- Volume 133, Issue 11, 1987

SUMMARY: Low concentrations of urea, which did not inhibit the synthesis of the catabolite nonrepressible enzyme alkaline phosphatase in Vibrio cholerae, or markedly affect its overall growth, specifically inhibited the expression of the tryptophanase operon in a temperature-dependent manner. However, in contrast to what is found in Escherichia coli, this urea-induced inhibition of tryptophanase synthesis in V. cholerae could be almost completely relieved by exogenously added cyclic AMP. The possible mechanism of the process is discussed.

SUMMARY: A thermostable α-amylase gene (amyT631) from Bacillus stearothermophilus A631 was cloned into pBR322 and recloned into pUB110: the resulting plasmid was designated pTUB607. To investigate the processing from preproenzyme to mature enzyme, amyT631 from pTUB607, after digestion with BAL31, was introduced into the B. subtilis α-amylase secretion vector pTUB285. Three chimaeric plasmids, pTUB613, pTUB616, and pTUB617, were isolated. The fused α-amylases expressed from the three plasmids seemed to be synthesized as preproenzymes. From analysis of the NH2-terminal amino acid sequences of purified extracellular α-amylases, the precursors of the fused enzymes appeared to be cleaved at first between amino acids 31 and 32 from the translation initiator Met (positions −11 and −10 with respect to the beginning of the mature enzyme), and processed to mature extracellular enzymes in which the NH2-terminal amino acid sequences were the same as that of the parental pTUB607 α-amylase, in spite of the lengths of the prosequences and the amino acid composition near the secondary cleavage sites being different in each enzyme.

SUMMARY: The respiratory system of Agrobacterium radiobacter NCIB 11883, a producer of succinoglucan exopolysaccharide under ammonia-limited conditions, was studied following growth in continuous culture (D = 0·045 h-1)under glucose, oxygen and ammonia limitation. The respiratory chain contained b- and c-type cytochromes, plus two terminal oxidases (aa 3 and co) under all growth conditions, and exhibited a low transhydrogenase activity. An inactive apoenzyme form of the quinoprotein glucose dehydrogenase was also present which could be activated by adding pyrroloquinoline quinone (PQQ) to cell suspensions. The activity of the terminal region of the respiratory chain increased approximately fourfold following growth under oxygen limitation, and this was accompanied by a significant increase in the concentration of cytochrome oxidaseco. Whole cells exhibited →H+/O quotients of 5·5-6·3 for the oxidation of endogenous substrate depending on the nature of the growth-limiting nutrient. It is concluded that the respiratory chain energy conservation system of this organism is not significantly modified during ammonia-limited growth to offset the increased energy demands for exopolysaccharide synthesis.

SUMMARY: Under carbon limitation the efficiency of aerobic energy conservation of Agrobacterium radiobacter was independent of the carbon source. Under nitrogen limitation the chemical composition of the exopolysaccharide produced was independent of carbon source; however, the rate of production was highest on glucose and decreased on other substrates in the order glucose, succinate, gluconate, xylose, sorbitol, glycerol and ethanol. When the observed yields of exopolysaccharide from glucose, gluconate and xylose were corrected for cell production they came very close to the theoretical values; however, the yields of exopolysaccharide decreased sharply when the carbon source was either more reduced or oxidized than glucose. Although the rate of exopolysaccharide production on glucose was greater than that on gluconate or xylose, the rate of ATP utilization for polymer synthesis was similar and accounted for most of the respiratory activity occurring in excess of that required for cell biosynthesis. The proportion of respiratory activity that is dissociated from polymer synthesis increased sharply with carbon sources that are significantly more reduced or oxidized than glucose. A mutant of A. radiobacter that was unable to synthesize exopolysaccharide had the same efficiency of growth as the parent strain. Under nitrogen-limited growth the respiratory activity in excess of growth requirements was similar to the polymer-producing strain, supporting the view that the rate of ATP turnover was similar in both strains. This suggests that the rate of ATP turnover rather than exopolysaccharide synthesis is physiologically important. It is proposed that the rate of ATP turnover in excess of that required for growth under nitrogen limitation (N-limited qo 2 × ATP/O quotient) is controlled to a level that is governed by the oxidation/reduction state and the Y ATP of the given substrate.

SUMMARY: Various conditions which decrease translational capacity and enhance the synthesis of ribosomal components were analysed with respect to the synthesis of heat-shock proteins in Escherichia coli: (a) deprivation of streptomycin from a streptomycin-dependent mutant, (b) addition of tetracycline to a partially tetracycline-resistant strain, and (c) nutritional shift-up conditions. In all cases, the rate of synthesis of the heat-shock proteins DnaK, GroEL and C62.5 decreased while the synthesis of ribosomal components increased. Thus inhibition of ribosome formation or a decrease in translational capacity do not induce the stress proteins, but have the opposite effect.

SUMMARY: Both NAD- and NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (G3PDH) (EC 1.2.1.12) activities were detected in glucose-grown cells of Pseudomonas aeruginosa strain PAO. After growth on gluconeogenic substrates such as citrate, the activity of the NAD-G3PDH was reduced severalfold in contrast to little change for the NADP-G3PDH. The two G3PDH activities could be separated by ammonium sulphate fractionation. PAGE revealed the presence of two G3PDH isoenzymes of 140 (NADP-specific) and 315 (NAD-specific) kDa. Slight differences were observed in the thermostabilities and pH optima of the two enzymes whereas the regulation of their activities by various compounds varied strongly. The NADP-G3PDH enzyme was activated by ATP, reduced NAD, and fructose 6-phosphate. It was inhibited by fructose 1,6-diphosphate and 6-phosphogluconate. The NAD-G3PDH enzyme was inhibited by ATP, reduced NAD, and 6-phosphogluconate; it was slightly activated by reduced NADP. The possible roles of these isoenzymes in the control of hexose catabolism and gluconeogenesis in P. aeruginosa are discussed.

SUMMARY: Purification of the sexual agglutinins of both mating types of Chlamydomonas moewusii yapensis(= C. moewusii syngen II) was achieved by a three-step procedure. Electron microscopy showed both agglutinins to be long, linear molecules. The mt+ agglutinin was a rigid molecule with one bulbous end and an average length of 251 nm. The mt-agglutinin was longer (average length 349 nm), had a more flexible conformation and lacked a bulbous end. The Mr was estimated to be 1·0 × 106 for the mt+ and 1·2 × 106 for the mt- agglutinin. The mt- agglutinin had a very high content of hydroxyproline (41%) and serine (14%); the mt+ agglutinin had a high content of glycine, serine and hydroxyproline (18, 16 and 12%, respectively). The main sugars in the agglutinins of both mating types were arabinose and galactose. The carbohydrate portion represented about 40% of the Mr in both agglutinins. The biological activity of both agglutinins could be destroyed by periodate treatment (although their sensitivities differed), indicating the involvement of carbohydrate residues. A differential susceptibility for exoglycosidases was observed: enzymic removal of terminal glucose residues abolished the biological activity of the mt+ agglutinin only, while removal of mannose residues selectively inactivated the mt- agglutinin. The similarities in form and composition of the agglutinins of several Chlamydomonas species suggest that these recognition molecules have a common ancestry, and that the species barriers are at least partially dependent on differences in the carbohydrate side-chains.

SUMMARY: Non-toxic concentrations of various substances were tested for their influence on the gliding motility of Mycoplasma mobile 163K. A significant inhibitory effect on motility was observed with agents acting on nucleic acid synthesis (mitomycin), protein synthesis (puromycin, chloramphenicol), energy metabolism (p-chloromercuribenzoate, iodoacetate) and with compounds reacting with the cytoplasmic membrane or contractile elements (albumin, cholesterol, EDTA, 2-propanol, procain, CaCl2, MgCl2, colchicin and KI). The surface-active compounds Triton X-100, Tego and SDS increased the gliding velocity significantly in some concentrations and incubation periods. The results suggest that the motility of M. mobile depends on a functional cytoplasmic membrane and that cytoskeletal elements are involved in the gliding mechanism.

SUMMARY: Proline-induced germ-tube formation and cell-cell aggregation in four strains of Candida albicans were completely inhibited when the pH of the medium was 5·0 or lower, whereas morphogenesis induced by N-acetylglucosamine (G1cNAc) was unaffected even at pH 4·5. The pH sensitivity of proline-induced germ-tube formation was not caused by a modulation of proline uptake, which was unchanged over the pH range 4·5-6·5. The proline uptake system was specific, constitutive and subject to ammonium repression, and only one permease was detected, with a K m of 179 μM. Cultures deprived of nitrogen in the presence of glucose were derepressed for proline uptake but the yeast-mycelial transition could not be mediated by either proline or G1cNAc. The inhibition of morphogenesis was reversed when the nitrogen starvation was relieved by the addition of ammonium ions, proline, or certain amino acids. These results indicate that the nitrogen status of the cells is critical for the morphogenesis of C. albicans.

SUMMARY: Among several parameters affecting the rate and amount of iron uptake by Saccharomyces cerevisiae, the oxidation state of iron appeared to be determinant. Iron presented as Fe(II) was taken up faster than Fe(III) and the kinetic parameters were different. Iron was taken up by the cells from different ferric chelates, at rates that did not depend on their stability constants, and uptake was strongly inhibited by an iron(II)-trapping reagent like ferrozine. Iron was physiologically reduced by a transplasmamembrane redox system, which was induced in iron-deficient conditions. We propose that iron must be reduced to be taken up by the cells in the same way as other divalent cations.

SUMMARY: Inhibition of autotrophic ammonia and nitrite oxidation by potassium ethyl xanthate (PEX) was studied in ammonium- and nitrite-limited chemostat cultures of Nitrosomonas europaea and Nitrobacter, respectively. Ammonia and nitrite oxidation were both stimulated by addition of low concentrations of PEX (0·1 μg ml-1), which significantly decreased steady-state substrate concentrations and increased steady-state cell concentrations. Addition of similar concentrations to exponentially growing cultures resulted in a slight stimulation of growth of Nitrobacter but did not affect Nitrosomonas. Inhibition of ammonia and nitrite oxidation by higher concentrations of PEX resulted in establishment of steady states without induction of lag phases, observed in batch culture, which would have resulted in wash-out. Establishment of steady states occurred more quickly and with less variation in substrate concentrations following addition of inhibitory rather than stimulatory concentrations of PEX. Steady-state data indicate that PEX may act as a competitive inhibitor of nitrite oxidation by Nitrobacter.