Growth of Mycobacterium leprae under low oxygen tension

Despite numerous attempts, Mycobacterium leprae has yet to be cultivated in vitro. This organism has been considered as microaerophilic. The effects of various known gas mixtures on the in vitro growth of M. leprae were investigated. A gas mixture containing 2.5% O2 and 10% CO2 was found to be more favourable for the growth of this mycobacterium on artificial medium. Growth was evaluated by three parameters namely cell counts, bacterial ATP and DNA. An optimal growth of M. leprae, as determined by all three parameters, on both liquid and solid media was obtained between 18 and 24 weeks of incubation under optimal gas mixture. Solid medium which contained egg-yolk was relatively more beneficial for in vitro growth than the liquid medium. The cultivated bacilli exhibited some important characteristics specific for M. leprae, including growth in mouse foot-pads. The bacilli gradually lost their power of adaptation to grow on artificial media and did not show any ATP or DNA after about 36 weeks of incubation.     

Influence of oxygen tension on the respiratory activity of Mycobacterium phlei

Growth of Mycobacterium phlei under low oxygen tension resulted in specific activities two to twenty times lower for formate dehydrogenase, malate dehydrogenase, beta-hydroxybutyrate dehydrogenase, lactate oxidase and NADH dehydrogenase than when cultures were grown under high aeration. An increase in fumarate reductase and succinate dehydrogenase occurred with M. phlei grown under low oxygen tension. Malate: vitamin K dehydrogenase and glucose-6-phosphate dehydrogenase activity were not significantly affected by the oxygen tension used to grow the bacteria, and neither culture contained a lactate dehydrogenase. With growth of M. phlei in conditions of low oxygen tension, cytochrome a was not detected, but cytochrome b was prominent in membranes and cytochrome c was present in the soluble fraction.     

Influence of culture medium on the resistance and response of Mycobacterium bovis BCG to reactive nitrogen intermediates

The aim of the present work was to evaluate the influence of the culture medium on the resistance and response of Mycobacterium bovis BCG to reactive nitrogen intermediates, in vitro. BCG was grown in Sauton, Dubos or Middlebrook 7H9 medium and exposed to sodium nitroprusside (SNP) for up to 7 days. The percentage of bacilli that survived was significantly lower in Middlebrook 7H9 than in Sauton or Dubos medium. Addition of SNP to Middlebrook 7H9 caused an increase in the RedOx potential in either the absence or the presence of BCG, while addition of the compound to Sauton medium gave rise to an increase in the RedOx potential only in the absence of bacteria, whereas a decrease in the RedOx potential was observed in the presence of BCG. The resistance of BCG to SNP in the different media did not correlate with the concentration of peroxynitrite in culture supernatants. BCG grown in different media showed a differential protein expression pattern, as assessed by two-dimensional gel electrophoresis. Exposure of BCG to sub-lethal concentrations of SNP in Middlebrook 7H9, but not in Sauton medium, revealed a differential expression of at least 38 protein species. Altogether these results demonstrate that the growth medium may have a remarkable influence on the resistance and the response of BCG to SNP and suggest that the different resistance of BCG in the two media is unlikely to be due to a differential antioxidant effect of the medium itself.     

Effects of oxygen tension on the establishment and lactate dehydrogenase activity of murine embryonic stem cells

The lack of a standardized culture environment for establishment of embryonic stem cell lines has hindered the orchestrated differentiation of cells and the application of this technology. Oxygen concentration has a profound effect on proliferation and differentiation of many cell types. This study tested the hypothesis that establishment dynamics, lactate dehydrogenase (LDH) isoforms, and mRNA expression patterns would be affected by the oxygen tension in the culture environment. Recovered (day 4) murine blastocysts were cultured in a gas environment of 6% CO(2) and either 20% or 5% O(2) (balance supplemented with N(2)). More (p < 0.05) blastocysts produced outgrowths in the low (79.3 +/- 0.1%) compared to the high (57.1 +/- 0.1%) O(2) groups, and more (p < 0.05) colonies in the low O(2) group (14/15; 93.3 +/- 0.1%) stained positive for alkaline phosphatase relative to the high O(2) group (9/15; 60.6 +/- 0.1%). Oxygen treatment had no effect on the activity of the oxioreductase lactate dehydrogenase. Interestingly, the stem cell lines in both treatments displayed multiple isoforms (III, IV, and V) of LDH, whereas the outgrowths displayed isoforms I and V. In contrast, two-cell embryos and blastocysts displayed only isoform I, and fibroblasts displayed isoforms IV and V. There were no treatment differences in mRNA expression of LDHalpha in the outgrowths, or established stem cells. LDH transition from the heart (I) to the muscle (V) isoform indicated an increase in glycolytic activity, consistent with the peri-hatching/implantation time period. Reduced O(2) environment had significant positive effects on the establishment and maintenance of murine stem cells, supporting the hypothesis, whereas the LDH isozyme transition was consistent among treatments.     

Growth requirements and the establishment of a chemically defined minimal medium inZymomonas mobilis

Summary A chemically defined minimal medium which fulfils the growth requirements of differentZymomonas mobilis strains has been established. The kinetics of ethanol production of the strains ATCC 10988, CU1, CP4 and 11163 grown on the minimal medium at different glucose concentrations were measured. All strains produced ethanol at rates similar to those on complete medium. The minimal medium described is suitable to study spontaneous metabolic deficiciencies and regulation of enzyme activities inZ.mobilis.     

Electrode measurement of oxygen tension with 1-ms time resolution

A continuous flow device utilizing a Clark oxygen electrode was constructed; this device had a dead time and resolution of 1 ms. Mixing was tested by observing the neutralization of acid with base, and at the maximal flow rate, the mixing was 94% complete within 1 ms and better than 98% complete within 2 ms after initial mixing. Observation of the oxygenation of hemoglobin gave data which agreed with previous data obtained by a stopped-flow optical experiment. The respiration of phosphorylating submitochondrial particles was measured utilizing this device. The burst of respiration in submitochondrial particles was triphasic, with a very rapid burst lasting some 60 ms, followed by a longer burst of respiration lasting more than 4 s.     

Electrode measurement of oxygen tension with 1-ms time resolution.

A continuous flow device utilizing a Clark oxygen electrode was constructed; this device had a dead time and resolution of 1 ms. Mixing was tested by observing the neurtralization of acid with base, and at the maximal flow rate, the mixing was 94% complete within 1 ms and better than 98% complete within 2 ms after initial mixing. Observation o of the oxygenation of hemoglobin gave data which agreed with previous data obtained by a stopped-flow optical experiment. The respiration of phosphorylating submitochondrial particles was measured utilizing this device. The burst of respiration in submitochondrial particles was triphasic, with a very rapid burst lasting some 60 ms, followed by a longer burst of respiration lasting more than 4 s.     

Role of protein kinase G in growth and glutamine metabolism of Mycobacterium bovis BCG

The survival of pathogenic mycobacteria in macrophages requires the eukaryotic enzyme-like serine/threonine protein kinase G. This kinase with unknown specificity is secreted into the cytosol of infected macrophages and inhibits phagosome-lysosome fusion. The pknG gene is the terminal gene in a putative operon containing glnH, encoding a protein potentially involved in glutamine uptake. Here, we report that the deletion of pknG did not affect either glutamine uptake or intracellular glutamine concentrations. In vitro growth of Mycobacterium bovis BCG lacking pknG was identical to that of the wild type. We conclude that in M. bovis BCG, glutamine metabolism is not regulated by protein kinase G.