Differentiation of a rapidly growing,scotochromogenic, polycyclic-aromatic-hydrocarbon-metabolizing strain of Mycobacterium sp. from other known Mycobacterium species

A rapidly-growing, acid-alcohol fast, scotochromogenic, polycyclic-aromatic-hydrocarbon-degrading Mycobacterium sp. isolate, Pyr-1, which was different from known Mycobacterium species based on biochemical tests, was further analyzed to compare its mycolic acids, cellular proteins, and nucleic acids with those of known species. Mass spectral analysis of the mycolic acids of Mycobacterium sp. Pyr-1 indicated that its mycolic acids were C₆₀H₁₂₀O₃ and C₆₂H₁₂₄O₃. The mycolic acid pattern from this bacterium was compared to those of 29 rapidly-growing, scotochromogenic species and 31 other species of Mycobacterium by reversed-phase high-performance liquid chromatography (HPLC). The mycolic acid pattern was unique, most closely resembling M. austroafricanum but also resembling M. parafortuitum and M. gilvum. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of soluble cellular proteins also readily differentiated this isolate from other species. The polypeptide pattern of Mycobacterium sp. Pyr-1 most closely resembled that of M. austroafricanum. Restriction enzyme analysis and Southern blot hybridization, however, revealed differences between the chromosomal DNA of our isolate and that of M. austroafricanum. The unique biochemical characteristics, mycolic acid pattern, polypeptide fingerprints, DNA restriction digest patterns, and DNA homology indicate that this strain is different from previously known species of mycobacteria. Since this bacterium is efficient in the metabolism of polycyclic aromatic hydrocarbons, its characteristics and relationships to other Mycobacterium species are reported here.     

Calmodulin-like protein and the phospholipids of Mycobacterium smegmatis

When Mycobacterium smegmatis TMC1546 was grown at different concentrations of glucose supplemented to a synthetic medium already containing 2% v/v glycerol, the following changes were observed. Amount of calmodulin-like protein (CAMLP), total and individual phospholipids (PLs) namely phosphatidylethanolamine, cardiolipin, phosphatidylglycerol and phosphatidylinositol mannosides and total lipids and growth increased up to 5% w/v but decreased at higher concentrations of glucose (7.5% w/v and above). Cyclic AMP content of the whole cells decreased continuously with increase in glucose concentration in the medium. Incorporation of 32Pi into total phospholipids was inhibited by two calmodulin antagonists trifluoperazine and phenothiazine (50% at 40 microM) and the calcium-specific chelator ethylene glycol bis (beta-aminoethyl ether) N,N,N',N'-tetraacetate (EGTA) 35% at 2 mM. Total lipids, CAMLP and growth of this organism are also modulated in a similar way in response to the glucose concentration in the growth medium. Taking these observations together it is suggested that CAMLP has some effect on the metabolism of PLs.     

Asymmetric distribution of phospholipids in membranes from Mycobacterium phlei

The distribution of phospholipids in the membranes of Mycobacterium phlei has been studied by the use of phospholipase C and trinitrobenzenesulfonic acid. In inverted membrane vesicles, whose external surface apparently corresponds topologically to the cytoplasmic surface of the membrane in intact cells, 80% of the phosphatidyl ethanolamine, 24% of diphosphatidyl glycerol, and 13% of phosphatidyl inositol are accessible to cleavage by phospholipase C. These results are in agreement with the finding that 70–75% of phosphatidyl ethanolamine in the membrane is accessible to chemical modification by trinitrobenzenesulfonic acid or dimethylsuberimidate at 4 °C. It can be inferred that in the inverted membrane the majority of phosphatidyl ethanolamine is present on the outer half of the lipid bilayer while inner half constitutes primarily other phospholipids namely phosphatidyl inositol and diphosphatidyl glycerol. Phospholipase C treatment of ETP membranes selectively impairs the active transport of Ca²⁺ without affecting the generation of a proton gradient, respiration, and coupled phosphorylation.     

NADH:ferredoxin reductase and NAD-reducing hydrogenase activities in Hydrogenobacter thermophilus strain TK-6

Abstract NADH:ferredoxin reductase (EC 1.18.1.3) and NAD-reducing hydrogenase (EC 1.12.1.2) activities were detected in the cytoplasm of Hydrogenobacter thermophilus TK-6. NADH:ferredoxin reductase activity was detected using metronidazole, an artificial electron acceptor, which reacts specifically with reduced ferredoxin. Soluble NAD-reducing hydrogenase activity was detected after extended preincubation. The lag disappeared when cell-free extract was incubated anaerobically for more than 30 min. The electron transport system of this chemolithoautotrophic bacterium is discussed.     

Resuscitation of dormant Mycobacterium tuberculosis by phospholipids or specific peptides

The presence of dormant tubercle bacilli presents a major problem for tuberculosis treatment. The culture supernatant of Mycobacterium tuberculosis was previously shown to resuscitate dormant bacilli in vitro. Here we report identification of active components as phospholipids and a tuberculosis protein Rv1174c. Remarkably, dormant bacilli from a one year old culture which failed to form any colonies could be resuscitated with peptides derived from Rv1174c and formed 10(5-7) colonies/ml. This finding represents the first unambiguous demonstration of resuscitation of dormant tubercle bacilli in vitro and may have implication for the study of mycobacterial dormancy and the design of novel strategies for improved treatment of tuberculosis.     

Genome sequence of the Mycobacterium abscessus strain M93

Mycobacterium abscessus is a rapid-growing species of nontuberculous mycobacteria that is frequently associated with opportunistic infections in humans. We report herein the draft genome sequence of M. abscessus strain M93.     

Synthesis of gatifloxacin derivatives and their biological activities against Mycobacterium leprae and Mycobacterium tuberculosis

Novel 3′-piperazinyl derivatives of the 8-hydrogeno and 8-methoxy-6-fluoro-1-cyclopropyl-4-quinolone-3-carboxylic acid scaffolds were designed, synthesized and characterized by ¹H, ¹³C and ¹⁹F NMR, and HRMS. The activity of these derivatives against pathogenic mycobacteria (M. leprae and M. tuberculosis), wild-type (WT) strains or strains harboring mutations implicated in quinolone resistance, were determined by measuring drug concentrations inhibiting cell growth (MIC) and/or DNA supercoiling by DNA gyrase (IC₅₀), or inducing 25% DNA cleavage by DNA gyrase (CC₂₅). Compound 4 (with a methoxy in R₈ and a secondary carbamate in R₃′) and compound 5 (with a hydrogen in R₈ and an ethyl ester in R₃′) displayed biological activities close to those of ofloxacin but inferior to those of gatifloxacin and moxifloxacin against M. tuberculosis and M. leprae WT DNA gyrases, whereas all of the compounds were less active in inhibiting M. tuberculosis growth and M. leprae mutant DNA gyrases. Since R₃′ substitutions have been poorly investigated previously, our results may help to design new quinolone derivatives in the future.