- Volume 143, Issue 12, 1997

The viability of cells and the expression of a reporter gene in Bacillus subtilis macrofibres have been examined using cytochemical approaches. Previous studies have shown that macrofibres grow at an exponential rate in fluid environments and undergo complex dynamic motions as they elongate but the behaviour of individual cells in them has never been examined. A fluorescence staining procedure that distinguishes living cells from dead cells was used in the experiments described to examine cells in both left- and right-handed macrofibres. Very few dead cells were found in individual fibres. Their locations appeared to be random, suggesting that neither the extreme shape deformation imposed upon cells in the loop ends of fibres, nor the entrapment of cells in the interior of a fibre compromise viability. In related experiments, initial studies of reporter gene expression during fibre morphogenesis were conducted using a strain previously studied as conventional colonies. Spatial and temporal differences in the appearance of the blue cleavage product of X-Gal were found, suggesting that differential gene expression may arise in macrofibres in contrast to the results found in colonies. Two conclusions may be drawn from the findings: (i) virtually all cells in macrofibres are viable and (ii) all the cells in macrofibres do not always behave in an identical manner.

The effect of Na+ (CI- or gluconate salt) on growth and dimorphic potential of the pathogenic yeast Candida albicans has been examined. Profiles of germ tube formation as a function of salt addition, pH and temperature indicated Na+ inhibition of germ tube outgrowth at high ambient pH (pH 8.0) which was exacerbated by replacement of CI- with gluconate (as an impermeant analogue). At acidic pH (pH 5.5) and permissive temperature (37 °), gluconate alone promoted the dimorphic transition. Rates of glucose-induced medium acidification and plasma membrane H+-ATPase activity have been measured to assess whether salt treatments could retard the cytoplasmic alkalinization known to precede germ tube formation. The precise site of Na+ action remains unclear but the anion effects may be interpreted in terms of anion-exchanger and channel activity acting to modulate cytosolic pH.

The expression and in situ activity of the membrane-bound and periplasmic nitrate reductases have been assayed in Paracoccus denitrificans Pd1222 grown under a range of aeration regimes in malate-limited or butyrate-limited chemostat cultures. In butyrate-limited cultures the expression of periplasmic nitrate reductase and the rate of in situ nitrate reduction were high at all oxygen concentrations measured between 0% and 100% air saturation. By contrast, in malate-limited cultures expression of the periplasmic nitrate reductase was low at 80-100% air saturation but increased to a maximum between 20% and 50% air saturation. Aerobic nitrate reduction was much higher in butyrate-limited than in malate-limited cultures, demonstrating a significant role for this process during butyrate metabolism. The rate of nitrate respiration increased in both the malate- and butyrate-limited cultures as aerobic metabolism switched completely to anaerobic metabolism. Expression of the membrane-bound nitrate reductase could be detected in butyrate-limited chemostat cultures maintained at an oxygen level of 100% air saturation. No membrane-bound nitrate reductase was detectable under similar conditions in malate-limited cultures but expression was detected at oxygen concentrations of 50% air saturation and below. Taken together, the results show that the nature of the carbon substrate and oxygen concentration can both influence expression of the periplasmic and membrane-bound nitrate reductases. The conditions under which expression of the periplasmic nitrate reductase and aerobic nitrate respiration are maximal can be rationalized in terms of a role for the periplasmic nitrate reductase in dissipating excess reductant generated during oxidative metabolism of reduced carbon substrates.

The broad-host-range, heat-tolerant Rhizobium strain BR816 produces sulphated Nod metabolites. Two ORFs highly homologous to the Sinorhizobium meliloti nodPQ genes were isolated and sequenced. It was found that Rhizobium sp. BR816 contained two copies of these genes; one copy was localized on the symbiotic plasmid, the other on the megaplasmid. Both nodP genes were interrupted by insertion of antibiotic resistance cassettes, thus constructing a double nodP1P2 mutant strain. However, no detectable differences in Nod factor TILC profile from this mutant were observed as compared to the wild-type strain. Additionally, plant inoculation experiments did not reveal differences between the mutant strain and the wild-type. It is proposed that a third, functionally homologous locus complements mutations in the Nod factor sulphation genes. Southern blot analysis suggested that this locus contains genes necessary for the sulphation of amino acids.