Summary: Mutants of Saccharomyces cerevisiae bearing lesions in the ergosterol biosynthetic pathway exhibit a pleiotropic drug-sensitive phenotype. This has been reported to result from an increased permeability of the membranes of the mutant strains to different drugs. As disruption of the yeast multidrug resistance protein, Pdr5p, results in a similar pleiotropic drug-sensitive phenotype, the possibility that Pdr5p may be functioning with a reduced efficiency in these altered sterol backgrounds was examined. To do this, the function of Pdr5p in isogenic strains of S. cerevisiae that have disruptions in the late stages of the ergosterol biosynthesis pathway (ERG6, ERG2, ERG3, ERG4) was studied. A reduced ability of Pdr5p to confer resistance to different drugs in these strains was observed, which did not appear to be dependent solely on the permeability of the membrane towards the drug. A simultaneous examination was made of how the lipid composition might be altering the efficiency of Pdr5p by similar studies in strains lacking phosphatidylserine synthase (encoded by CHO1). The results indicated that the drug sensitivity of the erg strains is, to a significant extent, a result of the reduced efficiency of the Pdr5p efflux pump, and that the membrane environment plays an important role in determining the drug resistance conferred by Pdr5p.
Summary: Clostridium sticklandii utilizes combinations of amino acids for growth by Stickland reactions. Proline is an efficient electron acceptor in these reactions and is reduced to 5-aminovalerate. Proline can be partly synthesized from ornithine by the action of ornithine aminotransferase and Δ1-pyrroline-5-carboxylate (PCA) reductase. Both enzymes were present in crude extracts of C. sticklandii in sufficient activity of 0·93 nkat (mg protein)-1 and 4·3 nkat (mg protein)-1, respectively, whereas enzymes involved in proline biosynthesis from glutamate were not detected. PCA reductase was purified to homogeneity in a three-step procedure involving ammonium sulfate precipitation, affinity chromatography with Procion Red and gel filtration on Sephadex GF200. The homogeneous enzyme was most likely an octamer of 230 kDa with a subunit size of 25 kDa as obtained by SDS-PAGE and 28·9 kDa as calculated from the sequence. Apparent K m values for PCA and NADH were 0·19 mM and 0·025 mM, respectively. The enzyme also catalysed in vitro the reverse reaction, the oxidation of proline, at alkaline pH values above 8 and higher substrate concentrations (apparent K m values: 1·55 mM for proline and 10·5 mM for NAD at pH 10·0). Studies with growing cells of C. sticklandii and [15N]proline revealed that proline is not oxidized in vivo because 15N was solely detected by HPLC-MS in 5-aminovalerate as the product of proline reduction. The proC gene encoding PCA reductase of C. sticklandii was cloned, sequenced and heterologously expressed in Escherichia coli. The enzyme exhibited high homologies to PCA reductases from different sources. Thus, C. sticklandii is able to synthesize the electron acceptor proline from ornithine (a degradation product of arginine) by action of ornithine aminotransferase and PCA reductase.
Summary: Continuous cultures of Chromatium vinosum DSM 185 were shifted from a high to a low irradiance (67 to 4 μE m-2 s-1) and vice versa (4 to 67 μE m-2 s-1). The kinetics of photoacclimation of the cultures were analysed during these transitions until steady state was reached. When irradiance was shifted from 4 to 67 μE m-2 s-1, bacteriochlorophyll synthesis halted for 4 h. During this period, pigments were progressively diluted in the newly formed biomass, resulting in a lower specific pigment content. The specific growth rate of the organisms did not change immediately after the shift, but rather underwent a gradual increase during the following 10 h. This transition was accompanied by a transient increase in the levels of glycogen, indicating that CO2 fixation rates increased immediately after the shift, and that unused photosynthate was stored as glycogen. The shift from a high to a low irradiance was characterized by an immediate drop in the specific growth rate to virtually zero, and by comparatively sharp decreases in the specific rates of sulfur and sulfide oxidation and in the specific rate of glycogen accumulation. The specific content of bacteriochlorophyll a increased during the first 10 h. During the same period the specific content of glycogen decreased.
Summary: Pseudomonas aeruginosa is able to persist during feast and famine in many different environments including soil, water, plants, animals and humans. The alternative sigma factor encoded by the rpoS gene is known to be important for survival under stressful conditions in several other bacterial species. To determine if the P. aeruginosa RpoS protein plays a similar role in stationary-phase-mediated resistance, an rpoS mutant was constructed and survival during exposure to hydrogen peroxide, high temperature, hyperosmolarity, low pH and ethanol was investigated. Disruption of the rpoS gene resulted in two- to threefold increase in the rate of kill of stationary-phase cells. The rpoS mutant also survived less well than the parental strain during the initial phas of carbon or phosphate-carbon starvation. However, after 25 d starvation the remaining population of culturable cells was not significantly different. Stationary-phase cells of the RpoS-negative strain were much more stress resistant than exponentially growing RpoS-positive cells, suggesting that factors other than the RpoS protein must be associated with stationary-phase stress tolerance in P. aeruginosa. Comparison of two-dimensional PAGE of the rpoS mutant and the parental strain showed four major modifications of protein patterns associated with the rpoS mutation.