Bdellovibrio bacteriovorus 6-5-S, an obligate bacterial parasite, was grown on cells of Spirillum serpens in 20 l of medium. Maximum numbers of the parasite were attained at 42–55 h, about 2–12 h after there were no viable host cells remaining. Growth of B. bacteriovorus occurred only under aerobic conditions. The organism is rich in catalase. Endogenous respiration of cells and oxidation of NADH by cell extracts is inhibited by cyanide and azide but not by carbon monoxide. Difference spectra indicate the parasite has a cytochrome system and likely couples oxidation to phosphorylation, although efforts to demonstrate fixation of inorganic 32P into organic phosphate failed. Glutamine, glutamate, and asparagine stimulate respiration, and cell extracts contain glutamate and alanine dehydrogenases. B. bacteriovorus 6-5-S also has a tricarboxylic acid cycle, as isocitric dehydrogenase, the α-ketoglutarate dehydrogenase system, succinyl-CoA synthetase, fumarase, and malate dehydrogenase were found in the soluble fraction of the cell extract, and a bound succinic dehydrogenase in the particulate fraction. The glyoxylate cycle may not be present since isocitric lyase could not be detected despite the presence of acetate in the growth medium. Tests on cell extracts for glucose kinase, glucose 6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase were also negative. However, aldolase, glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase, and lactic acid dehydrogenase were present although at rather low levels, indicating that the parasite can obtain energy from the substrate-linked phosphorylations of the glycolytic system.
A halophilic coccus, Micrococcus halodenitrificans n. sp., isolated from meat curing brines is described. This organism grows optimally in media containing between 4.4 and 8.8% sodium chloride as determined by viable counts and manometric methods. The viable count decreases in media containing 2.2% or less sodium chloride. As salt concentrations increase above 8.8%, the length of the lag phase increases and the rate of growth decreases. The organism exhibits a specific sodium chloride requirement for growth. However, it continues to respire in the presence of sodium bromide.
Bdellovibrio bacteriovorus 6-5-S releases at least two enzymes into the culture fluid when grown on autoclaved cells of Spirillum serpens VHL. One of the enzymes is bacteriolytic and the other is proteolytic. The lytic enzyme was purified by a factor of 3000 using ammonium sulfate fractionation, Sephadex G-100 gel filtration, and the anion exchanger DEAE-Sephadex. The lytic enzyme degrades peptidoglycan of S. serpens by hydrolyzing the diaminopimelic acid – alanine bond in the tetrapeptide chain. Ca2+ or Mg2+ exerted little or no effect on the activity of the lytic enzyme. The enzyme had a molecular weight of 40 000 as indicated by gel filtration. Crude preparations were unstable at 4C. The second enzyme, a protease that digested Azocoll, was purified by a factor of 7 by ammonium sulfate fractionation followed by gel filtration with Sephadex G-100. The protease was eluted in the void volume from a Sephadex G-100 column and therefore may have a molecular weight of at least 100 000. Its activity was enhanced by additions of Ca2+ and Mg2+. The enzyme was stable at 4C.
CROTHERS, S. F., and J. ROBINSON. 1971. Changes in the permeability of Esclrericlria coli during parasitization by Bdellovibrio bacteriovorrrs. Can. J. Microbiol. 17: 689-697. Bdellovibrio bacteriovorlrs strain 6-54 comp!eted a typical infection cycle when incubated with E. coli ML 35 (lac i-z+y-) in 0.025 M Hepes buffer, pH 7.8, supplemented with 0.002 M CaC12.2H20. Growth of this strain of B. bacteriovor~rs was optimal over the range of pH 7.5-8.5. N o growth occurred at pH 6.5. The broad pH range may occur because a buffer per se is not required for growth and multiplication. The parasite failed to multiply in a two-membered culture unsupplemented with Ca2+ or Mgz+.Growth and multiplication of B. hncteriovor~rs in a two-membered culture were assessed by various parameters, including decrease in absorbance at 520 mp and release of materials absorbing at 260 mp and at 280 mp. The onset of lysis of the host cell was accompanied by an increase in the release of materials absorbing at 260 mp and at 280 mp. The ratio of the absorbance of these materials at 280 and 260 mp increased at the same time, from which it may be inferred that probably amino acids or proteins were being released.No a-galactosidase could be detected in the culture fluid of the two-membered culture. The infected E. coli cells were more permeable than uninfected cells to o-nitrophenyl-a-D-galactoside, and to the fluorescent dye 8-anilino-I-naphthalenes:~lfonic acid.
Bdellovibrio bacteriovorus, strain 6-5-S, multiplied in the presence of washed suspensions of Escherichia coli, Spirillum serpens VHL, and Bacillus megaterium which had been autoclaved for 5 min at 121C. No intracellular life cycle was observed. Bdellovibrio bacteriovorus was also able to multiply in an extract from autoclaved E. coli cells after the cells had been removed by centrifugation. Growth of the parasite was dependent upon the addition of Ca2+ and Mg2+ to the buffer solution. The growth rate and yield of B. bacteriovorus on autoclaved cells were not affected by the initial concentration of the parasite. During multiplication of B. bacteriovorus, amino acids, amino sugars, and reducing sugars were released into the culture fluid.
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