- Volume 131, Issue 6, 1985

The ferric enterobactin receptor protein (81 kDal) of Escherichia coli O 1 11 was purified by preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis and used to raise polyclonal antiserum in rabbits. This antiserum was used in conjunction with the immunoblot technique to examine the degree of antigenic homology of the ferric enterobactin receptor protein among 17 pathogenic and laboratory strains of E. coli. Both the molecular weight and the antigenic properties of the enterobactin receptor were highly conserved. However, the laboratory strain C and a pathogenic enteroinvasive strain, E. coli O164, were unusual in not producing the 81 kDal protein. The antiserum also recognized an 81 kDal protein from iron-restricted Salmonella typhimurium and an 83 kDal protein from iron-restricted Klebsiella pneumoniae.

The R6-5 plasmid-specified outer membrane protein, TraT protein, has previously been shown to mediate resistance to bacterial killing by serum. Colony hybridization with a 700 bp DNA fragment carrying most of the traT gene was used to examine the prevalence of traT in Gram-negative bacteria, particularly strains of Escherichia coli, isolated from clinical specimens. traT was found in isolates of E. coli, Salmonella, Shigella and Klebsiella, but not in Pseudomonas, Aeromonas or Plesiomonas, nor in the few isolates of Enterobacter, Proteus, Acinetobacter, Citrobacter, Serratia or Yersinia that were examined. It was detected in a significantly higher proportion of the E. coli strains isolated from the blood of patients with bacteraemia/septicaemia or from faeces of patients with enteric infections (50–70%) than in that of strains isolated from normal faeces (20–40%). The incidence of traT in strains isolated from cases of urinary tract infections was variable. traT was found to be frequently associated with production of the K1 capsule and with the carriage of ColV plasmids, but not with the carriage of R plasmids, nor with serum resistance or the production of haemolysin.

p-Aminobenzoic acid synthase (PABA synthase) of Streptomyces griseus catalyses the conversion of chorismic acid to p-aminobenzoic acid (PABA), a precursor of the aromatic p-aminoacetophenone moiety of candicidin, a polyene macrolide antibiotic. This enzyme uses glutamine or ammonia as amino donors for PABA formation. Enzyme extracts converted [14C]chorismic acid to labelled PABA. PABA synthase was present in S. griseus IMRU 3570 only during the antibiotic producing phase. No detectable levels of the enzyme were found in cell-free extracts of nonproducing mutants of S. griseus obtained after UV mutagenesis. PABA synthase activity was found also in Streptomyces coelicolor var. aminophilus, producer of the polyene macrolide antibiotic fungimycin, but it was not present in extracts of several other streptomycetes that do not produce aromatic polyene macrolide antibiotics. PABA synthase (amidotransferase) activity was partially purified by DEAE-Bio-gel and Sephacryl S-200 filtrations. The estimated molecular weight was 50000. PABA synthase was repressed by aromatic amino acids and PABA but not by anthranilic acid. Inorganic phosphate strongly repressed but did not inhibit PABA synthase activity.

The sources of conductance changes during reduction of trimethylamine oxide to trimethylamine by Escherichia coli with formate as electron donor and in the presence of phosphate buffer were investigated. Theoretical considerations and experimental results suggest that the major source of conductance change is the conversion of dihydrogen phosphate to hydrogen phosphate. This transformation contributes almost twice as much to the total conductance change as does the conversion of uncharged trimethylamine oxide to charged trimethyl-ammonium.

Cells of Saccharomyces cerevisiae exhibiting respirative glucose metabolism in continuous culture were able to use ethanol as a co-substrate. The ethanol uptake rate was dependent on the residual respirative capacity of the cells. The activities of gluconeogenic enzymes and of malate dehydrogenase were higher in cells degrading glucose respiratively than in cells metabolizing glucose respiro-fermentatively, but were lower than in cells growing on ethanol only. The pattern of distribution of the mitochondrial cytochromes was similar but the differences were less distinct. In synchronously growing cells, the activities of gluconeogenic enzymes and of malate dehydrogenase oscillated, with activities increasing during the budding phase. The increase was preceded by the appearance of ethanol in the culture medium.

In a medium containing sulphite and sulphate, Saccharomyces cerevisiae TC8 produced H2S from sulphite but not from sulphate which, under these conditions, was not taken up. Production of H2S started with the onset of the stationary phase of growth, and both were triggered by the depletion of ammonium ions or the addition of cycloheximide to cultures. Addition of ammonium sulphate to stationary-phase cultures stopped H2S production and stimulated growth. Inclusion of 1 mhl-methionine in the medium halved the rate of H2S production, while lowering the concentration of sulphite also decreased the rate of production. Sulphite from a pyruvate-sulphite complex was also metabolized to give H2S. Maximum rates of H2S production, induced by including different concentrations of ammonium ions in the medium or by adding cycloheximide to cultures, closely correlated with the sulphite reductase activity in extracts of organisms.

Rhizobium leguminosarum MNF3841 grown on glucose/NH4Cl constitutively transported l-asparagine, l-aspartate, l-glutamate, l-glutamine, glycine, l-leucine, l-methionine and l-phenylalanine. Transport rates were increased 1.5-4-fold by growth on glucose/l-glutamate. Uptake of L-glutamate, l-glutamine, l-asparagine and l-leucine was inhibited to varying extents by a broad range of l-amino acids. Analogues of l-glutamate in which the amino group or α-hydrogen was methylated inhibited L-glutamate transport much less effectively. Also while 2-and 3-amino acids interfered with l-glutamate uptake, d-glutamate did not. Inhibition by 2,4-dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone and cyanide indicated that amino acid transport was active. The ratio of the intracellular to extracellular concentration of l-leucine after 5 min accumulation was 768. Cells loaded with l-[14C]leucine exhibited exchange not only with external l-leucine but also with l-glutamate. The apparent K m for l-glutamate transport was 0·081 μm. Both l-aspartate and l-alanine were competitive inhibitors of l-glutamate uptake with apparent K i values of 0·164 μm and 2·3 μm, respectively. These results suggest that there is an extremely high affinity carrier for l-glutamate that is not only very sensitive to inhibition by l-aspartate but also capable of being inhibited by a broad range of amino acids at an order of magnitude higher concentration.

Mutants of Azotobacter chroococcum which showed unusually O2-sensitive N2 fixation when grown on sugars were isolated following mutagenesis with nitrosoguanidine or the transposon Tm1. These mutants we called Fos– (inability to fix N2 on sugars). Aerotolerant growth of Fos– mutants was, in general, restored by carboxylic acids, Ca2+ or a combined-N source. Two main groups of mutants were distinguished: (1) regulatory mutants which failed to synthesize nitrogenase under aerobic conditions and which were complemented by the Klebsiella pneumoniae nifA gene product; (2) mutants affected at different steps in intermediary metabolism. The latter strains exhibited low respiration rates or high apparent K s(O2) values (when grown on sucrose) compared with the parent strain. They seemed to be defective in respiratory protection of nitrogenase. The restoration of aerotolerant N2-dependent growth of Fos– mutants by carboxylic acids was correlated with their ability to induce a decrease in the apparent K s(O2) value; however, in both the mutant and the parent strains maximum respiration rates did not change significantly. The Ca2+ requirement for diazotrophic growth in A. chroococcum seems to be related to the capability of these organisms to fix N2 in air. Ca2+ might be required for high fluxes in the tricarboxylic acid cycle and might act at the level of phosphoenolpyruvate carboxylase, which is involved in the replenishment of the tricarboxylic acid cycle and/or at the level of tricarboxylic acid cycle enzymes per se. It is suggested that respiratory protection requires the maintenance of high respiratory rates even at low O2 tension.

Methanosarcina barkeri strain FR-19 lysed spontaneously in substrate-depleted cultures. The addition of 0.3 m-sucrose prevented complete lysis and resulted in the formation of osmotically sensitive UV-fluorescent spheres. Electron microscopic examination showed that complete degradation of the cell wall occurred before the release of true protoplasts, which were stabilized by sucrose and glucose (0·2–0·3 m) but not by glycerol. Exponentially increasing methane production and regeneration of protoplasts could not be demonstrated.

A method of transposon mutagenesis using Tn5 has been developed for the facultative methylotroph Methylobacterium AM1 using the IncP-10 plasmid R91-5. Auxotrophic mutants and mutants involving the metabolism of methanol have been isolated.

Journal of General Microbiology (1985), 131, 437-449

p. 442, first paragraph: the first two sentences should read ‘During purification, RNA polymerase activity was found to be completely dependent on added template after the ammonium sulphate precipitation (Fraction III), and linearly dependent on the amount of enzyme protein added up to 300 μ;m ml-1 with both calf thymus DNA and herring sperm DNA as templates. Some of the requirements of the RNA polymerase activity are shown in Table 1.’