- Volume 138, Issue 9, 1992

A variety of cobalamin analogues synthesized chemically or microbiologically were used to study the corrinoid specificities of cell growth and the cobalamin uptake system in Euglena gracilis z. Although Euglena could not utilize most of the analogues for cell growth, benzimidazolyl cobamide, cyanocobalamin-O 5-phosphate, and cyanocobalamin-b-, -d-and -e-monocarboxylates had effects similar to that of cyanocobalamin on cell growth. It is suggested that Euglena cells have the ability to synthesize ‘complete cobalamin’ from the acid derivatives (amidation reaction) and/or the phosphate derivative (dephosphorylation reaction). Inhibition of uptake of radiolabelled cyanocobalamin caused by addition of various analogues indicates that both the α-lower axial ligand (the cobalt-coordinated nucleotide) and the (b)-propionamide side-chain of the cobalamin molecule are essential for the cobalamin uptake system in Euglena. These results indicate that there are different corrinoid specificities for cell growth and the cobalamin uptake system in E. gracilis z.

The uptake (transport and metabolism) of urea was studied in a strain of the marine bacterium Deleya venusta, measuring the uptake of [14C]urea in vivo and the urease reaction in vitro. Urea uptake in vivo was sodium-dependent and exhibited a K m value of 1.4 μM for urea, a broad pH optimum between pH 6.0 and 8.5, a distinct temperature optimum at 35°C and a requirement for energy. Urease activity in vitro exhibited a K m value of 0.86 mM for urea and showed maximum activities at pH 8.5 and 60°C; the enzyme was neither dependent on the presence of sodium, nor inhibited by metabolic inhibitors. Synthesis of the urea uptake system was subject to nitrogen control; ammonium resulted in a repression of the system, whereas high uptake rates were observed after growth with nitrate or incubation of the cells in the absence of a nitrogen source. The uptake reaction in vivo, but not the urease activity in vitro, was decreased greatly in the presence of ammonium. This inhibition was relieved by methionine sulphoximine (MSX), a potent inhibitor of glutamine synthetase; in mutant strains impaired in this enzyme no inhibition of urea uptake by ammonium was observed. These results suggest that glutamine formed from ammonium rather than ammonium itself regulates urea uptake activity in D. venusta.

Bacillus licheniformis NCIB 6346 utilized glucose in preference to maltose when both sugars were present in the growth medium. Addition of glucose to a culture growing on maltose resulted in inhibition of maltose uptake and an immediate cessation of maltose metabolism. The mechanism of maltose transport was examined in whole cells and cell extracts. Phosphoenolpyruvate did not stimulate phosphorylation of maltose, indicating the absence of a phosphotransferase system. However, the presence of a maltose phosphorylase enzyme was suggested by phosphorylation of the sugar in the presence of inorganic phosphate. Maltose accumulation was strongly inhibited by proton conducting uncouplers, and was driven by an artificial transmembrane pH gradient, inside alkaline. These results imply that maltose is transported by a proton symport mechanism in this bacterium.

The regulation of expression of the porin genes of Escherichia coli by acid pH was investigated using reporter gene fusions. The ompC-lacZ gene fusion was expressed in response to acidification of the external medium. The kinetics of β-galactosidase synthesis under acid-induction differed significantly from those obtained under conditions of osmotic stress. The latter led to rapid induction without a lag, followed by establishment of a rate that was equal to the growth rate; acid-induction was frequently preceded by a short lag period, was relatively slow and did not achieve a rate that was in balance with the growth rate. Further, induction of the ompC gene at acid external pH was dependent upon the presence of glucose as sole carbon source; growth with either glycerol or succinate as sole carbon source reduced induction of ompC at acid pH. Osmotic induction was independent of carbon source. The induction of the ompC gene at acid pH was also reduced by addition of cAMP to the growth medium. The porins are known to be subject to catabolite repression and our data are consistent with the exposure to acidic pH resulting in progressive changes in the state of catabolite repression. Acidification of the cytoplasm also provoked a rapid induction of the ompC-lacZ gene fusion. The kinetics of induction resembled the response to osmotic upshock. This response was independent of the identity of the carbon source supplied for growth. The contribution of changes in cytoplasmic pH to the induction of ompC at acid pH is discussed.

The oral spirochaete Treponema denticola ATCC 33520 was grown at a mean generation time of 10 h in anaerobic continuous culture in a serum- and carbohydrate-free medium at pH 7.0. The extracellular proteolytic activities of this spirochaete were then investigated by incubating washed cells with 68 2-naphthylamide derivatives of the Extended API System. Chymotrypsin-like, trypsin-like, elastase-like and iminopeptidase activities were demonstrated. The phenylalanine peptidase or chymotrypsin-like activity of T. denticola ATCC 33520, estimated with N-succinyl-L-phenylalanyl-L-leucyl-L-phenylalanine-thiobenzyl ester (SPLP) had a pH optimum at pH 8.5, a specific activity of 36.6 nmol min−1(mg dry wt)−1and was inhibited only slightly by HgCl2. The trypsin-like activity, estimated with benzoyl-DL-arginine-7-amido-4-methylcoumarin (BAMC), had a pH optimum at pH 9, and a specific activity of 0.3 nmol min−1(mg dry wt)−1; inhibition by HgCl2 indicated the involvement of active thiol groups. The activity should preferably be termed arginine peptidase activity, according to the carboxy-terminal amino acid of the test substrate. The extracellular proline peptidase activity, estimated with L-proline-7-amido-4-methylcoumarin. HBr (PRAMC), had an activity of 1.5 nmol min−1(mg dry wt)−1, an optimum at pH 8.5 and the properties of a thiol protease. The main cell-bound and extracellular active peptidase activities of fast-growing cells of T. denticola ATCC 33520 are phenylalanine peptidase, proline peptidase, arginine peptidase and an oligopeptide-dependent alanine peptidase activity. The cell-bound peptidase activities are of potential importance for invasion and multiplication in the junctional epithelium and the destruction process in periodontal pockets with an anaerobic and alkaline environment.

Streptomyces bikiniensis HH1, an A-factor-deficient mutant that did not form aerial mycelium on agar medium unless supplemented with A-factor, produced spores abundantly within colonies. The spores formed on reproductive branches morphologically similar to aerial hyphae except that they did not emerge from the surface of the colonies. The spores were morphologically more heterogeneous than those formed when A-factor was added.