Hugo L. David

Effect of Isoniazid on the In Vivo Mycolic Acid Synthesis, Cell Growth, and Viability of Mycobacterium tuberculosis

When an actively growing culture of the H37Ra strain of Mycobacterium tuberculosis was exposed to isoniazid at a concentration of 0.5 μg/ml, the cells began to lose their ability to synthesize mycolic acids immediately. After 60 min, the cells had completely lost this ability. The synthesis of the three mycolate components—α-mycolate, methoxymycolate, and β-mycolate—was inhibited. The viability of the isoniazid-treated cells was unaffected up to about 60 min of exposure, after which time there was a gradual decline in the viability to about 18% after 180 min. Correspondingly, growth of the drug-treated cells slowed down and stopped after 24 hr. The inhibition of the synthesis of mycolic acids was reversible if the drug was removed before the loss of viability set in. Incubation of the viable cells in the absence of the drug for 24 hr restored the mycolate synthesis. These results strongly suggest that the inhibition of the synthesis of the mycolic acids is closely associated with the primary mechanism of action of isoniazid on the tubercle bacilli. The sequence of events which leads to the loss of viability of cells exposed to isoniazid is described.

Effect of Temperature on the Rate of the Transparent to Opaque Colony Type Transition in Mycobacterium avium

The results of drug susceptibility tests were found to be affected by changes that occur spontaneously in populations of Mycobacterium avium maintained in the laboratory. Because the transparent colony type variant was resistant to antituberculosis chemotherapeutic agents and the opaque colony type variant was usually susceptible to these agents, the transition of transparent to opaque colony type was investigated. The rate of the transition was found to be temperature dependent and, in agreement with a previous report, was found to be about 10−4 to 10−5 per generation at 37 C. Reversion was found to occur at a rate of 10−6 to 10−7 at 37 C. The mutation rate from susceptibility to resistance to rifampin, kanamycin, and erythromycin was about 10−8 to 10−9 mutations per bacterium per generation. Judged from our data, the high rate of the transparent to opaque variation was not caused either by mutator effects or by the occurrence of extrachromosomal genes in these bacteria, but could have been due to selective mechanisms still incompletely understood.

Antibacterial action of colistin (polymyxin E) against Mycobacterium aurum.

Mycobacterium aurum was susceptible to the antibiotic colistin (polymyxin E),which had an MIC of 5 micrograms/ml and an apparent bactericidal effect at concentrations above 50 micrograms/ml. Treatment of actively growing cells with sublethal concentrations of colistin (15 micrograms/ml) resulted in synchronized cell division once the antibiotic was removed. Under conditions of synchronized cell growth, one cycle of DNA replication lasted 120 min and one cycle of cell division lasted about 180 min. Although the antibiotic treatment during synchronization experiments did not produce apparent changes in the bacterial envelope, it was accompanied by the accumulation of a polysaccharide-like substance in the bacterial cytoplasm which gradually decreased after the removal of antibiotic and by an increase in the number of mesosomes at 3 h after antibiotic removal. This step was closely linked to the doubling time of bacteria. Lethal concentrations of colistin of 50 and 100 micrograms/ml, which caused about 90 and 99% cell death, respectively, produced significant cytoplasmic membrane injuries, patchy appearance of the cell wall outer polysaccharide layer, and little cell lysis. These data indicate that the cytoplasmic membrane is a site of action of colistin and raise a question as to whether an outer bilayer exists in mycobacteria, at least functionally. Images

Isolation and characterization of uridine diphosphate-N-glycolylmuramyl-L-alanyl-γ-D-glutamyl-meso-α,α′-diaminopimelic acid from Mycobacterium tuberculosis

Abstract When growing cells of the H37Ra strain of Mycobacterium tuberculosis are exposed to 1.0 m M D-cycloserine, an intracellular peptide-containing nucleotide accumulates. This incomplete precursor for the synthesis of cell wall was isolated and characterized as UDP-N-glycolylmuramyl-L-Ala-γ-D-Glu- meso -DAP.

Characterization ofcar α,car Lep, andCrt I genes controlling the biosynthesis of carotenes inMycobacterium aurum

The genes controlling the biosynthesis of carotenes inMycobacterium aurum responsible for its yellow pigmentation were previously cloned (FEMS Microbiol Lett 1992, 90:239–244). In this study, the genescrt I, car α, andcar Lep, controlling respectively the formation of lycopene from phytoene (phytoene desaturase), α-carotene from lycopene, and leprotene from lycopene, were localized.

Partial physical and functional map of aMycobacterium aurum carotenogenesis operon

The genes controlling the biosynthesis of the carotenes inMycobacterium aurum were clustered in a 10.83-kb segment. Fragments generated by endonuclease digestions of the segment were cloned into a pHLD69 shuttle vector. The plasmids so constructed were used to transform a colorless (albino)M. aurum mutant (strain A11), a brick-red mutant accumulating large amounts of lycopene (strain NgR9), the buff-coloredMycobacterium smegmatis MC2-155, and the buffcoloredMycobacterium tuberculosis H37Ra. From the endonuclease digestion patterns and the phenotypes of the transformed strains, the partial physical and functional maps of a carotenogenesis operon were established. This investigation also showed that the genes controlling the conversion of lycopene into the xanthophylls were not located in the 10.83-kb segment.