- Volume 130, Issue 11, 1984

Lysates of guinea pig or human red blood cells (RBC) contain far more of the factors that induce resistance in gonococci to complement-mediated killing by fresh human serum than do plasma or serum. As was previously found with serum, most of the resistance-inducing activity of guinea pig RBC lysates was found in ultrafiltrates with molecular weights of less than 5000. In contrast, and as with human serum, most of the resistance-inducing activity of human RBC lysates did not pass ultrafilters which removed molecules of less than 5000 daltons, although some active material of low molecular weight was present.

The growth of four clinical strains of Paracoccidioides brasiliensis was investigated using the chemically defined medium of McVeigh and Morton. Emphasis was placed upon controlling conditions of inoculum preparation, age of inoculum used, and the homogeneity of samples used for analysis. The medium was evaluated for its ability to support growth of the yeast phase of P. brasiliensis at 37 °C. Cultures were followed for 240 h and growth patterns were determined by measuring optical density, dry weight, nucleic acids and protein. The medium is excellent for growing the yeast phase of P. brasiliensis. The exponential phase lasted an average of 135 h and the stationary phase 72 h; a decline began after 207 h. This defined medium supports abundant growth of the yeast phase of P. brasiliensis and may thus prove useful in the preparation of yeast-phase antigens.

The kinetics of cell proliferation of a population of a small-size mutant of Saccharomyces cerevisiae were compared with those of a wild-type cell population, using time-lapse cinephotomicrography. The mean size of small-size mutant cells was approximately half that of wild-type cells at corresponding points in the cell cycle. The cycle times of small-size mutant cells were much more variable, especially for daughter cells, than those of wild-type cells. The difference in the variability of cycle times of the two strains was mainly due to the different degree of variability of their respective unbudded periods. Like wild-type cells, daughter cells of the small-size mutant were smaller and had a longer cycle time than parent cells. The small-size mutant retains a single size control over the cell cycle.

Saccharomyces cerevisiae Y185, grown anaerobically in media containing ergosterol and palmitoleic, oleic or linoleic acids, synthesized phospholipids extensively enriched in the exogenously supplied fatty acid. A study was made of the effect of solute concentration on rates of accumulation of nine amino acids by organisms enriched in different fatty-acyl residues. Data were fitted using computer-aided statistical analysis to three equations to derive kinetic constants for accumulation. Analysis of data for two of the amino acids, namely l-threonine and l-histidine, showed different kinetics in organisms enriched in different fatty-acyl residues. Woolf-Hofstee plots for accumulation of l-threonine, as well as l-serine, showed abrupt changes in curvature at low concentrations with differently enriched organisms. Data for accumulation of both amino acids gave a significant fit to the model describing accumulation by one transport system without diffusion. Data for accumulation of l-histidine as well as l-aspartic acid best fitted a model describing accumulation by one transport system and diffusion. Values for K T and the diffusion constant, but not V max, differed only for accumulation of L-histidine in organisms with different fatty-acyl enrichments. A third model, describing accumulation by two separable transport systems, best fitted data for accumulation of l-glutamic acid and L-methionine. Data for accumulation of l-leucine, l-isoleucine and l-valine could not be fitted to any of the models. Woolf-Hofstee plots for accumulation of l-leucine and l-isoleucine by organisms enriched in oleyl or linoleyl residues were superimposable, although similar plots for accumulation of l-valine differed in shape.

Nitrogen fixation (acetylene reduction) by Desulfovibrio gigas was displayed weakly and capriciously in lactate/sulphate media and sequential or continuous diazotrophic cultures were not successfully established. Washed cells from lactate-grown N-limited chemostat populations at 28 C showed reproducible anaerobic derepression of acetylene reduction, accompanied by limited growth, in low-N buffer, if sodium pyruvate + sodium sulphate were provided. Hydrogenase activity was not affected by the concentrations of acetylene used. Optimum concentrations of cells, pyruvate + sulphate, casein hydrolysate, Na2MoO4 and FeSO4 were established: peak activities of ~10 nmol C2H2 reduced min−1 (mg bacterial protein)−1 occurred with 10% (v/v) C2H2 after about 48 h; Ni2+, M2+ or derepression under Ar did not influence activity. NH+ 4 or air prevented derepression. An oxidant, usually sulphate, was essential. Thiosulphate was a poor substitute for sulphate; sulphite was apparently ineffective. Lactate, fumarate or H2 did not replace pyruvate as derepression substrate.

Pyruvate-derepressed populations showed reversible inactivation when exposed briefly to air. Activity was substantially inhibited by 10 or 100 μm-NH+ 4, reversibly at low NH+ 4 concentrations.

Chlorella cultures were grown in a tubular loop reactor which facilitated both irradiation of the culture and gas mixing compared with a conventional stirred vessel with vortex aeration. Measurements of the inhibition of maximum specific growth rate (proportional to photosynthetic rate) in the tubular reactor showed that CO2 behaves as a typical inhibitory substrate at partial pressures (PCO2 ) up to 0·6 atm. The PCO2 for 50% reduction in maximum specific growth rate was 0·36 atm. At 0·6 atm there was a discontinuity in the inhibitory effect with a sharp increase in the inhibitory effect at higher PCO2 values. Cultures rapidly adjusted to step changes in the PCO2 up to 0·6 atm. At a PCO2 of 1 atm inhibition was complete but the inhibitory effect was readily reversed.

The effects of cinnamic, propionic, benzoic and sorbic acids on the growth and intracellular pH of Escherichia coli were investigated. The data suggest that the potency of weak acids as food preservatives is related to their capacity to reduce specifically the intracellular pH. The data also suggest that although both the undissociated and dissociated forms of the acid cause the intracellular pH to fall, growth inhibition is due predominantly to the undissociated acid.

The obligate intracellular rickettsia, Coxiella burnetii, was shown to possess an energy dependent proline transport system which displayed a high degree of specificity and was highly dependent on pH. Transport was maximal at pH 3·0 to 4·5, a pH range approximating that of the host cell phagolysosome where the agent replicates. Transport was inhibited by the uncouplers carbonyl cyanide m-chlorophenylhydrazone and dinitrophenol, but not by sodium arsenite. In the presence of glutamate, a preferred energy source, proline uptake was enhanced more than two-fold. This enhancement of proline uptake was greatly decreased in the presence of sodium arsenite. The addition of glutamate decreased the apparent K m for proline transport from 45 μm to 15 μm, with the V max increasing from 3μ6 pmol s−1 (mg dry wt)−1 to 4·8 pmol s−1 (mg dry wt)−1. Two proline analogues, furoic acid and azetidine-2-carboxylic acid, were effective inhibitors of proline transport. d-Proline, 4-hydroxyproline, glycine and proline amide inhibited transport minimally, while no inhibition was seen with succinate, pyruvate or glutamate.

The uptake of α-aminoisobutyrate (AIB) by washed cell suspensions of bloodstream forms of Trypanosoma brucei brucei has been shown to be an energy-dependent process. No metabolism of AIB was detected under conditions leading to a 100-fold accumulation of AIB within the organism. Kinetic studies revealed that AIB uptake involved two components; that operating at low substrate concentrations had an apparent K m of 4·6 mm. Experiments with ionophores such as gramicidin and carbonylcyanide m-chlorophenylhydrazone were consistent with the AIB uptake system operating as a H+-symporter responding to the electrochemical gradient of H+, the major component of which was the membrane potential.

The design, fabrication and use of a flow cell that allows rapid displacement of media viewed by short working distance, high power objectives are described. The cell has a small internal depth (about 0·04 cm), small volume (about 0·05 ml), and is chemically inert. It has been tested extensively in studies of tethered bacteria.

The Szybalski wedge plate technique was modified to generate two-dimensional diffusion gradients for two environmental variables: pH value and NaCl concentration were selected in this study. After experimentation, pH values chosen ranged from 3·9 to 8·1 and salt concentrations from 23 to 79 gl−1. The pH gradients were slightly sigmoidal whilst the salt gradients were approximately linear. The reproducibility of the technique was assessed and found to be satisfactory in replicate growth experiments. A wide range of growth patterns was observed for a large variety of different bacterial types. The growth boundary and the pattern details are sufficiently distinct to suggest that the technique can distinguish between closely related species or strains.

A hitherto unidentified volatile substance(s) is known to induce macrocyst formation in a strain (Dm 7) of Dictyostelium mucoroides and in a mutant (MF 1) derived from it. The properties of this substance suggested that it might be ethylene, and here it is shown that this is indeed the case. The addition of ethylene to MF 1 cells, in conditions otherwise favouring sorocarp formation, induced the formation of macrocysts. Conversely, the addition of mercury perchlorate, an absorbent of ethylene, inhibited macrocyst formation and induced sorocarp formation under conditions otherwise favouring macrocyst formation. Two inhibitors of ethylene synthesis, aminooxyacetic acid and aminoethoxyvinyl glycine, also inhibited macrocyst formation. Production and release of ethylene by D. mucoroides cells was confirmed by gas chromatography.

Growing hyphae of Achlya bisexualis were found to generate a longitudinal pH gradient in the surrounding medium; the medium adjacent to the tip was slightly more alkaline than the bulk phase, while that near distal parts was acidic. The profile of external pH paralleled that of electric current, as measured with a vibrating probe; the apical alkaline zone corresponded to the region of current inflow. In organisms grown in complete medium, both current flow and apical alkalinization were inhibited when amino acid uptake was blocked, either by removing amino acids from the medium or by raising the external pH to 8·5. Achlya could, however, adapt to a medium deficient in organic nutrients; elongating hyphae again generated both the pH profile and the transcellular electric current. It is proposed that both the pH profile and the electric current are manifestations of a transcellular proton current, which arises from the segregation of proton pumps from proton leaks. Symport of protons with amino acids may be one mechanism by which protons enter the hyphal apex.

Thirty-five Xanthomonas campestris pv. oryzae,fourteen X. campestris pv. oryzicola strains and six ‘brown blotch’ pathogens of rice, all of different geographical origin, were studied by numerical analysis of 133 phenotypic features and gel electrophoregrams of soluble proteins, %G + C determinations and DNA:rRNA hybridizations. The following conclusions were drawn. (i) The Xanthomonas campestris pathovars oryzae and oryzicola display clearly distinct protein patterns on polyacrylamide gels and can be differentiated from each other by four phenotypic tests. (ii) Both pathovars are indeed members of Xanthomonas which belongs to a separate rRNA branch of the second rRNA superfamily together with the rRNA branches of Pseudomonas fluorescens, Marinomonas, Azotobacter, Azomonas and Frateuria. (iii)‘Brown blotch’ strains are considerably different from X. campestris pv. oryzae and oryzicola. They are not members of the genus Xanthomonas, but are more related to the generically misnamed Flavobacterium capsulatum, Pseudomonas paucimobilis, Flavobacterium devorans and ‘Pseudomonas azotocolligans’ belonging in the fourth rRNA superfamily. (iv) No correlation was found between the virulence, pathogenic groups or geographical distribution of X. campestris pv. oryzae or pv. oryzicola strains and any phenotypic or protein electrophoretic property or clustering.

An electron microscopic study was undertaken of the entry of specific antibody into neutrophils containing surviving intracellular highly virulent group B streptococci after phagocytosis of the organisms had occurred. Electron micrographs are presented to demonstrate that specific antibody gains access to the ingested bacteria. This antibody binds to the surface of the streptococci, which subsequently permits the neutrophil to kill these organisms.