Mycolicibacterium nivoides sp. nov isolated from a peat bog.

A fast-growing, non-chromogenic, acid-fast-staining bacterium (DL90T) was isolated from a peat bog in northern Minnesota. On the basis of 16S rRNA gene sequence similarity (99.8 % identity with Mycolicibacterium septicum and 98 % with Mycolicibacterium peregrinum) and chemotaxonomic data (fatty acid content), strain DL90T represents a member of the genus Mycolicibacterium. Physiological tests (growth curves, biofilm formation, antibiotic sensitivity, colony morphologies and heat tolerance) and biochemical analysis (arylsulfatase activity and fatty acid profiles) distinguish DL90T from its closest relative M. septicum. Phylogenomic reconstruction of the 'Fortuitium-Vaccae' clade, digital DNA-DNA hybridization (DDH) values of 61 %, and average nucleotide identity (ANI) values of approximately 95 % indicate that DL90T is likely to be diverged from M. septicum. Thus, we propose that DL90T represents a novel species, given the name Mycolicibacterium nivoides with the type strain being isolate DL90T (=JCM 32796T=NCCB 100660T).

Mycobacterium sarraceniae sp. nov. and Mycobacterium helvum sp. nov., isolated from the pitcher plant Sarracenia purpurea.

Several fast- to intermediate-growing, acid-fast, scotochromogenic bacteria were isolated from Sarracenia purpurea pitcher waters in Minnesota sphagnum peat bogs. Two strains (DL734T and DL739T) were among these isolates. On the basis of 16S rRNA gene sequences, the phylogenetic positions of both strains is in the genus Mycobacterium with no obvious relation to any characterized type strains of mycobacteria. Phenotypic characterization revealed that neither strain was similar to the type strains of known species of the genus Mycobacterium in the collective properties of growth, pigmentation or fatty acid composition. Strain DL734T grew at temperatures between 28 and 32 °C, was positive for 3-day arylsulfatase production, and was negative for Tween 80 hydrolysis, urease and nitrate reduction. Strain DL739T grew at temperatures between 28 and 37 °C, and was positive for Tween 80 hydrolysis, urea, nitrate reduction and 3-day arylsulfatase production. Both strains were catalase-negative while only DL739T grew with 5 % NaCl. Fatty acid methyl ester profiles were unique for each strain. DL739T showed an ability to survive at 8 °C with little to no cellular replication and is thus considered to be psychrotolerant. Therefore, strains DL734T and DL739T represent two novel species of the genus Mycobacterium with the proposed names Mycobacterium sarraceniae sp. nov. and Mycobacterium helvum sp. nov., respectively. The type strains are DL734T (=JCM 30395T=NCCB 100519T) and DL739T (=JCM 30396T=NCCB 100520T), respectively.

A novel mechanism of growth phase-dependent tolerance to isoniazid in mycobacteria.

Tuberculosis remains one of the most deadly infectious diseases worldwide and is a leading public health problem. Although isoniazid (INH) is a key drug for the treatment of tuberculosis, tolerance to INH necessitates prolonged treatment, which is a concern for effective tuberculosis chemotherapy. INH is a prodrug that is activated by the mycobacterial enzyme, KatG. Here, we show that mycobacterial DNA-binding protein 1 (MDP1), which is a histone-like protein conserved in mycobacteria, negatively regulates katG transcription and leads to phenotypic tolerance to INH in mycobacteria. Mycobacterium smegmatis deficient for MDP1 exhibited increased expression of KatG and showed enhanced INH activation compared with the wild-type strain. Expression of MDP1 was increased in the stationary phase and conferred growth phase-dependent tolerance to INH in M. smegmatis. Regulation of KatG expression is conserved between M. smegmatis and Mycobacterium tuberculosis complex. Artificial reduction of MDP1 in Mycobacterium bovis BCG was shown to lead to increased KatG expression and susceptibility to INH. These data suggest a mechanism by which phenotypic tolerance to INH is acquired in mycobacteria.

Mycobacterium minnesotense sp. nov., a photochromogenic bacterium isolated from sphagnum peat bogs.

Several intermediate-growing, photochromogenic bacteria were isolated from sphagnum peat bogs in northern Minnesota, USA. Acid-fast staining and 16S rRNA gene sequence analysis placed these environmental isolates in the genus Mycobacterium, and colony morphologies and PCR restriction analysis patterns of the isolates were similar. Partial sequences of hsp65 and dnaJ1 from these isolates showed that Mycobacterium arupense ATCC BAA-1242(T) was the closest mycobacterial relative, and common biochemical characteristics and antibiotic susceptibilities existed between the isolates and M. arupense ATCC BAA-1242(T). However, compared to nonchromogenic M. arupense ATCC BAA-1242(T), the environmental isolates were photochromogenic, had a different mycolic acid profile and had reduced cell-surface hydrophobicity in liquid culture. The data reported here support the conclusion that the isolates are representatives of a novel mycobacterial species, for which the name Mycobacterium minnesotense sp. nov. is proposed. The type strain is DL49(T) (=DSM 45633(T) = JCM 17932(T) = NCCB 100399(T)).

Ex vivo Platforms to Study the Primary and Recall Immune Responses to Intracellular Mycobacterial Pathogens and Peptide-Based Vaccines.

Progress in the study of the immune response to pathogens and candidate vaccines has been impeded by limitations in the methods to study the functional activity of T-cell subsets proliferating in response to antigens processed and presented by antigen presenting cells (APC). As described in this review, during our studies of the bovine immune response to a candidate peptide-based vaccine and candidate rel deletion mutants in Mycobacterium avium paratuberculosis (Map) and Mycbacterium bovis (BCG), we developed methods to study the primary and recall CD4 and CD8 T-cell responses using an ex vivo platform. An assay was developed to study intracellular killing of bacteria mediated by CD8 T cells using quantitative PCR to distinguish live bacteria from dead bacteria in a mixed population of live and dead bacteria. Through use of these assays, we were able to demonstrate vaccination with live rel Map and BCG deletion mutants and a Map peptide-based vaccine elicit development of CD8 cytotoxic T cells with the ability to kill intracellular bacteria using the perforin-granzyme B pathway. We also demonstrated tri-directional signaling between CD4 and CD8 T cells and antigen-primed APC is essential for eliciting CD8 cytotoxic T cells. Herein, we describe development of the assays and review progress made through their use in the study of the immune response to mycobacterial pathogens and candidate vaccines. The methods obviate some of the major difficulties encountered in characterizing the cell-mediated immune response to pathogens and development of attenuated and peptide-based vaccines.

Small acid-soluble proteins with intrinsic disorder are required for UV resistance in Myxococcus xanthus spores.

Bacterial sporulation in Gram-positive bacteria results in small acid-soluble proteins called SASPs that bind to DNA and prevent the damaging effects of UV radiation. Orthologs of Bacillus subtilis genes encoding SASPs can be found in many sporulating and nonsporulating bacteria, but they are noticeably absent from spore-forming, Gram-negative Myxococcus xanthus. This is despite the fact that M. xanthus can form UV-resistant spores. Here we report evidence that M. xanthus produces its own unique group of low-molecular-weight, acid-soluble proteins that facilitate UV resistance in spores. These M. xanthus-specific SASPs vary depending upon whether spore formation is induced by starvation inside cell aggregations of fruiting bodies or is induced artificially by glycerol induction. Molecular predictions indicate that M. xanthus SASPs may have some association with the cell walls of M. xanthus spores, which may signify a different mechanism of UV protection than that seen in Gram-positive spores.

Role of phase variation in the resistance of Myxococcus xanthus fruiting bodies to Caenorhabditis elegans predation.

The phenomenon of phase variation between yellow and tan forms of Myxococcus xanthus has been recognized for several decades, but it is not known what role this variation may play in the ecology of myxobacteria. We confirm an earlier report that tan variants are disproportionately more numerous in the resulting spore population of a M. xanthus fruiting body than the tan vegetative cells that contributed to fruiting body formation. However, we found that tan cells may not require yellow cells for fruiting body formation or starvation-induced sporulation of tan cells. Here we report three differences between the yellow and tan variants that may play important roles in the soil ecology of M. xanthus. Specifically, the yellow variant is more capable of forming biofilms, is more sensitive to lysozyme, and is more resistant to ingestion by bacteriophagous nematodes. We also show that the myxobacterial fruiting body is more resistant to predation by worms than are dispersed M. xanthus cells.

Deletion of the histone-like protein (Hlp) from Mycobacterium smegmatis results in increased sensitivity to UV exposure, freezing and isoniazid.

Adaptation to environmental stress is an important survival characteristic of any bacterial species. As a soil-dwelling saprophyte, Mycobacterium smegmatis is exposed to factors such as UV light and rounds of freezing and thawing that occur in temperate climates. Numerous studies in Escherichia coli have linked histone-like proteins to stress resistance and adaptation. We hypothesized that the 'histone-like' protein Hlp might likewise be involved in the stress response of M. smegmatis. The hlp gene was inactivated and the M. smegmatis Δhlp strain was found to be more susceptible to UV light and to the stress created by repeated cycles of freezing and thawing. In addition, loss of Hlp altered the colony morphology and allowed the organism to grow dispersed in the absence of a detergent, suggesting changes in the cell wall composition. As cell wall changes could affect permeability to certain antibiotics, the susceptibility of M. smegmatis Δhlp to kanamycin, rifamipicin, ethambutol and isoniazid (INH) was tested. M. smegmatis Δhlp was more susceptible to INH, but loss of Hlp did not affect susceptibility to the other antibiotics tested. This suggests that the increased sensitivity of M. smegmatis Δhlp to INH was unlikely to be the result of alterations in cell permeability.

Inactivation of lsr2 results in a hypermotile phenotype in Mycobacterium smegmatis.

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(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(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(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(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(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.

Demonstration of allelic exchange in the slow-growing bacterium Mycobacterium avium subsp. paratuberculosis, and generation of mutants with deletions at the pknG, relA, and lsr2 loci.

Mycobacterium avium subsp. paratuberculosis is the causative pathogen of Johne's disease, a chronic inflammatory wasting disease in ruminants. This disease has been difficult to control because of the lack of an effective vaccine. To address this need, we adapted a specialized transduction system originally developed for M. tuberculosis and modified it to improve the efficiency of allelic exchange in order to generate site-directed mutations in preselected M. avium subsp. paratuberculosis genes. With our novel optimized method, the allelic exchange frequency was 78 to 100% and the transduction frequency was 1.1 x 10(-7) to 2.9 x 10(-7). Three genes were selected for mutagenesis: pknG and relA, which are genes that are known to be important virulence factors in M. tuberculosis and M. bovis, and lsr2, a gene regulating lipid biosynthesis and antibiotic resistance. Mutants were successfully generated with a virulent strain of M. avium subsp. paratuberculosis (M. avium subsp. paratuberculosis K10) and with a recombinant K10 strain expressing the green fluorescent protein gene, gfp. The improved efficiency of disruption of selected genes in M. avium subsp. paratuberculosis should accelerate development of additional mutants for vaccine testing and functional studies.

Electron microscopy analysis of Mycobacterium tuberculosis cell division.

The ultrastructure of Mycobacterium tuberculosis cells undergoing division was examined by electron microscopy. Two features of cell division were observed and are described here. First, cells are capable of undergoing a type of "snapping" postfission movement. This movement is likely due to a multi-layered cell wall in which the inner layer participates in septum formation while the outer layer ruptures first on one side. A second feature related to cell division is the ability of dividing cells to form transient branching structures.

The wag31 gene of Mycobacterium tuberculosis is positively regulated by the stringent response.

The stringent response of Mycobacterium tuberculosis is coordinated by Rel and is required for full virulence in animal models. A serological-based approach identified Wag31(Mtb) as a protein that is upregulated in M. tuberculosis in a rel-dependent manner. This positive regulation was confirmed by analysis of M. tuberculosis mRNA expression. Mycobacterium smegmatis was used to confirm that the expression of wag31(Mtb) from its native promoter is positively regulated by the stringent response. Furthermore, elevated wag31(Mtb) expression in M. smegmatis drastically alters the cell-surface hydrophobic properties.

Identification of major sporulation proteins of Myxococcus xanthus using a proteomic approach.

Myxococcus xanthus is a soil-dwelling, gram-negative bacterium that during nutrient deprivation is capable of undergoing morphogenesis from a vegetative rod to a spherical, stress-resistant spore inside a domed-shaped, multicellular fruiting body. To identify proteins required for building stress-resistant M. xanthus spores, we compared the proteome of liquid-grown vegetative cells with the proteome of mature fruiting body spores. Two proteins, protein S and protein S1, were differentially expressed in spores, as has been reported previously. In addition, we identified three previously uncharacterized proteins that are differentially expressed in spores and that exhibit no homology to known proteins. The genes encoding these three novel major spore proteins (mspA, mspB, and mspC) were inactivated by insertion mutagenesis, and the development of the resulting mutant strains was characterized. All three mutants were capable of aggregating, but for two of the strains the resulting fruiting bodies remained flattened mounds of cells. The most pronounced structural defect of spores produced by all three mutants was an altered cortex layer. We found that mspA and mspB mutant spores were more sensitive specifically to heat and sodium dodecyl sulfate than wild-type spores, while mspC mutant spores were more sensitive to all stress treatments examined. Hence, the products of mspA, mspB, and mspC play significant roles in morphogenesis of M. xanthus spores and in the ability of spores to survive environmental stress.

Expression, production and release of the Eis protein by Mycobacterium tuberculosis during infection of macrophages and its effect on cytokine secretion.

The eis gene of Mycobacterium tuberculosis has been shown to play a role in the survival of the avirulent Mycobacterium smegmatis within the macrophage. In vitro and in vivo analysis of Deltaeis deletion mutants and complemented strains showed no effect on survival of M. tuberculosis in U-937 macrophages or in a mouse aerosol infection model, respectively. Further studies were done in an attempt to determine the role of eis in M. tuberculosis intracellular survival and to define a phenotypic difference between wild-type and the Deltaeis deletion mutant. Bioinformatic analysis indicated that Eis is an acetyltransferase of the GCN5-related family of N-acetyltransferases. Immunofluorescence microscopy and Western blot analysis studies demonstrated that Eis is released into the cytoplasm of M. tuberculosis-infected U-937 macrophages. Eis was also found in the extravesicular fraction and culture supernatant of M. tuberculosis-infected macrophages. The effect of Eis on human macrophage cytokine secretion was also examined. Eis modulated the secretion of IL-10 and TNF-alpha by primary human monocytes in response both to infection with M. tuberculosis and to stimulation with recombinant Eis protein. These results suggest that Eis is a mycobacterial effector that is released into the host cell to modulate inflammatory responses, possibly via transcriptional or post-translational means.

CbgA, a protein involved in cortex formation and stress resistance in Myxococcus xanthus spores.

CbgA plays a role in cortex formation and the acquisition of a subset of stress resistance properties in Myxococcus xanthus spores. The cbgA mutant produces spores with thin or no cortex layers, and these spores are more sensitive to heat and sodium dodecyl sulfate than their wild-type counterparts.

Scanning electron microscopy analysis of aged Mycobacterium tuberculosis cells.

Most electron microscopy studies of Mycobacterium tuberculosis ultrastructure were performed in the 1950s and 1960s and lack high resolution by modern standards. This study was performed to re-evaluate the fine structure of M. tuberculosis using modern scanning electron microscopy. Bacteria were grown in rich medium with a constant supply of oxygen for several weeks. Results show that surface bleb-like structures accumulate as cultures age. The most unusual feature of aging M. tuberculosis cultures is that they develop extracellular fibrils, which could play roles of adhering cells to surfaces and to one another.

The relA homolog of Mycobacterium smegmatis affects cell appearance, viability, and gene expression.

The modification of metabolic pathways to allow for a dormant lifestyle appears to be an important feature for the survival of pathogenic bacteria within their host. One regulatory mechanism for persistent Mycobacterium tuberculosis infections is the stringent response. In this study, we analyze the stringent response of a nonpathogenic, saprophytic mycobacterial species, Mycobacterium smegmatis. The use of M. smegmatis as a tool for studying the mycobacterial stringent response was demonstrated by measuring the expression of two M. tuberculosis genes, hspX and eis, in M. smegmatis in the presence and absence of rel(Msm). The stringent response plays a role in M. smegmatis cellular and colony formation that is suggestive of changes in the bacterial cell wall structure.

The role of RelMtb-mediated adaptation to stationary phase in long-term persistence of Mycobacterium tuberculosis in mice.

Long-term survival of nonreplicating Mycobacterium tuberculosis (Mtb) is ensured by the coordinated shutdown of active metabolism through a broad transcriptional program called the stringent response. In Mtb, this response is initiated by the enzymatic action of RelMtb and deletion of relMtb produces a strain (H37RvDeltarelMtb) severely compromised in the maintenance of long-term viability. Although aerosol inoculation of mice with H37RvDeltarelMtb results in normal initial bacterial growth and containment, the ability of this strain to sustain chronic infection is severely impaired. Significant histopathologic differences were noted in lungs and spleens of mice infected with H37RvDeltarelMtb compared with controls throughout the course of the infection. Microarray analysis revealed that H37RvDeltarelMtb suffers from a generalized alteration of the transcriptional apparatus, as well as specific changes in the expression of virulence factors, cell-wall biosynthetic enzymes, heat shock proteins, and secreted antigens that may alter immune recognition of the recombinant organism. Thus, RelMtb is critical for the successful establishment of persistent infection in mice by altering the expression of antigenic and enzymatic factors that may contribute to successful latent infection.

Analysis of the immune response to Mycobacterium avium subsp. paratuberculosis in experimentally infected calves.

Johne's disease of cattle is widespread and causes significant economic loss to producers. Control has been hindered by limited understanding of the immune response to the causative agent, Mycobacterium avium subsp. paratuberculosis, and lack of an effective vaccine and sensitive specific diagnostic assays. The present study was conducted to gain insight into factors affecting the immune response to M. avium subsp. paratuberculosis. A persistent proliferative response to M. avium subsp. paratuberculosis purified protein derivative and soluble M. avium subsp. paratuberculosis antigens was detected in orally infected neonatal calves 6 months postinfection (p.i.) by flow cytometry (FC). CD4(+) T cells with a memory phenotype (CD45R0(+)) expressing CD25 and CD26 were the predominant cell type responding to antigens. Few CD8(+) T cells proliferated in response to antigens until 18 months p.i. gammadelta T cells did not appear to respond to antigen until 18 months p.i. The majority of WC1(+) CD2(-) and a few WC1(-) CD2(+) gammadelta T cells expressed CD25 at time zero. By 18 months, however, subsets of gammadelta T cells from both control and infected animals showed an increase in expression of CD25, ACT2, and CD26 in the presence of the antigens. Two populations of CD3(-) non-T non-B null cells, CD2(+) and CD2(-), proliferated in cell cultures from some control and infected animals during the study, with and without antigen. The studies clearly show multicolor FC offers a consistent reliable way to monitor the evolution and changes in the immune response to M. avium subsp. paratuberculosis that occur during disease progression.