- Volume 143, Issue 2, 1997

The core-forming lipoate acetyltransferase (E2p) subunits of the pyruvate dehydrogenase (PDH) complex of Escherichia coli contain three tandemly repeated lipoyl domains although one lipoyl domain is apparently sufficient for full catalytic activity in vitro. Plasmids containing IPTG-inducible aceEF-IpdA operons which express multilip-PDH complexes bearing one N-terminal lipoyl domain and up to seven unlipoylated (mutant) domains per E2p chain, were constructed. Each plasmid restored the nutritional lesion of a strain lacking the PDH complex and expressed a sedimentable PDH complex, although the catalytic activities declined significantly as the number of unlipoylated domains increased above four per E2p chain. It was concluded that the extra domains protrude from the 24-meric E2p core without affecting assembly of the E1p and E3 subunits, and that the lipoyl cofactor bound to the outermost domain can participate successfully at each of the three types of active site in the assembled complex. Physiological studies with two series of isogenic strains expressing multilip-PDH complexes from modified chromosomal pdh operons (pdhR-aceEF-IpdA) showed that three lipoyl domains per E2p chain is optimal and that only the outermost domain need be lipoylated for optimal activity. It is concluded that the reason for retaining three lipoyl domains is to extend the reach of the outermost lipoyl cofactor rather than to provide extra cofactors for catalysis.

To investigate the role of the buoyancy provided by gas vesicles in the facultative anaerobe Halobacterium salinarium PHH1, the growth of a gas-vacuolate (Gv+) strain in competition with two gas-vesicle-defective (Gvdef) mutants was examined. The Gv+ strain synthesized gas vesicles throughout its growth cycle, and floated up to form a thick surface scum during the exponential growth phase in static culture. Mutant Gvdef1 produced significantly fewer gas vesicles than the Gv+ strain in corresponding stages of growth, although in late stationary phase a small proportion of cells floated up to the surface of static cultures. Mutant Gvdef2 had a much lower gas vesicle content in shaken culture and produced negligible amounts of gas vesicles in static culture. The Gv+ and the two Gvdef strains grew equally well in shaken cultures, but in static cultures, where steep vertical gradients of oxygen concentration were established, Gvdef1 was outgrown by the Gv+ strain. Gvdef2 outcompeted the Gv+ strain in shallow static cultures, perhaps because Gvdef2 carried a smaller protein burden, which offset the benefits of buoyancy. This selection for Gvdef2 was lost in deeper static cultures, although it could be restored by aerating static cultures from below. The results support the hypothesis that the role of buoyancy in halobacteria is to maintain cells at the more aerated surface of brine pools.

Reported differences in antibiotic production dynamics resulting from altering the growth-limiting nutrient (growth-dissociated production in carbon-limited culture and apparent growth-associated production in nitrogen-limited culture) are due to the different effects on growth kinetics. The substrate affinity for nitrate is significantly lower than that for glucose, resulting in nitrogen limitation effectively occurring throughout the culture. Glucose limitation occurs later in the culture, coinciding with the induction of antibiotic production. Induction occurs at the start of nitrogen-limited culture so that production appears to be growth-associated. Evidence that this hypothesis is consistent with production kinetics in cyclic fed batch culture was also obtained.

Alginate production and degree of polymerization were affected when the highly mucoid Pseudomonas aeruginosa 8821M was grown with growth-inhibitory concentrations of Cu2+ (supplied as CuCl2; 1-5 mM). The inhibition of alginate biosynthesis was consistent with the decreased activity in Cu2+-stressed cells of phosphomannose isomerase/GDP-mannose pyrophosphorylase (encoded by algA), phosphomannomutase (encoded by algC) and GDP-mannose dehydrogenase (encoded by algD). However, in cells grown with concentrations of CuCl2 below 2 mM, the steady-state mRNA levels from algA, algC, algD and from the regulatory gene algR1 increased moderately. This observation is consistent with the suggested linkage between the control of alginate gene expression and the global regulation involved in the oxidative stress response. At highly inhibitory concentrations the levels of the four alginate gene transcripts decreased from maximal values. The bell-shaped curves, representing the effect of Cu2+ concentration on mRNA levels from the four alginate genes, exhibited similar patterns but did not concur. The decrease of the specific activity of enzymes necessary for GDP-mannuronic acid synthesis in Cu2+-grown cells was correlated with changes in gene expression, with the inhibitory effect of Cu2+ on enzyme activities and with Cu2+-induced oxidative inactivation of enzymes, especially the particularly sensitive phosphomannose isomerase activity.