Search

Background Microbiological cultures are the mainstay of the diagnosis of tuberculosis (TB). False-positive TB results lead to significant unnecessary therapeutic and economic burden and are frequently caused by laboratory cross-contamination. The aim of this meta-analysis was to quantify the prevalence of laboratory cross-contamination. Methods Through a systematic review of five electronic databases, we identified studies reporting rates of laboratory cross-contamination, confirmed by molecular techniques in TB cultures. We evaluated the quality of the identified studies using the National Institute of Health (NIH) Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies, and conducted a meta-analysis using standard methodology recommended by the Cochrane Collaboration. Results Based on 31 eligible studies evaluating 29,839 TB cultures, we found that 2% (95% confidence intervals [CI] 1-2%) of all positive TB cultures represent false-positive results secondary to laboratory cross-contamination. More importantly, we evaluated the rate of laboratory cross-contamination in cases where a single-positive TB culture was available in addition to at least one negative TB culture, and we found a rate of 15% (95% CI 6-33%). Moreover, 9.2% (91/990) of all patients with a preliminary diagnosis of TB had false-positive results and received unnecessary and potentially harmful treatments. Conclusions Our results highlight a remarkably high prevalence of false-positive TB results as a result of laboratory cross-contamination, especially in single-positive TB cultures, leading to the administration of unnecessary, harmful treatments. The need for the adoption of strict technical standards for mycobacterial cultures cannot be overstated., Author(s): Aleksandra Barac [sup.1] [sup.2] , Hannah Karimzadeh-Esfahani [sup.3] , Mahya Pourostadi [sup.4] , Mohammad Taghi Rahimi [sup.5] , Ehsan Ahmadpour [sup.6] [sup.7] , Jalil Rashedi [sup.8] , Behroz Mahdavipoor [...]

A hybrid crystal structure of Mycobacterium smegmatis mycocerosic acid synthase, a multienzyme involved in the biosynthesis of mycobacterial branched-chain fatty acids, exemplifies the organization of fully-reducing polyketide synthases. A polyketide synthase structure revealed Mycocerosic acid synthase (MAS) is a mycobacterial polyketide synthase (PKS) responsible for the biosynthesis of C.sub.20-C.sub.28 branched-chain fatty acids, key components of the specialized mycobacterial cell wall. These authors report a hybrid crystal structure of MAS from Mycobacterium smegmatis. Many commonly used drugs are either naturally occurring polyketides or their derivatives. This work represents the first high-quality model of a full PKS module, and it could be used to help dissect and re-engineer other PKSs to make new, non-natural polyketides. Polyketide synthases (PKSs) are biosynthetic factories that produce natural products with important biological and pharmacological activities.sup.1,2,3. Their exceptional product diversity is encoded in a modular architecture. Modular PKSs (modPKSs) catalyse reactions colinear to the order of modules in an assembly line.sup.3, whereas iterative PKSs (iPKSs) use a single module iteratively as exemplified by fungal iPKSs (fiPKSs).sup.3. However, in some cases non-colinear iterative action is also observed for modPKSs modules and is controlled by the assembly line environment.sup.4,5. PKSs feature a structural and functional separation into a condensing and a modifying region as observed for fatty acid synthases.sup.6. Despite the outstanding relevance of PKSs, the detailed organization of PKSs with complete fully reducing modifying regions remains elusive. Here we report a hybrid crystal structure of Mycobacterium smegmatis mycocerosic acid synthase based on structures of its condensing and modifying regions. Mycocerosic acid synthase is a fully reducing iPKS, closely related to modPKSs, and the prototype of mycobacterial mycocerosic acid synthase-like.sup.7,8 PKSs. It is involved in the biosynthesis of C.sub.20-C.sub.28 branched-chain fatty acids, which are important virulence factors of mycobacteria.sup.9. Our structural data reveal a dimeric linker-based organization of the modifying region and visualize dynamics and conformational coupling in PKSs. On the basis of comparative small-angle X-ray scattering, the observed modifying region architecture may be common also in modPKSs. The linker-based organization provides a rationale for the characteristic variability of PKS modules as a main contributor to product diversity. The comprehensive architectural model enables functional dissection and re-engineering of PKSs., Author(s): Dominik A. Herbst [sup.1] , Roman P. Jakob [sup.1] , Franziska Zähringer [sup.1] [sup.3] , Timm Maier [sup.1] Author Affiliations: (1) Department Biozentrum, University of Basel, Basel, Switzerland (3) [...]

Mycobacterium caprae is a cluster within the M. tuberculosis complex (online Technical Appendix, www.cdc.gov/EID/content/17/3/532-Techapp.pdf). This pathogen has been recognized mainly in central Europe, where it has been occasionally isolated from [...]

Lipoarabinomannan (LAM) is a major glycolipid in the mycobacterial cell envelope. LAM consists of a mannosylphosphatidylinositol (MPI) anchor, a mannan core and a branched arabinan domain. The termini of the arabinan branches can become substituted with one to three [alpha](1 [right arrow] 2)-linked mannosyl residues, the mannose cap, producing ManLAM. ManLAM has been associated with a range of different immunomodulatory properties of Mycobacterium tuberculosis during infection of the host. In some of these effects, the presence of the mannose cap on ManLAM appears to be crucial for its activity. So far, in the biosynthesis of the mannose cap on ManLAM, two enzymes have been reported to be involved: a mannosyltransferase that adds the first mannosyl residue of the mannose caps to the arabinan domain of LAM, and another mannosyltransferase that elongates the mannose cap up to three mannosyl residues. Here, we report that a third gene is involved, MMAR_2380, which is the Mycobacterium marinum orthologue of Rv1565c. MMAR_2380 encodes a predicted transmembrane acyltransferase. In M. marinum [DELTA]MMAR_2380, the LAM arabinan domain is still intact, but the mutant LAM lacks the mannose cap. Additional effects of mutation of MMAR_2380 on LAM were observed: a higher degree of branching of both the arabinan domain and the mannan core, and a decreased incorporation of [1,2-[sup.14]C]acetate into the acyl chains in mutant LAM as compared with the wild-type form. This latter effect was also observed for related lipoglycans, i.e. lipomannan (LM) and phosphatidylinositol mannosides (PIMs). Furthermore, the mutant strain showed increased aggregation in liquid cultures as compared with the wild-type strain. All phenotypic traits of M. marinum AMMAR_2380, the deficiency in the mannose cap on LAM and changes at the cell surface, could be reversed by complementing the mutant strain with MMAR2380. Strikingly, membrane preparations of the mutant strain still showed enzymic activity for the arabinan mannose-capping mannosyltransferase similar to that of the wild-type strain. Although the exact function of MMAR_2380 remains unknown, we show that the protein is essential for the presence of a mannose cap on LAM. DOI 10.1099/mic.0.037507-0

Nanopore sequencing has the potential to become a direct, fast, and inexpensive DNA sequencing technology. The simplest form of nanopore DNA sequencing utilizes the hypothesis that individual nucleotides of single-stranded DNA passing through a nanopore will uniquely modulate an ionic current flowing through the pore, allowing the record of the current to yield the DNA sequence. We demonstrate that the ionic current through the engineered Mycobacterium smegmatis porin A, MspA, has the ability to distinguish all four DNA nucleotides and resolve single-nucleotides in single-stranded DNA when double-stranded DNA temporarily holds the nucleotides in the pore constriction. Passing DNA with a series of double-stranded sections through MspA provides proof of principle of a simple DNA sequencing method using a nanopore. These findings highlight the importance of MspA in the future of nanopore sequencing. bionanotechnology | next generation sequencing | single-molecule | stochastic sensing | protein pore www.pnas.org/cgi/doi/10.1073/pnas.1001831107

Proteins responsible for outer membrane transport across the unique membrane structure of Mycobacterium spp. are attractive drug targets in the treatment of human diseases caused by the mycobacterial pathogens, Mycobacterium tuberculosis, M. bovis, M. leprae and M. ulcerans. In contrast with Escherichia coil, relatively few outer-membrane proteins (OMPs) have been identified in Mycobacterium spp., largely due to the difficulties in isolating mycobacterial membrane proteins and our incomplete understanding of secretion mechanisms and cell wall structure in these organisms. To further expand our knowledge of these elusive proteins in mycobacteria, we have improved upon our previous method of OMP prediction in mycobacteria by taking advantage of genomic data from seven mycobacteria species. Our improved algorithm suggests 4333 sequences as putative OMPs in seven species with varying degrees of confidence. The most virulent pathogenic mycobacterial species are slightly enriched in these selected sequences. We present examples of predicted OMPs involved in horizontal transfer and paralogy expansion. Analysis of local secondary structure content allowed identification of small domains predicted to perform as OMPs; some examples show their involvement in events of tandem duplication and domain rearrangements. We discuss the taxonomic distribution of these discovered families and architectures, often specific to mycobacteria or the wider taxonomic class of Actinobacteria. Our results suggest that OMP functionality in mycobacteria is richer than expected and provide a resource to guide future research of these understudied proteins. DOI 10.1099/mic.0.040089-0

DNA--protein interactions that occur during transcription initiation play an important role in regulating gene expression. To initiate transcription, RNA polymerase (RNAP) binds to promoters in a sequence-specific fashion. This is followed by a series of steps governed by the equilibrium binding and kinetic rate constants, which in turn determine the overall efficiency of the transcription process. We present here the first detailed kinetic analysis of promoter--RNAP interactions during transcription initiation in the [[sigma].sup.A]-dependent promoters [P.sub.rrnAPCL1], [P.sub.rrnB] and [P.sub.gyr] of Mycobacterium smegmatis. The promoters show comparable equilibrium binding affinity but differ significantly in open complex formation, kinetics of isomerization and promoter clearance. Furthermore, the two rrn promoters exhibit varied kinetic properties during transcription initiation and appear to be subjected to different modes of regulation. In addition to distinct kinetic patterns, each one of the housekeeping promoters studied has its own rate-limiting step in the initiation pathway, indicating the differences in their regulation. DOI 10.1099/mic.0.038620-0

Class A penicillin-binding proteins (PBPs) are large, bifunctional proteins that are responsible for glycan chain assembly and peptide cross-linking of bacterial peptidoglycan. Bacteria in the genus Mycobacterium have been reported to have only two class A PBPs, PonA1 and PonA2, that are encoded in their genomes. We report here that the genomes of Mycobacterium smegmatis and other soil mycobacteria contain an additional gene encoding a third class A penicillin-binding protein, PonA3, which is a paralog of PonA2. Both the PonA2 and PonA3 proteins contain a penicillin-binding protein and serine/threonine protein kinase-associated (PASTA) domain that we propose may be involved in sensing the cell cycle and a C-terminal proline-rich region (PRR) that may have a role in protein-protein or protein-carbohydrate interactions. We show here that an M. smegmatis [DELTA]ponA2 mutant has an unusual antibiotic susceptibility profile, exhibits a spherical morphology and an altered cell surface in stationary phase, and is defective for stationary-phase survival and recovery from anaerobic culture. In contrast, a [DELTA]ponA3 mutant has no discernible phenotype under laboratory conditions. We demonstrate that PonA2 and PonA3 can bind penicillin and that PonA3 can partially substitute for PonA2 when ponA3 is expressed from a constitutive promoter on a multicopy plasmid. Our studies suggest that PonA2 is involved in adaptation to periods of nonreplication in response to starvation or anaerobiosis and that PonA3 may have a similar role. However, the regulation of PonA3 is likely different, suggesting that its importance could be related to stresses encountered in the environmental niches occupied by M. smegmatis and other soil-dwelling mycobacteria. doi: 10.1128/JB.00025-10

LysB, a mycobacteriophage Ms6-encoded protein, was previously identified as a lipolytic enzyme able to hydrolyse the ester bond in lipase and esterase substrates. In the present work, we show that LysB can hydrolyse lipids containing mycolic acids from the outer membrane of the mycobacterial cell wall. LysB was shown to hydrolyse the mycolic acids from the mycolylarabinogalactan--peptidoglycan complex where the mycolates of the inner leaflet of the outer membrane are covalently attached to an arabinosyl head group. In addition, treatment of the extractable lipids from Mycobacterium smegmatis, Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra with LysB showed that trehalose 6,6'-dimycolate (TDM), a trehalose diester of two mycolic acid molecules, was hydrolysed by the enzyme. We have also determined the structures of the mycolic acid molecules that form the M. smegmatis TDM. The identification of a phage-encoded enzyme that targets the outer membrane of the mycobacterial cell wall enhances our understanding of the mechanism of mycobacteriophage lysis. DOI 10.1099/mic.0.032821-0

Carbon monoxide dehydrogenase (CO-DH) is an enzyme catalysing the oxidation of CO to carbon dioxide in Mycobacterium sp+ strain JC1 DSM 3803. Cloning of the genes encoding CO-DH from the bacterium and sequencing of overlapping clones revealed the presence of duplicated sets of genes for three subunits of the enzyme, cutB1C1A1 and cutB2C2A2, in operons, and a cluster of genes encoding proteins that may be involved in CO metabolism, including a possible transcriptional regulator. Phylogenetic analysis based on the amino acid sequences of large subunite of CO-DH suggested that the CO-DHs of Mycobacterium sp. JC1 and other mycobacteria are distinct from those of other types of bacteria. The growth phenotype of mutant strains lacking cutA genes and of a corresponding complemented strain showed that both of the duplicated sets of CO-DH genes were functional in this bacterium. Transcriptional fusions of the cutB genes with lacZ revealed that the cutBCA operons were expressed regardless of the presence of CO and were further inducible by CO. Primer extension analysis indicated two promoters, one expressed in the absence of CO and the other induced in the presence of CO. This is believed to be the first report to show the presence of multiple copies of CO-DH genes with identical sequences and in close proximity in carboxydobacteria, and to present the genetic evidence for the function of the genes in mycobacteria. DOI 10.1099/mic.0.034769-0

Rifampicin and its derivatives are at the forefront of the current standard chemotherapeutic regimen for active tuberculosis; they act by inhibiting the transcription activity of prokaryotic RNA polymerase. Rifampicin is believed to interact with the [beta] subunit of RNA polymerase. However, it has been observed that protein--protein interactions with RNA polymerase core enzyme lead to its reduced susceptibility to rifampicin. This mechanism became more diversified with the discovery of RbpA, a novel RNA polymerase-binding protein, in Streptomyces coelicolor that could mitigate the effect of rifampicin on RNA polymerase activity. MsRbpA is a homologue of RbpA in Mycobacterium smegmatis. On deciphering the role of MsRbpA in M. smegmatis we found that it interacts with RNA polymerase and increases the rifampicin tolerance levels, both in vitro and in vivo. It interacts with the [beta] subunit of RNA polymerase. However, it was found to be incapable of rescuing rifampicin-resistant RNA polymerases in the presence of rifampicin at the respective [IC.sub.50]. DOI 10.1099/mic.0.033670-0

Mycobacterium sp. strain JC1 is able to grow on methanol as a sole source of carbon and energy using methanol: N,N'-dimethyl-4-nitrosoaniline oxidoreductase (MDO) as a key enzyme for primary methanol oxidation. Purified MDO oxidizes ethanol and formaldehyde as well as methanol. The Mycobacterium sp. strain JC1 gene for MDO (mdo) was cloned, sequenced, and determined to have an open reading frame of 1272 bp. Northern blot and promoter analysis revealed that redo transcription was induced in cells grown in the presence of methanol. Northern blotting together with RT-PCR also showed that the redo gene was transcribed as monocistronic rnRNA. Primer extension analysis revealed that the transcriptional start site of the mdo gene is located 21 bp upstream of the mdo start codon. An mdo-deficient mutant of Mycobacterium sp. strain JC1 did not grow with methanol as a sole source of carbon and energy. DOI 10.1099/mic.0.034124-0

The genus Mycobacterium, which is a member of the high G+C group of Gram-positive bacteria, includes important pathogens, such as M. tuberculosis and M. leprae. A recent publication in PNAS reported that M. marinum and M. bovis bacillus Calmette--Guerin produce a type of spore known as an endospore, which had been observed only in the low G+C group of Gram-positive bacteria. Evidence was presented that the spores were similar to endospores in ultrastructure, in heat resistance and in the presence of dipicolinic acid. Here, we report that the genomes of Mycobacterium species and those of other high G+C Gram-positive bacteria lack orthologs of many, if not all, highly conserved genes diagnostic of endospore formation in the genomes of low G+C Gram-positive bacteria. We also failed to detect the presence of endospores by light microscopy or by testing for heat-resistant colony-forming units in aged cultures of M. marinum. Finally, we failed to recover heat-resistant colony-forming units from frogs chronically infected with M. marinum. We conclude that it is unlikely that Mycobacterium is capable of endospore formation. sporulation | tuberculosis www.pnas.org/cgi/doi/10.1073/pnas.0911299107

Oxidative damage to DNA results in the occurrence of 7,8-dihydro-8-oxoguanine (8-oxoG) in the genome. In eubacteria, repair of such damage is initiated by two major base-excision repair enzymes, MutM and MutY. We generated a MutY-deficient strain of Mycobacterium smegmatis to investigate the role of this enzyme in DNA repair. The MutY deficiency in M. smegmatis did not result in either a noteworthy susceptibility to oxidative stress or an increase in the mutation rate. However, rifampicin-resistant isolates of the MutY-deficient strain showed distinct mutations in the rifampicin-resistance-determining region of rpoB. Besides the expected C to A (or G to T) mutations, an increase in A to C (or T to G) mutations was also observed. Biochemical characterization of mycobacterial MutY (M. smegmatis and M. tuberculosis) revealed an expected excision of A opposite 8-oxoG in DNA. Additionally, excision of G and T opposite 8-oxoG was detected. MutY formed complexes with DNA containing 8-oxoG : A, 8-oxoG : G or 8-oxoG : T but not 8-oxoG : C pairs. Primer extension reactions in cell-free extracts of M. smegmatis suggested error-prone incorporation of nucleotides into the DNA. Based on these observations, we discuss the physiological role of MutY in specific mutation prevention in mycobacteria. DOI 10.1099/mic.0.033621-0

Mycobacterium lepromatosis is a newly discovered leprosy-causing organism. Preliminary phylogenetic analysis of its 16S rRNA gene and a few other gene segments revealed significant divergence from Mycobacterium leprae, a well-known cause of leprosy, that justifies the status of M. lepromatosis as a new species. In this study we analyzed the sequences of 20 genes and pseudogenes (22,814 nucleotides). Overall, the level of matching of these sequences with M. leprae sequences was 90.9%, which substantiated the species-level difference; the levels of matching for the 16S rRNA genes and 14 protein-encoding genes were 98.0% and 93.1%, respectively, but the level of matching for five pseudogenes was only 79.1%. Five conserved protein-encoding genes were selected to construct phylogenetic trees and to calculate the numbers of synonymous substitutions (dS values) and nonsynonymous substitutions (dN values) in the two species. Robust phylogenetic trees constructed using concatenated alignment of these genes placed M. lepromatosis and M. leprae in a tight cluster with long terminal branches, implying that the divergence occurred long ago. The dS and dN values were also much higher than those for other closest pairs of mycobacteria. The dS values were 14 to 28% of the dS values for M. leprae and Mycobacterium tuberculosis, a more divergent pair of species. These results thus indicate that M. lepromatosis and M. leprae diverged ~10 million years ago. The M. lepromatosis pseudogenes analyzed that were also pseudogenes in M. leprae showed nearly neutral evolution, and their relative ages were similar to those of M. leprae pseudogenes, suggesting that they were pseudogenes before divergence. Taken together, the results described above indicate that M. lepromatosis and M. leprae diverged from a common ancestor after the massive gene inactivation event described previously for M. leprae. doi: 10.1128/JB.00762-09

The >120 recognized species of nontuberculous mycobacteria (NTM) share common features: 1) they are facultative pathogens; 2) evidence of human-to-human transmission is lacking; 3) some NTM species are ubiquitous and [...]

Mycolic acids are essential for the survival, virulence and antibiotic resistance of the human pathogen Mycobacterium tuberculosis. Inhibitors of mycolic acid biosynthesis, such as isoniazid and ethionamide, have been used as efficient drugs for the treatment of tuberculosis. However, the increase in cases of multidrug-resistant tuberculosis has prompted a search for new targets and agents that could also affect synthesis of mycolic acids. In mycobacteria, the acyl-CoA carboxylases (ACCases) provide the building blocks for de novo fatty acid biosynthesis by fatty acid synthase (FAS) I and for the elongation of FAS I products by the FAS II complex to produce meromycolic acids. By generating a conditional mutant in the accD6 gene of Mycobacterium smegmatis, we demonstrated that AccD6 is the essential carboxyltransferase component of the ACCase 6 enzyme complex implicated in the biosynthesis of malonyI-CoA, the substrate of the two FAS enzymes of Mycobacterium species. Based on the conserved structure of the AccD5 and AccD6 active sites we screened several inhibitors of AccD5 as potential inhibitors of AccD6 and found that the ligand NCI-172033 was capable of inhibiting AccD6 with an [IC.sub.50] of 8 [micro]M. The compound showed bactericidal activity against several pathogenic Mycobacterium species by producing a strong inhibition of both fatty acid and mycolic acid biosynthesis at minimal inhibitory concentrations. Overexpression of accD6 in M. smegmatis conferred resistance to NCI-172033, confirming AccD6 as the main target of the inhibitor. These results define the biological role of a key ACCase in the biosynthesis of membrane and cell envelope fatty acids, and provide a new target, AccD6, for rational development of novel anti-mycobacterial drugs.

The human pathogen Mycobacterium ulcerans produces a polyketide metabolite called mycolactone with potent immunomodulatory activity. M. ulcerans strain Agy99 has a 174 kb plasmid called pMUM001 with three large genes (mlsA1, 51 kb; mlsA2, 7.2 kb; mlsB, 43 kb) that encode type I polyketide synthases (PKS) required for the biosynthesis of mycolactone, as demonstrated by transposon mutagenesis. However, there have been no reports of transfer of the mls locus to another mycobacterium to demonstrate that these genes are sufficient for mycolactone production because in addition to their large size, the mls genes contain a high level of internal sequence repetition, such that the entire 102 kb locus is composed of only 9.5 kb of unique DNA. The combination of their large size and lack of stability during laboratory passage makes them a challenging prospect for transfer to a more rapidly growing and genetically tractable host. Here we describe the construction of two bacterial artificial chromosome Escherichia coli/ Mycobacterium shuttle vectors, one based on the pMUM001 origin of replication bearing mlsB, and the other based on the mycobacteriophage L5 integrase, bearing mlsA1 and m/sA2. The combination of these two constructs permitted the two-step transfer of the entire 174 kb pMUM001 plasmid to Mycobacterium marinum, a rapidly growing non-mycolactone-producing mycobacterium that is a close genetic relative of M. ulcerans. To improve the stability of the mls locus in M. marinum, recA was inactivated by insertion of a hygromycin-resistance gene using double-crossover allelic exchange. As expected, the ArecA mutant displayed increased susceptibility to UV killing and a decreased frequency of homologous recombination. Southern hybridization and RT-PCR confirmed the stable transfer and expression of the m/s genes in both wild-type M. marinum and the recA mutant. However, neither mycolactone nor its predicted precursor metabolites were detected in either strain. These experiments show that it is possible to successfully manipulate and stably transfer the large mls genes, but that other bacterial host factors appear to be required to facilitate mycolactone production.

D-Alanine is a structural component of mycobacterial peptidoglycan. The primary route of D-alanine biosynthesis in eubacteria is the enantiomeric conversion from L-alanine, a reaction catalysed by D-alanine racemase (Air). Mycobacterium smegmatis air insertion mutants are not dependent on D-alanine for growth and display a metabolic pattern consistent with an alternative pathway for D-alanine biosynthesis. In this study, we demonstrate that the M. smegmatis alr insertion mutant TAM23 can synthesize D-alanine at lower levels than the parental strain. The insertional inactivation of the air gene also decreases the intracellular survival of mutant strains within primary human monocyte-derived macrophages. By complementation studies, we confirmed that the impairment of air gene function is responsible for this reduced survival. Inhibition of superoxide anion and nitric oxide formation in macrophages suppresses the differential survival. In contrast, for bacteria grown in broth, both strains had approximately the same susceptibility to hydrogen peroxide, acidified sodium nitrite, low pH and polymyxin B. In contrast, TAM23 exhibited increased resistance to lysozyme. D-Alanine supplementation considerably increased TAM23 viability in nutritionally deficient media and within macrophages. These results suggest that nutrient deprivation in phagocytic cells combined with killing mediated by reactive intermediates underlies the decreased survival of air mutants. This knowledge may be valuable in the construction of mycobacterial auxotrophic vaccine candidates.

Cultivation of Mycobacterium smegmatis cells in a nitrogen-limited minimal medium (SR-1) followed by prolonged storage at room temperature without shaking resulted in the gradual accumulation of morphologically distinct ovoid forms characterized by (i) low metabolic activity; (ii) elevated resistance to antibiotics and to heat treatment; and (iii) inability to produce colonies on standard agar plates (non-platable cells). Detailed microscopic examination confirmed that ovoid cells possessed an intact cell envelope, specific fine structure and large electron-transparent bodies in the cytoplasm. Cell staining with Nile red and analysis of the lipid content by TLC revealed the presence of significant amounts of apolar lipids in these bodies. The ovoid forms could be stored for significant periods (up to 5 months) and resuscitated afterwards in a modified Sauton's medium. Importantly, resuscitation of ovoid cells was accompanied by their transformation into the typical rod-shaped cells. We suggest that the observed ovoid cells represent dormant forms, resembling morphologically distinct cells of Mycobacterium tuberculosis previously isolated from tuberculosis patients and infected animals.

Cyclic di-GMP (c-di-GMP) plays an important role in bacterial adaptation to enable survival in changing environments. It orchestrates various pathways involved in biofilm formation, changes in the cell surface, host colonization and virulence. In this article, we report the presence of c-di-GMP in Mycobacterium smegmatis, and its role in the long-term survival of the organism. M. smegmatis has a single bifunctional protein with both GGDEF and EAL domains, which show diguanylate cyclase (DGC) and phosphodiesterase (PDE)-A activity, respectively, in vitro. We named this protein MSDGC-1. Deletion of the gene encoding MSDGC-1 did not affect growth and biofilm formation in M. smegmatis, but long-term survival under conditions of nutritional starvation was affected. Most of the proteins that contain GGDEF and EAL domains have been demonstrated to have either DGC or PDE-A activity. To gain further insight into the regulation of the protein, we cloned the individual domains, and tested their respective activities. MSDGC-1, the full-length protein, is required for activity, as its GGDEF and EAL domains are inactive when separated.

Mycobacteriophage L5 is a temperate phage with a broad host range among the fast- and slow-growing mycobacteria such as Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium ulcerans. L5 switches off host protein synthesis during the early stage of lytic growth, as was previously shown by protein expression profiling. Also, lethal genetic elements have been identified in L5 based on the fact that transformants could not be obtained with these genes. Using an inducible mycobacterial shuttle vector, we have identified three ORFs within an early operon of mycobacteriophage L5 which encode gene products (gp) toxic to the host M. smegmatis when expressed. These ORFs, coding for gp77, gp78 and gp79, presumably function as shut-off genes during early stages of phage replication. There is evidence that cell division is affected by one of the proteins (gp79). The transcription of the cytotoxic polypeptides is directed by a promoter situated in ORF83 and transcription control is achieved through the phage repressor gp71, which is shown by co-expression of this protein. The findings presented here should provide useful tools for the molecular genetics of mycobacteria. Further analysis of these and other mycobacteriophage-derived toxic polypeptides, together with the identification of their cellular targets, might provide a tool for the rapid identification of promising drug targets in emerging and re-emerging mycobacterial pathogens.

Buruli ulcer is a necrotizing skin disease caused by Mycobacterium ulcerans and associated with exposure to aquatic habitats. To assess possible transmission of M. ulcerans by aquatic biting insects, we [...]

Mycobacterial species are characterized by the presence of lipid-rich, hydrophobic cell envelopes. These cell envelopes contribute to properties such as roughness of colonies, aggregation of cells in liquid culture without detergent, and biofilm formation. We describe here a mutant strain of Mycobacterium smegmatis, called DL1215, which demonstrates marked deviations from the above-mentioned phenotypes. DL1215 arose spontaneously from a strain deficient for the stringent response (M. smegmatis [DELTA][rel.sub.Msm] strain) and is not a reversion to a wild-type phenotype. The nature of the spontaneous mutation was a single base-pair deletion in the lsr2 gene, leading to the formation of a truncated protein product. The DL1215 strain was complicated by having both inactivated [rel.sub.Msm] and lsr2 genes, and so a single lsr2 mutant was created to analyze the gene's function. The lsr2 gene was inactivated in the wild-type M. smegmatis [mc.sub.2]155 strain by allelic replacement to create strain DL2008. Strain DL2008 shows characteristics unique from those of both the wild-type and [DELTA][rel.sub.Msm] strains, some of which include a greatly enhanced ability to slide over agar surfaces (referred to here as 'hypermotility'), greater resistance to phage infection and to the antibiotic kanamycin, and an inability to form biofilms. Complementation of the DL2008 mutant with a plasmid containing lsr2 (pLSR2) reverts the strain to the [mc.sup.2]155 phenotype. Although these phenotypic differences allude to changes in cell surface lipids, no difference is observed in glycopeptidolipids, polar lipids, apolar lipids, or mycolic acids of the cell wall.

During 1994-2005, we isolated Mycobacterium microti from 5 animals and 4 humans. Only 1 person was immunocompromised. Spoligotyping showed 3 patterns: vole type, llama type, and a new variant llama [...]

Mycobacteria can tolerate relatively high concentrations of triphenylmethane dyes such as malachite green and methyl violet. To identify mycobacterial genes involved in the decolorization of malachite green, a transposon mutant library of Mycobacterium smegmatis [mc.sup.2] 155 was screened for mutants unable to decolorize this dye. One of the genes identified was MSMEG_5126, an orthologue of Mycobacterium bovis fbiC encoding a 7,8-didemethyl-8hydroxy-5-deazariboflavin (FO) synthase, which is essential for the biosynthesis of the electron carrier coenzyme [F.sub.420]. The other gene identified was MSMEG_2392, encoding an alanine-rich protein with a DUF121 domain. The minimum inhibitory concentrations (MICs) for malachite green and methyl violet of the six fbiC mutants and two MSMEG_2392 mutants were one-third and one-fifth, respectively, of the MIC of the parent strain M. smegmatis [mc.sup.2] 155. Representative fbiC and MSMEG_2392 mutant strains were also sensitive to oxidative stress caused by the redox-cycling agents plumbagin and menadione, and the sensitivity was reversed in the complemented strains. HPLC analysis of representative fbiC and MSMEG_2392 strains revealed that, while the fbiC mutant lacked both coenzyme [F.sub.420] and FO, the MSMEG_2392 mutant contained FO but not coenzyme [F.sub.420]. These results indicate that MSMEG_2392 is involved in the biosynthesis of coenzyme [F.sub.420].

Mycobacteriophage Tweety is a newly isolated phage of Mycobacterium smegmatis. It has a viral morphology with an isometric head and a long flexible tail, and forms turbid plaques from which stable lysogens can be isolated. The Tweety genome is 58 692 bp in length, contains 109 protein-coding genes, and shows significant but interrupted nucleotide sequence similarity with the previously described mycobacteriophages Llij, PMC and Che8. However, overall the genome possesses mosaic architecture, with gene products being related to other mycobacteriophages such as Che9d, Omega and Corndog. A gene encoding an integrase of the tyrosine-recombinase family is located close to the centre of the genome, and a putative attP site has been identified within a short intergenic region immediately upstream of int. This Tweety attP-int cassette was used to construct a new set of integration-proficient plasmid vectors that efficiently transform both fast- and slow-growing mycobacteria through plasmid integration at a chromosomal locus containing a [tRNA.sup.Lys] gene. These vectors are maintained well in the absence of selection and are completely compatible with integration vectors derived from mycobacteriophage L5, enabling the simple construction of complex recombinants with genes integrated simultaneously at different chromosomal positions.

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.

The cell envelopes of several species of nontuberculous mycobacteria, including the Mycobacterium avium complex, contain glycopeptidolipids (GPLs) as major glycolipid components. GPLs are highly antigenic surface molecules, and their variant oligosaccharides define each serotype of the M. avium complex. In the oligosaccharide portion of GPLs, the fucose residue is one of the major sugar moieties, but its biosynthesis remains unclear. To elucidate it, we focused on the 5.0-kb chromosomal region of the M. avium complex that includes five genes, two of which showed high levels of similarity to the genes involved in fucose synthesis. For the characterization of this region by deletion and expression analyses, we constructed a recombinant Mycobacterium smegmatis strain that possesses the rtfA gene of the M. avium complex to produce serovar 1 GPL. The results revealed that the 5.0-kb chromosomal region is responsible for the addition of the fucose residue to serovar 1 GPL and that the three genes mdhtA, merA, and gtfD are indispensable for the fucosylation. Functional characterization revealed that the gtfD gene encodes a glycosyltransferase that transfers a fucose residue via 1 [right arrow] 3 linkage to a rhamnose residue of serovar 1 GPL. The other two genes, mdhtA and merA, contributed to the formation of the fucose residue and were predicted to encode the enzymes responsible for the synthesis of fucose from mannose based on their deduced amino acid sequences. These results indicate that the fucosylation pathway in GPL biosynthesis is controlled by a combination of the mdhtA, merA, and gtfD genes. Our findings may contribute to the clarification of the complex glycosylation pathways involved in forming the oligosaccharide portion of GPLs from the M. avium complex, which are structurally distinct.

Phosphate import is required for the growth of mycobacteria and is regulated by environmental inorganic phosphate ([P.sub.i]) concentrations, although the mechanism of this regulation has not been characterized. The expression of genes involved in [P.sub.i] acquisition is frequently regulated by two-component regulatory systems (2CRs) consisting of a sensor histidine kinase and a DNA-binding response regulator. In this work, we have identified the senX3-regX3 2CR as a [P.sub.i]-dependent regulator of genes involved in phosphate acquisition in Mycobacterium smegmatis. Characterization of senX3 mutants with different PhoA phenotypes suggests a dual role for SenX3 as a phosphatase or a phosphodonor for the response regulator RegX3, depending upon [P.sub.i] availability. Expression of PhoA activity required phosphorylation of RegX3, consistent with a role for phosphorylated RegX3 (RegX3~P) as a transcriptional activator of phoA. Furthermore, purified RegX3~P bound to promoter sequences from phoA, senX3, and the high-affinity phosphate transporter component pstS, demonstrating direct transcriptional control of all three genes. DNase I footprinting and primer extension analyses have further defined the DNA-binding region and transcriptional start site within the phoA promoter. A DNA motif consisting of an inverted repeat was identified in each of the promoters bound by RegX3~P. Based upon our findings, we propose a model for [P.sub.i]-regulated gene expression mediated by SenX3-RegX3 in mycobacteria.

Bacterial nonhomologous end joining (NHEJ) is a recently described DNA repair pathway best characterized in mycobacteria. Bacterial NHEJ proteins LigD and Ku have been analyzed biochemically, and their roles in linear plasmid repair in vivo have been verified genetically; yet the contributions of NHEJ to repair of chromosomal DNA damage are unknown. Here we use an extensive set of NHEJ- and homologous recombination (HR)-deficient Mycobacterium smegmatis strains to probe the importance of HR and NHEJ in repairing diverse types of chromosomal DNA damage. An M. smegmatis [DELTA]recA [DELTA]ku double mutant has no apparent growth defect in vitro. Loss of the NHEJ components Ku and LigD had no effect on sensitivity to UV radiation, methyl methanesulfonate, or quinolone antibiotics. NHEJ deficiency had no effect on sensitivity to ionizing radiation in logarithmic- or early-stationary-phase cells but was required for ionizing radiation resistance in late stationary phase in 7H9 but not LB medium. In addition, NHEJ components were required for repair of I-SceI mediated chromosomal double-strand breaks (DSBs), and in the absence of HR, the NHEJ pathway rapidly mutates the chromosomal break site. The molecular outcomes of NHEJ-mediated chromosomal DSB repair involve predominantly single-nucleotide insertions at the break site, similar to previous findings using plasmid substrates. These findings demonstrate that prokaryotic NHEJ is specifically required for DSB repair in late stationary phase and can mediate mutagenic repair of homing endonuclease-generated chromosomal DSBs.

Mycobacterium vanbaalenii PYR-1 is capable of degrading a wide range of high-molecular-weight polycyclic aromatic hydrocarbons (PAHs), including fluoranthene. We used a combination of metabolomic, genomic, and proteomic technologies to investigate fluoranthene degradation in this strain. Thirty-seven fluoranthene metabolites including potential isomers were isolated from the culture medium and analyzed by high-performance liquid chromatography, gas chromatography-mass spectrometry, and UV-visible absorption. Total proteins were separated by one-dimensional gel and analyzed by liquid chromatography-tandem mass spectrometry in conjunction with the M. vanbaalenii PYR-1 genome sequence (http://jgi.doe.gov), which resulted in the identification of 1,122 proteins. Among them, 53 enzymes were determined to be likely involved in fluoranthene degradation. We integrated the metabolic information with the genomic and proteomic results and proposed pathways for the degradation of fluoranthene. According to our hypothesis, the oxidation of fluoranthene is initiated by dioxygenation at the C-1,2, C-2,3, and C-7,8 positions. The C-1,2 and C-2,3 dioxygenation routes degrade fluoranthene via fluorene-type metabolites, whereas the C-7,8 routes oxidize fluoranthene via acenaphthylene-type metabolites. The major site of dioxygenation is the C-2,3 dioxygenation route, which consists of 18 enzymatic steps via 9-fluorenone-l-carboxylic acid and phthalate with the initial ring-hydroxylating oxygenase, NidA3B3, oxidizing fluoranthene to fluoranthene cis-2,3-dihydrodiol. Nonspecific monooxygenation of fluoranthene with subsequent O methylation of dihydroxyfluoranthene also occurs as a detoxification reaction.

Many processes that are essential for mycobacterial growth are poorly understood. To facilitate genetic analyses of such processes in mycobacteria, we and others have developed regulated expression systems that are repressed by a tetracycline repressor (TetR) and induced with tetracyclines, permitting the construction of conditional mutants of essential genes. A disadvantage of these systems is that tetracyclines function as transcriptional inducers and have to be removed to initiate gene silencing. Recently, reverse TetR mutants were identified that require tetracyclines as corepressors. Here, we report that one of these mutants, TetR r1.7, allows efficient repression of lacZ expression in Mycobacterium smegmatis in the presence but not the absence of anhydrotetracycline (atc). TetR and TetR r1.7 also allowed efficient silencing of the essential secA1 gene, as demonstrated by inhibition of the growth of a conditional mutant and dose-dependent depletion of the SecA1 protein after the removal or addition, respectively, of atc. The kinetics of SecA1 depletion were similar with TetR and TetR r1.7. To test whether silencing of secA1 could help identify substrates of the general secretion pathway, we analyzed the main porin of M. smegmatis, MspA. This showed that the amount of cell envelope-associated MspA decreased more than 90-fold after secA1 silencing. We thus demonstrated that TetR r1.7 allows the construction of conditional mycobacterial mutants in which the expression of an essential gene can be efficiently silenced by the addition of atc and that gene silencing permits the identification of candidate substrates of mycobacterial secretion systems.

Elucidation of the transmission, epidemiology, and evolution of Mycobacterium ulcerans, the causative agent of Buruli ulcer, is hampered by the striking lack of genetic diversity of this emerging pathogen. However, [...]

Neutrophils contain antimicrobial peptides with antituberculous activity, but their contribution to immune resistance to tuberculosis (TB) infection has not been previously investigated to our knowledge. We determined differential white cell [...]

Modified [beta]-cyclodextrins have been shown previously to enhance sterol conversion to 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) by growing Mycobacterium spp. The enhancement effect was mainly attributed to steroid solubilization by the formation of inclusion complexes with modified cyclodextrins. In this work, the influence of randomly methylated [beta]-cyclodextrin (MCD) on the growth, AD- and ADD-producing activity, cell wall (CW) composition and ultrastructure of sterol-transforming Mycobacterium sp. VKM Ac-1816D was studied. The specific growth rate of the strain on glycerol increased in the presence of MCD (20-100 mM). Washed cells grown in the presence of MCD (20-40 mM) expressed 1.6-fold higher ADD-producing activity than did the cells grown without MCD, and their adhesiveness differed. Electron microscopy showed MCD-mediated CW exfoliation and accumulation of membrane-like structures outside the cells, while preserving cells intact. The analysis of CW composition revealed both a decrease in the proportion of extractable lipids and a considerable shift in fatty acid profile resulting from MCD action. The MCD-mediated enhancement of mycolic and fatty acids content was observed outside the cells. The total secreted protein level rose 2.4-fold, and the extracellular 3-hydroxysteroid oxidase activity 3.2-fold. The composition of the CW polysaccharide was not altered, while the overall proportion of the carbohydrates in the CW of the MCD-exposed mycobacteria increased. The results showed that the multiple mechanisms of MCD-mediated intensification of sterol to AD(D) conversion by mycobacteria include not only solubilization of steroids, but also the increase of CW permeability for both steroids and soluble nutrients, disorganization of the lipid bilayer and the release of steroid-transforming enzymes weakly associated with the CW.

Chronic tuberculosis represents a major health problem for one-third of the world's population today. A key question relevant to chronic tuberculosis is the physiological status of Mycobacterium tuberculosis during this important stage of infection. To examine the molecular bases of chronic tuberculosis and the role of host immunity in mycobacterial growth, we determined the mycobacterial transcriptional profiles during chronic and reactivation phases of murine tuberculosis using in vivo microarray analysis (IVMA). Following 28 days of aerosol infection, mycobacterial counts remained stable, although the bacilli were metabolically active with a 50% active transcriptome. The expression of genes involved in lipid and carbohydrate pathways was significantly enriched during the middle stage of chronic tuberculosis, suggesting a nutrient-rich microenvironment. A total of 137 genes were significantly regulated in mid-chronic tuberculosis (45 and 60 days) compared to an early stage (14 days) of infection. Additional sets of genes, including the virulence regulator virS, were up-regulated during the reactivation stage, indicating their possible roles in mycobacterial resurgence. Interestingly, a set of potential transcriptional regulators was significantly induced at the late stage of chronic tuberculosis. Bioinformatic analysis identified a large number of genes that could be regulated by one of the potential transcriptional regulators encoded by rv0348, including the sigF operon. Taken together, IVMA provided a better definition of the transcriptional machinery activated during chronic and reactivation stages of tuberculosis and identified a novel transcriptional regulator. A similar approach can be adopted to study key stages of intracellular pathogens.

We describe mycobacterial phospholipase A activity (MPLA) and, using reverse genetics, have associated this activity with putative mycobacterial cutinase. PLAs, which hydrolyze fatty acids on phospholipids, play a significant role in human inflammatory states and disease pathogenesis. In prokaryotes, the recognition of their role in virulence is more recent. Cutinases are serine esterases whose primary substrate is cutin, the waxy exterior layer of plants. Mycobacterium tuberculosis has maintained seven putative cutinases, though it should not encounter cutin; we demonstrate that known cutinases and MPLA cleave phospholipids in a PLA-type manner and also hydrolyze Tween. We analyzed cutinase motifs in mycobacteria and found the motif very prevalent. All mycobacteria tested had MPLA activity. These studies suggest an alternative use for putative cutinases by the M. tuberculosis group that is likely related to MPLA activity and lipid metabolism.

The adaptation of the tubercle bacillus to the host environment is likely to involve a complex set of gene regulatory events and physiological switches in response to environmental signals. In order to deconstruct the physiological state of Mycobacterium tuberculosis in vivo, we used a chemostat model to study a single aspect of the organism's in vivo state, slow growth. Mycobacterium bovis BCG was cultivated at high and low growth rates in a carbon-limited chemostat, and transcriptomic analysis was performed to identify the gene regulation events associated with slow growth. The results demonstrated that slow growth was associated with the induction of expression of several genes of the dormancy survival regulon. There was also a striking overlap between the transcriptomic profile of BCG in the chemostat model and the response of M. tuberculosis to growth in the macrophage, implying that a significant component of the response of the pathogen to the macrophage environment is the response to slow growth in carbon-limited conditions. This demonstrated the importance of adaptation to a low growth rate to the virulence strategy of M. tuberculosis and also the value of the chemostat model for deconstructing components of the in vivo state of this important pathogen.

A multigene sequencing approach was used for the identification and molecular typing of environmental mycobacteria of the fast growing subgroup by isolating strain from hemodialysis water and clinical samples. The results indicate high genetic diversity among the isolates obtained, which might be new species of the genus.

Phosphorus is an essential nutrient, but how phosphates cross the mycobacterial cell wall is unknown. Phosphatase activity in whole cells of Mycobacterium smegmatis was significantly lower than that in lysed cells, indicating that access to the substrate was restricted. The loss of the outer membrane (OM) porin MspA also reduced the phosphatase activity in whole cells compared to that in lysed cells. A similar result was obtained for M. smegmatis that overexpressed endogenous alkaline phosphatase, indicating that PhoA is not a surface protein, contrary to a previous report. The uptake of phosphate by a mutant lacking the porins MspA and MspC was twofold lower than that by wild-type M. smegmatis. Strikingly, the loss of these porins resulted in a severe growth defect of M. smegmatis on low-phosphate plates. We concluded that the OM of M. smegmatis represents a permeability barrier for phosphates and that Msp porins are the only OM channels for the diffusion of phosphate in M. smegmatis. However, phosphate diffusion through Msp pores is rather inefficient as shown by the 10-fold lower permeability of M. smegmatis for phosphate compared to that for glucose. This is likely due to the negative charges in the constriction zone of Msp porins. The phosphatase activity in whole cells of Mycobacterium bovis BCG was significantly less than that in lysed cells, indicating a similar uptake pathway for phosphates in slow-growing mycobacteria. However, porins that could mediate the diffusion of phosphates across the OM of M. bovis BCG and Mycobacterium tuberculosis are unknown.

It had been assumed that production of the cytotoxie polyketide mycolactone was strictly associated with Mycobacterium ulcerans, the causative agent of Buruli ulcer. However, a recent study has uncovered a broader distribution of mycolactone-producing mycobacteria (MPM) that includes mycobacteria cultured from diseased fish and frogs in the United States and from diseased fish in the Red and Mediterranean Seas. All of these mycobacteria contain versions of the M. ulcerans pMUM plasmid, produce mycolactones, and show a high degree of genetic relatedness to both M. ulcerans and Mycobacterium marinum. Here, we show by multiple genetic methods, including multilocus sequence analysis and DNA-DNA hybridization, that all MPM have evolved from a common M. marinum progenitor to form a genetically cohesive group among a more diverse assemblage of M. marinum strains. Like M. ulcerans, the fish and frog MPM show multiple copies of the insertion sequence IS2404. Comparisons of pMUM and chromosomal gene sequences demonstrate that plasmid acquisition and the subsequent ability to produce mycolactone were probably the key drivers of speciation. Ongoing evolution among MPM has since produced at least two genetically distinct ecotypes that can be broadly divided into those typically causing disease in ectotherms (but also having a high zoonotic potential) and those causing disease in endotherms, such as humans.