- Volume 141, Issue 11, 1995

Summary: In order to examine the role of glutaminase in Rhizobium etli, we isolated and characterized a R. etli glutaminase mutant (LM16). This mutant was selected for its impaired ability to grow on glutamine as nitrogen and carbon source while retaining the ability to grow on other nitrogen and carbon sources. The mutant showed very low levels of glutaminase activity under various growth conditions in comparison with the wild-type strain. With glutamine as the only nitrogen and carbon source, LM16 showed poor growth, with a very high content of glutamine, low glutamate content, and reduced ammonium excretion and 14CO2 evolution from [U-14C]glutamine compared to the wild-type strain. This indicates that the main role of R. etli glutaminase is in the use of glutamine as carbon source. R. etli glutaminase also plays a role in maintaining the balance between glutamate and glutamine, as shown by the accumulation of glutamine and the low glutamate content of the mutant under different growth conditions. These results also indicate that glutaminase participates in a glutamine cycle in which it degrades glutamine which is then resynthesized by glutamine synthetase. The higher glutamine and lower glutamate content found in bacteroids of LM16 in comparison with bacteroids of the wild-type strain indicate that glutamine degradation by glutaminase plays an important role during the symbiosis between R. etli and Phaseolus vulgaris.

Summary: Streptococcus mutans is able to synthesize extracellular glucans from sucrose which contribute to adherence of these bacteria. Extracellular dextranase can partially degrade the glucans, and may therefore affect virulence of S. mutans. In order to isolate mutants unable to produce dextranase, a DNA library was constructed by inserting random Sau3AI-digested fragments of chromosomal DNA from S. mutans into the BamHI site of the streptococcal integration vector pVA891, which is able to replicate in Escherichia coli but does not possess a streptococcal origin of replication. The resultant plasmids were introduced into S. mutans LT11, allowing insertional inactivation through homologous recombination. Two transformants were identified which did not possess dextranase activity. Integration of a single copy of the plasmid into the chromosome of these transformants was confirmed by Southern hybridization analysis. Chromosomal DNA fragments flanking the plasmid were recovered using a marker rescue technique, and sequenced. Comparison with known sequences using the blastx program showed 56% homology at the amino acid level between the sequenced gene fragment and dextranase from Streptococcus sobrinus, strongly suggesting that the S. mutans dextranase gene (dexA) had been inactivated. The colony morphology of the dextranase mutants when grown on Todd-Hewitt agar containing sucrose was altered compared to the parent strain, with an apparent build-up of extracellular polymer. The mutants were also more adherent to a smooth surface than LT11 but there was no apparent difference in sucrose-dependent cell-cell aggregation. In contrast to LT11, neither the dexA mutants nor a mutant in the dexB gene, which encodes a dextran glucosidase, were able to ferment dextran to produce acid, supporting an earlier hypothesis that both enzymes are required for metabolism of dextran. From the results obtained by inactivating the dexA gene, a role for dextranase is suggested in controlling the amount and nature of extracellular glucans, in adherence of S. mutans, and in the utilization of glucans as a carbohydrate source.

Summary: Saccharomyces cerevisiae was more resistant to cadmium when the growth medium contained excess iron. Cadmium reduced the amount of iron taken up by cells during growth, and the cell ferrireductase activity was also strongly inhibited. These effects depended on the YAP1 and CAD1/YAP2 gene dosage. The growth rate of cells in iron-deficient conditions and their ferrireductase activity in the absence of added cadmium were also strongly affected by the dosage of YAP1 and CAD1/YAP2 genes. Our results suggest an indirect influence of these genes on iron metabolism, possibly via modification of the cell redox status.

Summary: The behavioural response to a gradient of oxygen (aerotaxis) has been characterized in the archaeon, Halobacterium salinarium. When the gas surrounding a drop of H. salinarium strain S9-P culture was changed abruptly from 10% (v/v) O2 to 100% N2, the bacteria transiently increased the frequency of reversing before they adapted and resumed random swimming. When the gas was returned to 10% O2 the bacteria responded by swimming smoothly for approximately 45 s. Aerotaxis was strongest when respiration in H. salinarium was highest and when bacteriorhodopsin and halorhodopsin were not contributing to the proton motive force. Starvation for methionine of the auxotrophic H. salinarium essentially abolished the step-down aerotactic response. Methanol production from demethylation of methyl-accepting chemotaxis proteins was transiently increased in H. salinarium S9-P by a step down or step up in oxygen concentration, as observed in methylation-dependent chemotaxis in H. salinarium. The taxis-negative and methyltransferase-deficient mutant, H. salinarium strain Pho72 did not exhibit changes in methanol release in response to aerotaxis or chemotaxis stimuli. This is the first report of an aerotactic response that is dependent on methylation of methyl-accepting chemotaxis proteins. Aerotaxis in Escherichia coli and Salmonella typhimurium is independent of transducer methylation.

Summary: The structural components in cell walls of three mutants of a facultative alkaliphile, Bacillus lentus C-125, defective in certain cell-wall components were characterized in detail. The cell walls of the wild-type C-125 were thick and increased in thickness when grown at high pH. Electron microscopy showed that triple layers developed when the bacteria were grown in an alkaline environment. In contrast, cell walls of teichuronopeptide (TUP)-defective mutants consisted of a single layer. For both the wild-type and mutants, the cell-wall concentrations of the acidic structural polymers teichuronic acid and TUP increased with respect to peptidoglycan as culture pH increased. For all four strains, the anion content of their cell walls was the greatest at high pH. The cell-wall density of the negatively charged compounds (uronic acids plus L-glutamic acid) was calculated as about 3 and 9 equivalents (I cell wall region)−1 for C-125 cells grown at pH 7 and 10, respectively. At high pH, the specific growth rates of the two TUP-defective mutants were much lower than those of the wild-type. It is concluded that increased levels of acidic polymers in the cell walls of alkaliphilic bacteria may be a necessary adaptation for growth at elevated pH.