- Volume 133, Issue 6, 1987

Summary: Lipopolysaccharide (LPS) from all six serotype strains of Haemophilus influenzae was similar in composition. The oligosaccharide, of each LPS, was composed of glucose, galactose, heptose and 2-keto-3-deoxyoctonic acid. The lipid A was composed of glucosamine, phosphate and the fatty acids 14:0 and 3-OH 14:0. Each LPS also contained ethanolamine and ethanolamine phosphate, and the oligosaccharides from two strains additionally contained small amounts of glucosamine. Although the LPS was similar in composition, different serotypes had quantitative differences, especially in the galactose content, which correlated with the antigenic specificity of their homologous antisera and with their mobility on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). A survey by SDS-PAGE showed that LPS from strains of the serotypes a, c and d was characteristically of lower Mr than the LPS from most (80%) serotype b strains.

Summary: Cell-free extracts of Ureaplasma urealyticum strains Pi and T960 (CX8) (serovars 6 and 8, respectively) metabolized inorganic pyrophosphate (PPi). The inorganic pyrophosphatase (PPase) activity was greatest with Mg2+ as cofactor, but Mn2+ acted as a poor substitute. The PPases of the two serovars differed electrophoretically. Although the highest PPase activity was obtained using PPi as substrate, the enzyme could also utilize to a lesser degree both tripolyphosphate and trimetaphosphate. No activity was observed against β-glycerophosphate, naphthyl phosphates, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, thiamin pyrophosphate, phosphoribosylpyrophosphate, ADP or ATP. Acid- and alkaline-phosphatase activities were observed with naphthyl phosphates as substrates, but they did not have the same electrophoretic mobility on gels as the PPase activity. U. urealyticum PPase was inhibited by oxidized glutathione, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, phenyl-glyoxal, p-chloromercuribenzoic acid, Mn2+, Zn2+ and Ca2+. Neither reduced glutathione, l-cysteine nor Co2+ enhanced activity. PPi can act as a substrate or regulator of certain metabolic reactions, and PPi metabolism can function in bacterial bioenergetics; its role in ureaplasmas is presently unclear.

Summary: Candida olea 148 secreted a single acid protease when cultured at acidic pH. In unbuffered medium, the culture pH eventually became alkaline and a single alkaline protease was produced. This was the only proteolytic enzyme produced when the organism was grown in buffered medium at alkaline pH. Both proteolytic enzymes were purified to homogeneity (as assessed by SDS-PAGE). The Mr of the acid protease was 30900, the isoelectric point 4·5; optimum activity against haemoglobin was at 42 °C and pH 3·3. This enzyme was inactivated at temperatures above 46 °C and was inhibited by pepstatin and diazoacetyl-norleucine methyl ester but was insensitive to inhibition by either 1,2-epoxy-3-(p-nitrophenoxy)-propane or compounds known to inhibit serine, thiol or metallo proteases. The acid protease contained 11% carbohydrate. The alkaline protease had an M r of 23400 and an isoelectric point of 5·4. The activity of this enzyme using azocoll as substrate was optimal at 40°C in the range pH 8·0-9·0. This enzyme was inactivated at temperatures above 42°C and was inhibited by phenylmethyl-sulphonyl fluoride and irreversibly inactivated by EDTA. The enzyme was also partially inhibited by DTT but was insensitive to either pepstatin or p-chloromercuribenzoic acid.

Summary: Carbon catabolite derepression induced changes in the pool of yeast mRNAs translatable in a protein-synthesizing reticulocyte system. Competition experiments with globin mRNA showed that the mRNA population obtained from derepressed cells possessed a higher translational efficiency than mRNA from repressed cells. The mRNAs that could account for the high translational efficiency of the derepressed mRNA were not detected in cells growing in glucose-rich medium. Analysis of protein synthesis in the presence of 7-methylguanosine 5′-phosphate indicated that the initiation factors recognizing the 5′-terminal structure of capped messengers interacted with lower affinity with the repressed than with some specific derepressed mRNAs.

Summary: The induction of endo-1,4-β-glucanase synthesis by Trichoderma reesei QM 9414 was investigated in conidia, mycelia and protoplasts. Cellulose induced endoglucanase synthesis only in conidia, but not in glucose-grown mycelia or protoplasts. Cellooligosaccharides and sophorose induced endoglucanase synthesis in mycelia, conidia and protoplasts. Only conidia exhibited detectable basal endoglucanase levels, whereas β-glucosidase activity was found in conidia, mycelia and protoplasts. The β-glucosidase was inhibited in vitro by nojirimycin and glucono-δ-lactone. Addition of either of these inhibitors to the induction medium blocked de novo synthesis of endo-1,4 β-glucanase with cellulose (conidia) or cellooligosaccharides (protoplasts and mycelia) as inducer, whereas induction by sophorose remained unaffected. The results are consistent with the assumption that basal constitutive levels of endoglucanase and β-glucosidase are involved in the induction of cellulase synthesis by cellulose in T. reesei.

Summary: Suspensions of intact, yeast-like cells of Sporothrix schenckii exhibited an acid phosphatase (EC 3.1.3.2) activity against p-nitrophenyl phosphate of about 5 IU (g dry wt)−1, without recourse to membrane perturbation. This extra-cytoplasmic acid phosphatase was reversibly and competitively inhibited by orthophosphate (K i=2 mm at pH 5) but unaffected by l(+)-tartrate (in contradistinction to some of the cytoplasmic acid phosphatases of the same organism). Inactivation by NaF of the extra-cytoplasmic isoenzyme was irreversible and followed first order kinetics; sensitivity to NaF was decreased by the presence of citrate, phosphate or substrate. Neither Km (0·3 mm at pH 5) nor V max for this enzyme in acetate buffer was greatly affected by pH in the range 3-5 but the first order rate constant for inactivation by NaF was strongly dependent on pH (maximum at pH 3·5). Crude cell-free extracts of yeast cells had nine electrophoretically distinct acid phosphatase activity bands and, on the basis of the pattern of inhibitors, the extra-cytoplasmic activity was identified as Y-I, an isoenzyme that barely penetrates standard polyacrylamide gel electropherograms. Additional evidence for the assignment came from selective inactivation of this isoenzyme by short treatments of intact cells with NaF under conditions that did not allow penetration of the plasma membrane by the inhibitor and did not kill the cells.

Summary: Glycollate inhibited growth of Pseudomonas aeruginosa in media containing either pyruvate or lactate as carbon sources. Glycollamide, but not glyoxylate, showed similar effects. Spontaneous mutants (L/G strains) were isolated that were able to grow on lactate medium in the presence of glycollate: their growth in pyruvate medium was still inhibited by glycollate. Synthesis of membrane-bound NAD+-independent D(−)- and L(+)-lactate dehydrogenases (iLDHs) was inducible by D- or L-lactate in the parent strain but was constitutive in the L/G strains. Glycollate inhibited induction of the synthesis of iLDHs in the parent strain growing in succinate medium but had no effect under the same conditions on strain L/G1. Glycollate was a competitive inhibitor of L(+)-iLDH (K i = 11 mM). No differences were found in the kinetic properties of L(+)-iLDH in cell-free extracts from strain L/G1 and the parent organism. Glycollate appears to inhibit growth on lactate medium predominantly through prevention of lactate induction of iLDH synthesis.

Summary: 13C nuclear magnetic resonance spectroscopy was used to study the biosynthesis and turnover of O-α-Dglucopyranosyl-(1→2)-glycerol (glucosylglycerol) in the marine cyanobacterium Synechococcus N100 during osmotic shock. There was a rapid increase in the level of this heteroside following hyperosmotic shock, with most of the solute being synthesized from extracellular carbon via photosynthetic CO2 fixation. However, changes in the 13C labelling patterns during the time course of the response suggested that up to 10% of the newly synthesized material may be derived from intracellular carbon. No increase in glucosylglycerol was observed when cells were subjected to hyperosmotic shock in the dark. These experiments indicated that a good yield of highly 13C-enriched glucosylglycerol, which could serve as a source of labelled glucose and glycerol, could be obtained simply and quickly by subjecting this cyanobacterium to hyperosmotic stress in the presence of 90% 13C-enriched bicarbonate.

Summary: Growth of Streptococcus salivarius ATCC 25975 in the presence of n-alkanols in the series methanol to decan-1-ol led to a decrease in the unsaturated to saturated fatty acid ratio. Each member of the set of n-alkanols which was examined over a range of concentrations possessed a point at which extracellular glucosyltransferase (GTF) production was minimal; increasing the concentration of the n-alkanol past this point stimulated GTF production. This effect was greatest with hexan-1 -ol although it was observed to a lesser extent with pentan-1 -ol and heptan-1-ol. Reduced cell-associated fructosyltransferase activity was observed with increasing concentrations of each n-alkanol. Growth in the presence of 25 mM-propan-1-ol gave rise to a fatty acid profile in which 55% of the fatty acids were of an odd chain length. S. salivarius ATCC 25975 was shown to be able to utilize ethanol in a similar manner to propan-1-ol by growing it in the presence of 400 mM-[14C]ethanol. Analysis of the membrane lipids at the stationary phase of growth indicated that 17·6 % of the carbon of the fatty acids was derived from ethanol. A leaky adh mutant, S. salivarius MJ 37501, was isolated. The leaky nature of the mutant enabled it to incorporate reduced levels of odd-chain-length fatty acids into its membrane lipids when grown in the presence of 100 mM-propan-1-ol, but not when grown in the presence of 25 mM-propan-1-ol. S. salivarius ATCC 25975 therefore metabolized propan-1-ol (and ethanol) via a constitutive alcohol dehydrogenase.

Summary: The time-course of 45Ca2+ influx into yeast cells was measured under non-steady-state conditions obtained by preincubating the cells in a Ca2+-free medium containing glucose and buffer. Two components were distinguished: a saturable component which reached a steady-state after about 40 s of 45Ca2+ uptake and a linear increase in cellular 45Ca2+ starting after 60-90s. Using differential extraction methods it was determined that after 20s of uptake, 45Ca2+ was localized in the cytoplasmic pool and in bound form with no 45Ca2+ in the vacuole. After 3 min most of the cellular 45Ca2+ was concentrated in the vacuole and in bound form. The initial rate of 45Ca2+ uptake under non-steady-state conditions thus measured 45Ca2+ transport across the plasma membrane without interference by vacuolar uptake. The effect of membrane potential (δψ) on this transport was investigated in cells depleted of ATP. A high δψ was produced by preincubating the cells with trifluoperazine (TFP) and subsequently washing the cells free from TFP. Substantial 45Ca2+ influx was measured in the absence of metabolic energy in cells with a high δψ. Below a threshold value of −69·5 mV the logarithms of the initial rate of 45Ca2+ influx and of the steady-state level of the first component were linear with respect to δψ. It is suggested that 45Ca2+ influx across the plasma membrane is mediated by channels which open when δψ is below a threshold value. The results indicated that Ca2+ influx across the plasma membrane was driven electrophoretically by δψ.

Summary: We have determined the nucleotide sequence of a 1500 bp fragment of the ColE9-J plasmid which encodes colicin E9 immunity and colicin E5 immunity and contains two lys genes. Open reading frames corresponding to the four genes have been located and their position confirmed by transposon mutagenesis of sub-clones of the ColE9-J plasmid. The E9imm gene shows 69% homology at both the nucleotide and the amino acid level to the previously sequenced E2 imm gene. The E5imm gene shows little homology to any other E colicin immunity gene which has been sequenced. The lys gene distal to the 3′ end of the E5imm gene shows considerable sequence homology to all other previously sequenced E colicin lys genes. The lys gene distal to the 3′ end of the E9imm gene is identical to the pColE2 and pColE3 lys genes for the first 59 nucleotides but encodes a much smaller gene product than any other lys gene which has been sequenced. The two lys genes sequenced here are exceptions to Shepherd's rule concerning the number of RNY codons in the three possible reading frames.

Summary: In Rhizobium ORS571, which is able to form both root and stem nodules on the tropical plant Sesbania rostrata and to grow in the free-living state at the expense of N2, two copies of the nifH gene, which codes for the nitrogenase Fe-protein, were characterized. One copy, nifH1, was localized in the nifHDK operon; the other, nifH2, was localized elsewhere. nifH1 and nifH2 differed in only six nucleotides and both encoded a polypeptide of 296 amino acids with a single change at position 282, a serine in nifH1 and a threonine in nifH2 At position 102, arginine in the sequence Gly-Arg-Gly-Val-Ile-Thr might, as in Rhodospirillum rubrum, be the site of ‘switch-off’ inactivation. Compared with other nifH genes, the highest SAB was found with Bradyrhizobium japonicum and Rhizobium sp. (Parasponia). A sequence identical (nifH1) or very similar (nifH2) to the nif consensus sequence was found upstream from the initiation codon. Homology with sequences upstream from Klebsiella pneumoniae nif promoters, which affect nifA-mediated activation, was also found in both cases. From mutants carrying either a single nifH1 or nifH2 deletion, or a double nifH1-nifH2 deletion, it appeared that both genes were functional ex planta and in Planta and that no other nifH copy seemed to be present in ORS571. The optimal expression of nifH1 and of nifH2 might depend on different physiological conditions.

Summary: A marine bacteriophage active against the marine luminous bacterium Vibrio fischeri MJ-1 was isolated from offshore waters in Ensenada, Baja California, Mexico, and was shown to be active in generalized transduction, transducing 14 of 17 different amino acid auxotrophs to prototrophy. For some of the amino acid auxotrophic markers, such as arginine and methionine, several different mutants could be transduced. The phage grew well at temperatures up to 27 °C, and produced high-titre lysates (1010 p.f.u. ml-1 or higher). Single-step growth analysis showed a latent period of 23 min at 25 °C with a burst size of 100. Phage adsorption was maximum at NaCl concentrations characteristic of the marine environment. No evidence for lysogeny was found.

Summary: Low levels of invertase (EC 3.2.1.26) activity were observed in most diploid strains of S. cerevisiae used in this work. There was no effect of mating type on invertase levels, and cell surface was not a limiting factor, because an increase in ploidy did not cause further decrease in specific invertase activity. Finally, some diploids showed the activity expected from the additive effects of different SUC genes, and haploid strains possessing two SUC genes expressed very variable invertase activities depending on the strain. This suggested the existence of one or more additional genes which control the levels of invertase. Genetic analysis of SUC5 strains provided evidence of the existence of a new gene, RPS5 which drastically reduced the specific invertase activity in strains possessing active SUC alleles. The recessive allele of this gene (rps5) allows expression of higher levels of invertase. We suggest that genes similar to RPS5 are responsible for the low levels of invertase activity observed in diploid strains of S. cerevisiae.

Summary: Cells of Escherichia coli K12 were sensitive to 100 mm-methylammonium when cultured under nitrogen limitation, and resistant when grown with an excess of either NH4C1 or glutamine. Glutamine synthetase activity was required for expression of the methylammonium-sensitive phenotype. Mutants were isolated which were resistant to 100 mm-methylammonium, even when grown under nitrogen limitation. P1 bacteriophage transduction and F′ complementation analysis revealed that the resistance-conferring mutations mapped either inside the glnA structural gene and/or elsewhere in the E. coli chromosome. Glutamine synthetase was purified from the wild-type and from some of the mutant strains. Strains carrying glnA-linked mutations that were solely responsible for the methylammonium-resistant phenotype yielded an altered enzyme, which was less active biosynthetically with either ammonium or methylammonium as substrate. Sensitivity to methylammonium appeared to be due to synthesis of γ-glutamylmethyl-amide by glutamine synthetase, which was synthesized poorly, if at all, by mutants carrying an altered glutamine synthetase enzyme.