- Volume 138, Issue 12, 1992

SUMMARY: Although Neisseria meningitidis can use haemoglobin as an iron source in vitro, the mechanism of haemoglobin-iron uptake is unknown. Using a biotinylated human haemoglobin probe in a solid-phase dot-binding assay, haemoglobin-binding activity was detected in total membranes derived from meningococci grown under iron-limited but not iron-sufficient conditions. In competition binding experiments, bovine and human haemoglobin could abrogate binding. In contrast, no binding inhibition was seen with ferric nitrate, protoporphyrin IX, and iron-loaded human transferrin. The ability of both haemin and catalase, a nonhaemoglobin haem-containing compound, to inhibit binding competitively suggested that the ligand recognized by the binding protein is the haem moiety. Scatchard plot analysis revealed a heterogeneous receptor population. Limited proteolysis with proteinase K abolished binding activity, suggesting a haemoglobin-protein interaction. Detection of activity in a whole-cell binding assay demonstrated that this haemin-binding protein was surface exposed. In a limited survey of meningococcal strains, the presence of haemoglobin-binding activity in all isolates indicated that expression of this binding protein is not serogroup specific.

SUMMARY: Malate degradation by Lactobacillus plantarum CECT 220 provides energy which enables this organism to remain viable for longer at low environmental pH values. Energy production was not coupled to H+-ATPase activity. This protective mechanism against acidic external pH is complemented by another system of ΔpH maintenance. The H+-ATPase did not seem to be involved in this system of pH maintenance as the system was not sensitive to N, N'-dicyclohexylcarbodiimide (DCCD) and was functional at very low pH values where ATPase activity was severely inhibited.

SUMMARY: The relation between respiration and luminescence of Vibrio harveyi was studied in the presence of the proton conductor CCCP (carbonyl cyanide m-chlorophenylhydrazone). CCCP affected both luminescence and oxygen uptake rate, but did not alter the cellular ATP level. In the absence of CCCP, luminescence and oxygen uptake rate were almost constant over the pH range 6.5–8.5. However, at 10 μM-CCCP, both were apparently pH-dependent, with maxima observed at pH 8.5 and minima at pH 6.5. In the presence of CCCP (0.1–10 μM), the changes in the luminescence were in close correlation with those in the oxygen uptake rate regardless of the pH. HQNO (2-heptyl-4-hydroxyquinoline-N-oxide) and NaCN inhibited the CCCP-resistant luminescence. From these results, it is concluded that the CCCP-resistant luminescence is respiratory-dependent.

SUMMARY: Secretion of the extracellular Rhizopus carboxyl proteinase (EC 3.4.23.6) by Rhizopus oligosporus is repressed in the presence of low-molecular-mass sources of nitrogen, sulphur and carbon. Proteinase is secreted when the medium is deficient in any one of these three nutrients. In the case of nitrogen metabolite repression, control is at the level of transcription. Induction of proteinase secretion by exogenous protein does not occur in any of the media examined.

SUMMARY: Incubation of Saccharomyces cerevisiae with the plant cytokinin N 6-(Δ2-isopentenyl)adenine (2iP) resulted in an induction of thermotolerance similar to that induced by sublethal temperatures. Intracellular cAMP levels did not change significantly either during incubation at a sublethal temperature or in the presence of 2iP or ethanol. This suggested that stress-induced thermotolerance is triggered by a mechanism independent of cAMP activation. However, measurement of stress-induced thermotolerance in two mutant strains (tpk1, tpk2, TPK3; tpk1, TPK2, tpk3) each deficient in two of the catalytic subunits of the cAMP-dependent protein kinase (cAPK), revealed that sublethal heat induces thermotolerance by a mechanism part-mediated by the catalytic subunits of cAPK. In contrast, 2iP and ethanol induced thermotolerance by a mechanism fully dependent on the catalytic subunits of cAPK for expression. Therefore, this implies there must be an alternative novel mechanism, other than cAMP, for activating cAPK during stress. Sublethal heating resulted in large increases in intracellular trehalose levels which correlated with the induction of thermotolerance. However, incubation in 2iP or ethanol had no significant effect. This suggests trehalose synthesis is either coincidental with heat stress or that different stress factors induce thermotolerance by alternative mechanisms. Incubation with protein synthesis inhibitors reduced the levels of trehalose synthesized during sublethal heating, suggesting that synthesis of trehalose-6-phosphate synthase during heat stress could be accounting for the increased trehalose levels.

SUMMARY: mRNA steady-state levels and activities of enzymes of intermediary carbon metabolism (hexokinase, phosphoglucoisomerase, phosphofructokinase, glucose-6-phosphate dehydrogenase, phosphoglucomutase) and glucose-regulated enzymes (pyruvate decarboxylase, pyruvate dehydrogenase, invertase, alcohol dehydrogenase) were determined in glucose-limited continuous cultures of an industrial strain of Saccharomyces cerevisiae at different dilution rates (D) ranging from 0.05 to 0.315 h-1. The activity of most enzymes measured remained constant over this range except for alcohol dehydrogenase I/II which decreased proportionally with increasing dilution rate. A decrease in phosphoglucomutase activity occurred with increasing dilution rate but reached a minimum at D 0.2 h-1 and from thereon remained constant. A decrease in pyruvate decarboxylase activity and a slight decrease in phosphoglucoisomerase activity was observed. At D 0.29/0.315 h-1, at the onset of the Crabtree effect, most glycolytic enzymes remained constant except for pyruvate decarboxylase and glucose-6-phosphate dehydrogenase which increased at D 0.315 h-1 and alcohol dehydrogenase I/II which decreased. The ADHI/II and PDC1 mRNA levels obtained at the different dilution rates were in accordance with the activity measurements. The mRNA level of HXK1 decreased with increasing dilution rates, whereas the transcription of HXK2 increased. Pyruvate dehydrogenase (PDA1) and PG.11 mRNA fluctuated but no significant change could be detected. These results indicate that there is no transcriptional or translational regulation of glycolytic flux between D 0.05 h-1 and 0.315 h-1 except at the branch point between oxidative and fermentative metabolism (pyruvate decarboxylase/pyruvate dehydrogenase) at D 0.315 h-1. Surprisingly regulation of the Crabtree effect does not seem to involve transcriptional regulation of PDA1. The concentrations of ATP and cAMP decreased slightly during the increase in dilution rate, but increased again at D 0.315 h-1. The concentrations of glucose 6-phosphate and fructose 6-phosphate did not increase when the dilution rate increased as expected from the activities of hexokinase, phosphoglucoisomerase and phosphofructokinase. Instead, a decrease in the glucose 6-phosphate and fructose 6-phosphate concentrations was observed. The concentration of glucose 1-phosphate also decreased with increasing dilution rate but increased again at D 0.29 and 0.315 h-1, whereas the fructose 1,6-diphosphate concentration increased from 0.05 h-1 to 0.315 h-1. These data indicate that glycolytic flux in S. cerevisiae is regulated mainly by allosteric regulation of glycolysis when growth rate is increased. Invertase was present (mRNA and activity) at every dilution rate which indicates that glucose-specific repression of enzyme systems is not present in glucose-limited continuous cultures, not even when the yeast produces ethanol. This also indicates that the Crabtree effect is not related to glucose repression.

SUMMARY: The effects of different gaseous regimes on the growth rate and extracellular enzyme location in vitro of colonies of Phlebia radiata, Phlebia rufa and Coriolus versicolor are reported. The two Phlebia species showed similar growth, extracellular enzyme and pH responses to gaseous composition (N2, O2 and CO2), but the responses of C. versicolor differed. Whilst maximum extension rates were obtained for all species under atmospheric gaseous composition, maximum biomass production occurred at 5% (v/v) O2 with 20% (v/v) CO2 for the Phlebia species and at 5% O2 with 60% CO2 for C. versicolor. The Phlebia species had a coenocytic margin (5–6 mm width) under atmospheric conditions, which increased in width with increasing percentage of CO2. Laccase and peroxidase activity were present throughout the septate region, but not in the coenocytic zone. With C. versicolor laccase and peroxidase activities appeared throughout the colony, but were more intense in the peripheral region, under all gaseous regimes. A laser densitometer, normally used to visualize proteins on electrophoresis strips, was used to estimate profiles of biomass and laccase-α-naphthol activity within colonies. Surface pH changed little in colonies of C. versicolor but dropped by over 1 pH unit from the margin inwards with the Phlebia species. The significance of these results is discussed in terms of ecological strategy and developmental versatility.

SUMMARY: The mechanism by which Lactobacillus fermentum strain 104-S adheres to porcine squamous epithelium was investigated by studying the adsorption to epithelial cells, and control surfaces, of radioactively labelled material released from the bacterial cells by water extraction. The released material was fractionated by gel filtration and the adsorption of pronase-sensitive and -resistant material in the various fractions to porcine gastric tissue and the control surfaces of polystyrene and immobilized bovine serum albumin (BSA) was determined. The fraction with affinity for the epithelium was characterized by enzymic degradation, periodate oxidation, lipid extraction, and protein and carbohydrate analyses. The adsorption pattern of radioactively labelled crude released material mimicked the adhesion of whole labelled cells to polystyrene and to gastric squamous tissue pieces. On fractionation, the pattern of adsorption to polystyrene and BSA was different from that obtained for the tissue pieces. Considerably less labelled pronase-stable material bound to surfaces of polystyrene and BSA, as compared with the tissue, suggesting that the pronase-resistant component has a tissue-specific affinity. After pronase treatment of the fraction of M r about 20000 (20 K) containing labelled components with affinity for the epithelium, only saccharides were detected. Radioactivity was lost after hydrolysis with HCl, and therefore this pronase-resistant labelled component must be a saccharide. It is concluded that protein moieties in the extract have an affinity for several surfaces, including polystyrene, and that saccharide moieties have a specific affinity for the gastric squamous epithelium.

SUMMARY: The heterocyst envelope of the N2-fixing cyanobacterium Anabaena flos-aquae thickened as exogenous O2 partial pressure (pO2) was increased from 5 to 40 kPa. The majority of the thickening occurred in the glycolipid layer area of the envelope. Such thickening appears to be an O2-induced mechanism for providing a greater O2 diffusion barrier against O2 inhibition of nitrogenase. Nitrogenase activity decreased at pO2 levels above ambient (20 kPa), indicating that a thicker envelope is not completely effective as a barrier to O2 diffusion. However, when cultures grown at 10 kPa and 40 kPa pO2 were transferred to ambient pO2, the 40 kPa pO2 cells showed higher nitrogenase activity 24 h after transfer compared to those grown at 10 kPa or ambient pO2, indicating O2-protection of nitrogenase by thicker heterocyst envelopes. Heterocyst frequency was lowest at 20 kPa O2.

SUMMARY: A periodicity in nitrogen fixation potential with respect to the light-dark regime was studied in the filamentous non-heterocystous cyanobacterium Trichodesmium sp. NIBB 1067. During a 12 h light/12 h dark cycle, potential nitrogenase activity measured by acetylene reduction in the light was insignificant in the dark period, but developed after illumination for 1 to 3 h. Maximum nitrogenase activity was found at the middle of the light period, and activity decreased near the end of the light period. Manipulation of the length of the light and dark periods, and use of the glutamine synthetase inhibitor L-methionine sulphoximine, led to the conclusion that (1) the periodicity in activity was not attributable to an endogenous rhythm, (2) development and maintenance of nitrogenase activity in Trichodesmium was regulated by the light period, and (3) the decrease in activity at the end of the light period was due to the accumulation of an intermediate(s) in nitrogen metabolism. The nitrogenase Fe- and MoFe-proteins were always present despite the changes in nitrogenase activity associated with the light-dark cycle. However, a change in apparent molecular mass of the Fe-protein on SDS-PAGE correlated with the change in nitrogenase activity. The results indicate that changes of nitrogenase activity in Trichodesmium under a light-dark regime can be attributed to activation and deactivation of the Fe-protein, and that the activation of the protein depends on light.