been developed for calculating the DNA homology. Analysis of DNA reassociation kinetics suggested the presence of one equivalent of the phage I3 genome within the M. smegmatis genome. Southern analysis revealed the presence of almost all of the phage I3 specific sequences within the host genome.     

Identification of antibody responses to the serotype-nonspecific molecular species of glycopeptidolipids in Mycobacterium avium infection

Glycopeptidolipids (GPLs) comprise a major surface glycolipid of Mycobacterium avium complex (MAC), and their unique oligosaccharide extensions are known to define MAC serotypes. Beside the mature form of “serotype-specific” GPLs (ssGPLs), those that share the backbone structure but lack the oligosaccharide extensions exist as abundantly in all MAC serotypes, but the presumption was that antibody responses might not be directed to these “serotype-nonspecific” GPLs (nsGPLs) due to the lack of the sugar chain epitope. Here, we show that IgG responses to nsGPLs indeed occur in MAC-infected guinea pigs. The pool of anti-nsGPL antibodies was distinct from that of anti-ssGPL antibodies in terms of requirements for the oligosaccharide and acetylation for their target recognition. Because nsGPLs are shared in virtually all MAC strains, but totally absent in Mycobacterium tuberculosis, this study suggests that detecting serum anti-nsGPL antibodies can potentially be useful for differential diagnosis of MAC infection and tuberculosis.     

Transfection of Mycobacterium smegmatis SN2 with mycobacteriophage I3 DNA

Mycobacterium smegmatis SN2 does not exhibit natural competence for the uptake of phage I3 DNA. Competence can artificially be induced by treatment with glycine or CaCl₂, and the combination of both is even more effective. The efficiency of transfection can be improved by inclusion of protamine sulphate and heterologous RNA in the system. From ³²P DNA uptake studies the major barrier for the entry of DNA has been found to be the complex cell wall. The efficiency of transfection calculated on the basis of fraction of DNA which has entered the cell is comparable to that of other bacterial systems. The phage development takes a longer time (7 h for one cycle) after transfection, as compared to infection (4 h).     

PCR-linked reverse DNA hybridization using oligonucleotide-specific probes of rpoB for identification of Mycobacterium avium and Mycobacterium intracellulare

A PCR-linked reverse DNA hybridization method using two different specific rpoB DNA probes (Avp and Intp) of Mycobacterium avium and Mycobacterium intracellulare, respectively, were evaluated for the differentiation and identification of M. avium and M. intracellulare culture isolates. Among the 504 culture isolates tested by this method, 48 strains showed positive results for M. avium and 60 strains showed positive results for M. intracellulare. The other 396 culture isolates showed negative results for both M. avium and M. intracellulare. These results were consistent with those obtained from partial rpoB (306 bp) sequence analysis and biochemical tests. The negative strains obtained by this DNA hybridization method were identified as M. tuberculosis (366 strains), M. peregrinum (11 strains), M. abscessus (9 strains), M. fortuitum (8 strains), and M. flavescens (2 strains) by rpoB DNA sequence analysis. Due to the high sensitive and specific result obtained by this assay, we suggest that this PCR-linked reverse DNA hybridization method using two different specific rpoB DNA probes of M. avium and M. intracellulare would be used for the rapid and precise method for differentiation and identification of M. avium and M. intracellulare.     

Characterising Mycobacterium tuberculosis Rv1510c protein and determining its sequences that specifically bind to two target cell lines

The process of Mycobacterium tuberculosis infection of the macrophage implies a very little-known initial recognition and adherence step, important for mycobacterial survival; many proteins even remain like hypothetical. The Rv1510c gene, encoding a putatively conserved membrane protein, was investigated by analysing the M. tuberculosis genome sequence data reported by Cole et al. and a previous report that used PCR assays to show that the Rv1510 gene was only present in M. tuberculosis. This article confirmed all the above and identified the transcribed gene in M. tuberculosis, Mycobacterium africanum, and in M. tuberculosis clinical isolates. Antibodies raised against peptides from this protein recognised a 44 kDa band, corresponding to Rv1510c theoretical mass (44,294 Da). Assays involving synthetic peptides covering the whole protein binding to U937 and A549 cell lines led to recognising five high activity binding peptides in the Rv1510 protein: 11094, 11095, 11105, 11108, and 11111. Their affinity constants and Hill coefficients were determined by using U937 cells. Cross-linking assays performed with some of these HABPs showed that they specifically bound to a U937 cell line 51 kDa protein, but not to Hep G2 or red blood cell proteins, showing this interaction's specificity.     

Effect of morphine on <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis> infection in mice and macrophages

The immunomodulatory effects of opioids are known in various infections. However, little is known about the effects of opioids in tuberculosis (TB). In the present study, we report the effects of morphine in Mycobacterium smegmatis infection in mice and macrophages. Morphine exerted a dose-dependent suppression of infection in vivo: 50 and 100 mg/kg morphine exerted significant (P<0.05) suppression whereas 5 mg/kg morphine showed no effect. Analogous to the in vivo effects, incubation of M. smegmatis-infected mouse peritoneal macrophages with morphine (100 μM) showed significant reduction in intramacrophage CFU counts. However, morphine did not show any direct antimycobacterial activity in broth dilution assay upto 100 μM concentration. Further, morphine-induced intramacrophage killing of M. smegmatis was abrogated by naloxone and aminoguanidine indicating the involvement of opioid-receptor activation and nitric oxide production in protective effects of morphine. In conclusion, morphine suppressed the progression of experimental TB in both mice and macrophage models.     

Mutations in the GTP-binding and synergy loop domains of Mycobacterium tuberculosis ftsZ compromise its function in vitro and in vivo

The Mycobacterium tuberculosis FtsZ (FtsZ(TB)), unlike other eubacterial FtsZ proteins, shows slow GTP-dependent polymerization and weak GTP hydrolysis activities [E.L. White, L.J. Ross, R.C. Reynolds, L.E. Seitz, G.D. Moore, D.W. Borhani, Slow polymerization of Mycobacterium tuberculosis FtsZ, J. Bacteriol. 182 (2000) 4028-4034]. In an attempt to understand the biological significance of these findings, we created mutations in the GTP-binding (FtsZ(G103S)) and GTP hydrolysis (FtsZ(D210G)) domains of FtsZ and characterized the activities of the mutant proteins in vitro and in vivo. We show that FtsZ(G103S) is defective for binding to GTP and polymerization activities, and exhibited reduced GTPase activity whereas FtsZ(D210G) protein is proficient in binding to GTP, showing reduced polymerization activity but did not show any measurable GTPase activity. Visualization of FtsZ-GFP structures in ftsZ merodiploid strains by fluorescent microscopy revealed that FtsZ(D210G) is proficient in associating with Z-ring structures whereas FtsZ(G103S) is not. Finally, we show that Mycobacterium smegmatis ftsZ mutant strains producing corresponding mutant FtsZ proteins are non-viable indicating that mutant FtsZ proteins cannot function as the sole source for FtsZ, a result distinctly different from that reported for Escherichia coli. Together, our results indicate that optimal GTPase and polymerization activities of FtsZ are required to sustain cell division in mycobacteria and that the same conserved mutations in different bacterial species have distinct phenotypes.     

A novel method based on high resolution melting (HRM) analysis for MIRU-VNTR genotyping of Mycobacterium tuberculosis

The mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) method is one of the most important methods that have been used in recent years for genotyping Mycobacterium tuberculosis. Agarose gel electrophoresis and capillary electrophoresis have been used to determine the size of amplicons, however, both of these methods have shortcomings. Here, we develop and evaluate a novel method for MIRU-VNTR typing based on high resolution melting (HRM) analysis. The MIRU40 locus was selected to evaluate different real-time PCR machines and the accuracy of our method; the Roche LightCycler 480 provided greatest consistency between the T_m value and repeat number and was used in subsequent evaluations. Our method gives greater accuracy in comparison with conventional agarose gel electrophoresis (98.9% vs. 90.9%, p = 0.017), and, with the help of fitting formulae, can be used to obtain the number of MIRU tandem repeats from the T_m value. To validate our method we analyzed 12 classical MIRU loci to genotype 88 clinical isolates. The number of MIRU tandem repeats was determined accurately, quickly and conveniently.     

Molecular characterization of a phenanthrene degradation pathway in Mycobacterium vanbaalenii PYR-1

Mycobacterium vanbaalenii PYR-1 is capable of degrading a number of polycyclic aromatic hydrocarbons (PAHs) to ring cleavage metabolites via multiple pathways. Genes for the large and small subunits of a pyrene dioxygenase, nidA and nidB, respectively, were previously identified in M. vanbaalenii PYR-1 [Appl. Environ. Microbiol. 67 (2001) 3577]. A library of the M. vanbaalenii PYR-1 genome was constructed in a fosmid vector to identify additional genes involved in PAH degradation. Twelve fosmid clones containing nidA were identified by Southern hybridization. Sequence analysis of one nidA-positive clone, pFOS608, revealed a number of additional genes involved in PAH degradation. At this locus, one putative operon contained genes involved in phthalate degradation, and another contained genes encoding a putative ABC transporter(s). A number of the genes found in this region are homologous to those involved in phenanthrene degradation via the phthalic acid pathway. The majority of phenanthrene degradation genes were located between putative transposase genes. In Escherichia coli, pFOS608 converted phenanthrene into phenanthrene cis-3,4-dihydrodiol, and converted 1-hydroxy-2-naphthoic acid into 2'-carboxybenzalpyruvate, 2-carboxybenzaldehyde, and phthalic acid. A subclone containing nidA and nidB converted phenanthrene into phenanthrene cis-3,4-dihydrodiol, suggesting that the NidAB dioxygenase is responsible for an initial attack on phenanthrene. This study is the first to identify genes responsible for the degradation of phenanthrene via the phthalic acid pathway in Mycobacterium species.