- Volume 129, Issue 4, 1983

Thiamin-grown cells of Saccharomyces carlsbergensis exhibited decreased activity of NAD-linked glutamate dehydrogenase (NAD-GDH) and, in contrast, increased activity of NADP-linked glutamate dehydrogenase (NADP-GDH), as compared with control cells. Normal levels of glutamate dehydrogenase (GDH) activities were restored by adding pyridoxine. δ-Aminolaevulinate also prevented the thiamin-induced decrease in NAD-GDH activity, but had no effect on the increase in NADP-GDH activity. NAD-GDH activity was decreased similarly under anaerobic conditions, but NADP-GDH activity was little affected. High concentrations of glucose brought about a decrease in NAD-GDH activity and an increase in NADP-GDH activity, as observed in the thiamin-grown cells. When glycerol was used as carbon source in place of glucose, the thiamin effect on GDH activities was not marked. These results suggest that GDH enzymes are independently controlled by thiamin. NAD-GDH activity is decreased mainly through the thiamin-induced respiratory deficiency, and the increase in NADP-GDH activity is due to the thiamin-enhanced glucose effect. Amino acid metabolism seemed not to be involved in the thiamin effect on GDH activities since the intracellular pools of NH4 + and glutamate were not altered by thiamin. The activities of glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase were not influenced greatly by thiamin, unlike the activity of δ-aminolaevulinate synthase.

Theoretical calculations of the NADPH requirement for yeast biomass formation reveal that this parameter is strongly dependent on the carbon and nitrogen source. The data obtained have been used to estimate the carbon flow over the NADPH-producing pathways in these organisms, namely the hexose monophosphate pathway and the NADP+-linked isocitrate dehydrogenase reaction. It was calculated that during growth of yeasts on glucose with ammonium as the nitrogen source at least 2% of the glucose metabolized has to be completely oxidized via the hexose monophosphate pathway for the purpose of NADPH synthesis. This figure increases to approximately 20% in the presence of nitrate as the nitrogen source. Not only during growth on glucose but also on other substrates such as xylose, methanol, or acetate the operation of the hexose monophosphate pathway as a source of NADPH is essential, since the NADP+-isocitrate dehydrogenase reaction alone cannot meet the NADPH demand for anabolism. NADPH production via these pathways requires an expenditure of ATP. Therefore, the general assumption made in calculations of the ATP demand for biomass formation that generation of NADPH does not require energy is, at least in yeasts, not valid.

Candida utilisCBS 621 was grown in chemostat cultures at D = 0.1 h−1 on glucose, xylose, gluconate, acetate, or ethanol as the growth-limiting substrate with ammonia or nitrate as the nitrogen source and analysed for NADPH-producing and NADPH-consuming enzyme activities.

Nitrate and nitrite reductases were strictly NADPH-dependent. For all carbon sources, growth with nitrate resulted in elevated levels of HMP pathway enzymes. NADP+-linked isocitrate dehydrogenase did not vary significantly with the NADPH requirement for biosynthesis. Growth on ethanol strongly enhanced activity of NADP+-linked aldehyde dehydrogenase. Neither NADP+-linked malic enzyme nor transhydrogenase activities were detectable under any of the growth conditions. The absence of transhydrogenase was confirmed by the enzyme profiles of cells grown on mixtures of glucose and formate.

It is concluded that the HMP pathway and possibly NADP+-linked isocitrate dehydrogenase are the major sources of NADPH in Candida utilis.

The DNA and protein content of individual Escherichia coli cells were measured at a rate of 104 cells per second with a sensitive microscope-based flow cytometer. DNA and protein were quantified by measuring the fluorescence from cells stained with a combination of the DNA-binding drugs Mithramycin and ethidium bromide and by scattered light, respectively. Separate experiments demonstrated that the light scatter signal was proportional to protein content. Dual parameter histograms (fluorescence/scattered light) of bacterial cultures gave detailed pictures of changes dependent upon the growth conditions and of the cell cycle kinetics. Effects of antibiotics could be readily detected and characterized after a few hours. The results demonstrate that flow cytometry is a promising method for application in experimental and clinical microbiology.

Exoprotease production by Pseudomonas aeruginosa ATCC 10145 was growth-associated when cultures were grown on complex substrates such as proteins but it occurred during the decelerating growth phase when the organism was grown on amino acids, mixtures of amino acids or simple carbon sources. NH4Cl and simple carbon sources caused repression. Exoprotease was produced in chemostat cultures in response to growth under any of the nutrient limitations studied (carbon, nitrogen or phosphate). Furthermore, by growing at rates less than approximately 0.1 h−1, the repression of enzyme production could be overcome to a large degree. At low growth rates there was an inverse relationship between growth rate and exoprotease production. Thus, exoprotease production was depressed by available energy sources and was increased in response to any nutrient limitation.

In Pseudomonas aeruginosa ATCC 10145 a negative correlation was observed between the protonmotive force (ΔP) and the amount of exoprotease produced, with a decrease in ΔP resulting in an increase in exoprotease. The two components of ΔP, the transmembrane pH gradient (ΔpH) and the membrane potential (Δψ) were examined independently and it was observed that d varied very little under the conditions which influenced the activities of exoprotease. However, a positive correlation existed between pH and exoprotease production although the intracellular Δψ varied very little with either changes in growth rate or changes in extracellular pH. It was observed that with a decrease in growth rate, dpH became more alkaline and increased exoprotease activities were recorded. Furthermore, an increase in extracellular pH to give an artificial alteration in dpH, and, consequently, a decrease in dP, increased exoprotease production, thus confirming the importance of dpH in exoprotease production.

A simple method of isolating bacteria that utilize cyanide as a source of nitrogen for growth has been developed. This involved supplying hydrogen cyanide as a vapour to glucose-containing minimal-salts agar plates. The bacteria isolated were Gram-negative, oxidase-positive rods producing a fluorescent green pigment and were tentatively identified as strains of Pseudomonas fluorescens. Three organisms were studied further and shown to be P. fluorescens biotype II. One of these (NCIB 11764) was grown in a glucose-containing fed-batch culture with either NH4Cl or KCN as the limiting nutrient. Cyanide-grown bacteria produced stoichiometric amounts of ammonia from cyanide when pulsed with cyanide under aerobic conditions. Stimulation of oxygen uptake was seen on addition of cyanide to suspensions of cyanide-grown but not ammonia-grown bacteria.

Phenazine production by Pseudomonas phenazinium was not reduced by high concentrations of phosphate during batch culture (cf. Pseudomonas aeruginosa) but was inhibited by high concentrations of (NH4)2SO4 (1 to 10 g 1−1). In carbon-, sulphate-, magnesium- and potassium-depleted medium phenazine production paralleled growth. In continuous culture, phenazine production occurred over a wide range of growth rates under various nutrient-limiting conditions. At low growth rates cell resources were conserved and cell mass was maintained at the expense of secondary metabolite production. Low oxygen tensions and the addition of phenylalanine (0.1 g 1−1) each inhibited phenazine production.

Five pure cultures of diatoms, Thalassiosira sp., Navicula sp., Nitzschia sp., and two closely related Chaetoceros spp., were isolated from enrichment cultures made from ice and water samples from the ice-edge in the Bering Sea. Four cultures were studied in detail. The isolates did not grow above 18 °C; optimum growth was from 10 to 14 °C. The Nitzschia sp. and Chaetoceros spp. grew reproducibly at 0 ± 0·2 °C, albeit with long generation times of 6 to 7 d. Generation times at 10 °C were 0·8 to 1·9 d. The long generation times at 0 °C appeared intrinsic; growth rates were not increased by addition of ammonia, complex organic hydrolysates or light and dark cycles. The elemental analysis and culture density of the algae were reasonable. For example, the elemental analysis of Nitzschia sp. grown at 0 °C was C, 34·51%; H, 5·00%; N, 5·12%; ash, 30·3%, very similar to the values for cells grown at 10 °C. Cell yields of 0·5 mg dry weight ml−1 were routinely achieved. These appear to be among the first pure cultures of psychrophilic diatoms and possibly of microalgae in general.

The obligate methanotroph Methylococcus capsulatus (Bath) was grown in batch and continuous culture with ammonia, nitrate or dinitrogen as the sole nitrogen source. Cell-free extracts from these cultures all contained the ammonia assimilation enzyme glutamine synthetase. During growth on ammonia, however, the glutamine synthetase was biosynthetically inactive. Isolation of glutamine synthetase from M. capsulatus (Bath) resulted in a 133-fold purification with a yield of 11%. The purified enzyme had a molecular weight of 617000 and a subunit size of 60000 Dal. The metal ion and nucleotide specificity of glutamine synthetase was studied using both a biosynthetic assay and an artificial assay - the γ-glutamyltransferase assay. Feedback inhibition by several end-products of glutamine metabolism was observed and their effects were cumulative Control of glutamine synthetase activity by adenylylation/deadenylylation was cera demonstrated by snake venom phosphodiesterase treatment of the enzyme isolated from cells grown with different nitrogen sources.

Ammonia assimilation was studied using continuous cultures of three obligate methanotrophs. The type X organism, Methylococcus capsulatus (Bath), assimilated ammonia during growth on dinitrogen or nitrate via the glutamine synthetase/glutamate synthase pathway but utilized the alanine dehydrogenase pathway when grown in the presence of excess ammonia. Repression and derepression of these ammonia assimilation enzymes was demonstrated during the switchover of continuous cultures from nitrogen-free (N2-fixing) medium to medium containing high concentrations of ammonia. The properties of alanine dehydrogenase and glutamate synthase in this organism are discussed. In the type I methanotroph, Methylomonas methanica S1, the pathway of ammonia assimilation was again dependent on the source of fixed nitrogen in the growth medium, but in the type II methanotroph, ‘Methylosinus trichosporium’ OB3b, ammonia was assimilated exclusively via the glutamine synthetase/glutamate synthase pathway.

The production by a number of strains of Pseudomonas syringae pv. syringae of compounds that inhibit the growth of Escherichia coli is reported. A high proportion of P. syringae pv. syringae strains from Syringa vulgaris and Citrus spp. produced the compounds responsible for this effect, whereas the proportion of producers amongst strains from Phaseolus spp., Prunus spp. or Pyrus communis was low, and amongst strains from Allium spp., Pisum sativum or Sorghum spp. was zero. This specificity in production may be a useful taxonomic feature.

The inhibition of E. coli was prevented non-specifically by several basic amino acids, but not by the tripeptide triglycine, indicating that the compound may enter E. coli by the basic amino acid uptake system. There is no obvious relationship between this toxin and phaseolotoxin, syringomycin or syringotoxin.

An examination of 20 strains of butyric acid-producing Clostridium species for phospholipid class compositions, plasmalogen content, and acyl and alk-1-enyl chains showed that the deoxyribonucleic acid homology groups I (Clostridium butyricum) and II (Clostridium beijerinckii) could be distinguished by their lipid compositions. The phospholipids of C. butyricum strains had ethanolamine as the major nitrogenous lipid polar head-group moiety, more octadecenoate plus C19-cyclopropane than hexadecenoate plus C17-cyclopropane acyl chains, and the predominant alk-1-enyl chain was C18-monounsaturated. Clostridium beijerinckii strains had N-methylethanolamine plus ethanolamine in phospholipid head-groups, more hexadecenoate plus C17-cyclopropane than octadecenoate plus C19-cyclopropane acyl chains, and the major alk-1-enyl chain was C16-saturated. Three species falling outside the two homology groups Clostridium fallax, Clostridium pseudofallax and Clostridium acetobutylicum had ethanolamine as the major phospholipid base, but these species could be distinguished from C. butyricum by their acyl and alk-1-enyl chain compositions. The lipid composition of Clostridium pasteurianum is even more distinct.