- Volume 140, Issue 9, 1994

We have characterized a new family of proteins (the ROK family) which includes six transcriptional repressors for sugar catabolic operons, three sugar kinases, and three unidentified open reading frames. Analyses of the aligned sequences and phylogenetic tree construction allow predictions regarding the functional nature of conserved domains and residues within these proteins as well as the pathway of evolutionary divergence that gave rise to the family.

We have established a reliable procedure for electroporation in the marine bacterium Vibrio alginolyticus. Plasmids carrying the P15A replicon were found to be stably maintained in the Vibrio cells, and chloramphenicol, kanamycin or tetracycline were used for selection. Since we found that the Vibrio cells excrete DNase into the culture medium, cells were subjected to osmotic shock before extensive washing in order to remove the DNase from the periplasmic space. This manipulation resulted in about a 10-fold increase in the efficiency of transformation. In addition, cells were washed in the presence of 5-10 mM Mg2+in order to stabilize the outer membrane. The efficiency of transformation was found to be optimal when cells were harvested at early stationary phase, and when electroporation was carried out at an electric field strength between 5-0 and 7.5 kV cm-1. Under optimal conditions, about 105transformants per μg of input DNA were reproducibly obtained, which is tolerable for cloning.

A gene encoding 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase was isolated from the maize fungal pathogen Ustilago maydis. This was accomplished by identifying cDNA and genomic clones that hybridized to an internal fragment of the gene, amplified from U. maydis genomic DNA by PCR. The nature of the gene was determined by nucleotide sequence analysis, and by comparing the derived amino acid sequence of the gene with HMG-CoA reductases from yeast, and from other organisms. The hydrophobic nature of the N-terminal region of the deduced protein sequence also supported the view that this gene encoded HMG-CoA reductase. A C-terminal-truncated fragment of the U. maydis HMG-CoA reductase gene was shown to be expressed in Escherichia coli in a catalytically active form. The expressed protein was also shown to be sensitive to an inhibitor of mammalian HMG-CoA reductase activity.

Aspergillus fumigatus and Aspergillus flavus are the most common cause of invasive mould infections worldwide and carry a high mortality. Corticosteroid therapy and Cushing's disease are associated with an increase in invasive aspergillosis. Corticosteroids impair immune function in mammals and, specifically, the conidicidal activity of human macrophages, which was thought to be sufficient explanation for this increased risk. However, we have found a 30-40% increase in growth rate of A. fumigatus and A. flavus exposed to pharmacological doses of hydrocortisone (a human glucocorticoid), suggesting an alternative or additional mechanism for the association. No significant effect was observed with other human steroids such as testosterone, oestradiol or progesterone, though a smaller (21%) but significant growth rate increase was obtained with the fungal sterol ergosterol. The presence of a ligand/receptor system is therefore possible in pathogenic Aspergillus spp. Although corticosterone-binding proteins have been identified in some yeast species, a demonstrable physiological effect has been lacking. Interruption of the putative ligand/receptor interaction could have a major effect on the growth and pathogenicity of A. fumigatus, providing opportunities for the development of alternative therapeutic strategies to those currently available.

Chlamydia trachomatis is a leading cause of sexually transmitted diseases and a number of strategies have been developed to produce vaccines to prevent its transmission. The purpose of this study was to map the neutralizing epitopes of C. trachomatis major outer-membrane protein (MOMP) serovar K by using anti-MOMP antibodies and synthetic peptides. Seven anti-MOMP monoclonal antibodies and three polyclonal antisera were produced and characterized. Their fine specificity was defined by direct binding assay on 15 peptides of 10 amino acid residues, overlapping by five residues, corresponding to the four variable domains (VDI-VDIV: residues 64-85, 139-160, 224-237 and 287-319) of MOMP serovar K. Our data confirmed that a neutralizing epitope is found in VDIV, defined by peptides K12 and K13. This epitope is 296TTLNPTIAG304, which has never been reported as a neutralizing epitope of serovar K. Another neutralizing epitope, defined by peptide K2, has been identified in VDI. This epitope is in the same position as 71VAGLEK76, a peptide with neutralizing activity found in serovar A, but they are not identical because antibodies against peptide K2 do not bind to this epitope. No neutralizing epitope was found in the two other variable domains (VDII and III). In summary, two neutralizing sites, one in variable domain I and one in variable domain IV, were identified in serovar K.

The sensitivity of the microaerophilic protozoan Trichomonas vaginalis to oxygen and products of its reduction, and the antioxidant defences employed by this organism, were investigated. Studies revealed that this amitochondrial flagellate is sensitive to oxygen tensions above those experienced in situ in the vagina (i.e. > 60 μM) and that metronidazole-resistant strains (CDC 85 and IR78) were more sensitive to elevated oxygen levels than a metronidazole-sensitive isolate (1910). In the presence of radical scavengers, inactivation of organisms at 60 μM oxygen was significantly lessened. Investigation of the antioxidant enzymes present in this organism revealed that activities of peroxide-reducing enzymes (e.g. catalase and general peroxidase) were not detectable, but that a cyanide-insensitive, azide-sensitive superoxide dismutase was present in cell extracts. Measurement of thiol-cycling enzymes indicated that NADPH could drive the reduction of oxidized glutathione (thiol reductase); however, the corresponding peroxidase activity was not detected. Analysis of thiols in whole cells of T. vaginalis indicated that glutathione was absent, but high levels of other thiols, propanethiol, methanethiol and H2S, were present. No significant differences were detected in thiol levels or antioxidant enzyme activities on comparison of metronidazole-sensitive and resistant strains. These results indicate that the sensitivity of T. vaginalis to oxygen above physiological levels is due to the lack of adequate peroxide-reducing enzymes and radical-scavenging mechanisms.

The synthesis and metabolism of yeast cell wall glucan were studied using a Saccharomyces cerevisiae construct in which radiolabelled galactose is metabolized to UDP-glucose and preferentially incorporated into glucan. Greater than 85% of the incorporated radiolabel was found within insoluble cell wall material. Our study also demonstrated that radiolabelled wall glucan is released from cells growing exponentially, and that the released radiolabel is reutilizable low molecular mass material. Size exclusion chromatography and enzymic analysis indicate that laminaribiose comprises approximately 50% of the released fraction. This is consistent with in vitro findings that laminaribiose is a by-product of a newly identified glucosyltransferase (R. P. Hartland, G. W. Emerson & P. A. Sullivan, 1991, Proc R Soc Lond B 246, 155-160) associated with fungal cell walls. Our results also suggest that pre-existing glucan undergoes less metabolic processes than newly synthesized material as evidenced by a decrease in released radiolabel over time. Pulse double labelling of glucan and total cellular protein indicate that glucan metabolism and protein synthesis (ps) are not tightly coupled although they do parallel each other during exponential growth. Inhibitors of glucan synthesis (gs) decrease the glucan to protein ratio. Measurement of ps allows normalization for non-specific decreases in the rate of cell wall synthesis due to general cessation of growth. Cilofungin and papulacandin B, two putative inhibitors of gs, inhibited galactose incorporation into glucan and thus showed a decrease in the glucan to protein ratio, although ps was affected. In contrast, cycloheximide, a known ps inhibitor, displayed an elevated ratio. This whole cell construct affords a simplified system for elucidating the synthesis and metabolic activity of the yeast cell wall and enables the discrimination between specific effectors of gs and ps.

The conidiation process of Neurospora crassa is characterized by three morphogenetic steps: hyphal adhesion, aerial hyphal formation, and production of conidia. Previous data indicated the occurrence of a hyperoxidant state at the onset of all three morphogenetic steps. Because glutamine synthetase (GS) and the biosynthetic glutamate dehydrogenase [GDH(NADP)] enzymes are susceptible to inactivation by reactive oxygen species, we followed these enzyme activities during conidiation and under different physiological conditions and related them to the hyperoxidant states and morphogenesis. Loss of GS activity occurred prior to all three morphogenetic steps, coinciding with an increase in total protein oxidation. Oxidized GS polypeptides were detected during hyphal adhesion. Loss of GDH(NADP) activity also occurred during hyphal adhesion and before aerial hyphal formation; the enzyme polypeptide and activity decreased in the adhered hyphae to low values and no GDH(NADP) was detected in aerial hyphae. The catabolic GDH [GDH(NAD)] behaved in an opposite manner, increasing its activity during hyphal adhesion and aerial hyphae development. These results are discussed with regard to cell differentiation and the conidiation process in N. crassa.

Adaptation of Aspergillus niger to short-term stress induced by three antifungal agents [amphotericin B (AMPH), miconazole (MCZ), and ketoconazole (KCZ)] was observed and evaluated quantitatively using individual hyphae. Spores were inoculated onto a poly-l-lysine-coated glass plate making up the base of a culture vessel. Potato dextrose broth (PDB) was added and the vessel incubated for 24 h at 28 °C. The growth rate of an arbitrarily selected test hypha was measured automatically. Exposure to AMPH (0.075 μg ml-1) stopped the growth of the hypha. After washing with PDB, the same concentration of AMPH was applied again. The growth of the test hypha was not inhibited. This phenomenon was defined as adaptation to the short-term stress of AMPH. Similarly, adaptation was observed with MCZ (0.01 μg ml-1) and KCZ (0.5 μg ml-1). The time required for the test hypha to restart growth after washing with PDB depended upon the concentration of MCZ or KCZ, but not upon the concentration of AMPH.

Exogenously supplied L-pipecolic acid was accumulated by Escherichia coli cells and protected them while growing at inhibitory osmolarity. Using specific uptake mutants and competitive assays, we established that the imino acid enters the cells through the ProP and ProU systems with K m values of 225 and 53 μM, respectively. Surprisingly, in spite of the requirement for the wild-type proX gene for osmoprotective ability, no binding activity of labelled pipecolate with the periplasmic protein encoded by proX could be detected. In an attempt to demonstrate whether the two porters (ProP and ProU) are the only carriers involved in osmoregulation, a variety of molecules known for their intracellular osmolarity-dependent accumulation in various organisms were investigated. N-Dimethylproline (proline betaine), N-dimethylglycine, homobetaine (β-alanine betaine), γ-butyrobetaine and dimethylsulfonio-propionate were found to be capable of promoting the growth of osmotically stressed E. coli. All of these molecules enter bacterial cells via ProP and ProU porters. None of the osmoprotectants except N-dimethylproline was able to bind the periplasmic protein encoded by proX, while this protein was necessary for their uptake. Apparently, ProP and ProU are the sole osmoporters involved in osmolyte influx into E. coli cells.

The degree of unsaturation of membrane lipids of Acanthamoeba castellanii at 30 °C decreased with batch-culture age. These changes were primarily attributable to a decline in microsomal Δ12-desaturase activity and in the relative proportion of linoleate. No change in the fatty acid composition, Δ12-desaturase activity or any increased incorporation of [1-14C]acetate into polyunsaturated fatty acids was observed following chilling of early- and mid-exponential-phase cultures to 15 °C. In contrast, chilling of late-exponential and stationary-phase cultures resulted in a rapid and marked increase in the synthesis of polyunsaturated fatty acids. Thus, after 12 h incubation at 15 °C, the relative proportions of oleate and linoleate in these older cultures were similar to those of early- and mid-exponential-phase cultures at 30 °C. Despite these differences, an approximately 9.5 h lag in cell division was evident following chilling of both mid-exponential and late-exponential/early-stationary-phase cultures, and the subsequent pattern of cell division over 60 h incubation was similar in both cases. Oxygen uptake rates in cultures of either age were also decreased approximately equally at 15 °C. In contrast, chilling of mid-exponential-phase cells resulted in only an approximately 44% reduction in the rate of phagocytosis of fluorescently-labelled latex beads, whereas an approximately 98% inhibition of phagocytosis by late-exponential/early-stationary-phase cells resulted at 15 °C. Furthermore, a gradual subsequent increase in the rate of phagocytosis at 15 °C was only observed in the older cultures and was correlated with increases in the fatty acid unsaturation index of these cells. Thus, 12 h after chilling, the cells from late-exponential/early-stationary-phase cultures had achieved rates of phagocytosis similar to those of chilled cells from mid-exponential growth and their fatty acid compositions were now similar. The results suggest a role of membrane lipid unsaturation in the control of phagocytotic activity of A. castellanii.

Streptococcus salivarius transports mannose by a phosphoenolpyruvate: sugar phosphotransferase system (PTS) which consists of a membrane Enzyme II and two forms of Enzyme III (IIIMan) with molecular masses of 38.9 kDa (IIIMan H) and 35.2 kDa (IIIMan L) respectively. Using a pseudorevertant (strain 57P) isolated from a IIIMan L-deficient spontaneous mutant unable to grow on mannose, we demonstrated that S. salivarius could also transport mannose by an inducible fructose PTS. This PTS phosphorylated fructose at the C-1 position with a high affinity (10 μM) and mannose at the C-6 position with a low affinity (200 μM). Derepression of this system in some IIIMan L-deficient mutants would explain their ability to grow on mannose.

This paper is concerned with the application of Metabolic Control Analysis to chemostat experiments. It deals with the peculiar problems which are presented when using a chemostat for such purposes. The paper shows how to apply a top-down approach to quantifying control in the chemostat exerted by both the ‘physical’ components (e.g. dilution rate) and the biological components. The analysis is considered at a number of levels, depending on the amount of information on the components of metabolism either known or accessible experimentally. A method is also presented for using the information obtained by this analysis to obtain a ‘virtual’ control coefficient, which is defined as a control coefficient which would be obtained if it were possible to clamp all metabolites ‘external’ to the micro-organism, including biomass, at their steady-state levels. This allows, amongst other things, control coefficients with respect to growth rate to be determined under chemostat conditions.

Periplasmic oxidation of glucose into gluconate and 2-ketogluconate in Klebsiella pneumoniae occurs via glucose dehydrogenase (GDH) and gluconate dehydrogenase (GaDH), respectively. Since, as is shown here, in the presence of glucose, gluconate and 2-ketogluconate are not further metabolized intracellularly the physiological function of this periplasmic route was studied. It was found that periplasmic oxidation of glucose could function as an alternative production route of ATP equivalents. Instantaneous activation of either GDH or GaDH reduced the rate of degradation of glucose via glycolysis and the tricarboxylic acid (TCA) cycle in vivo. Furthermore, aerobic, magnesium- and phosphate-limited chemostat cultures with glucose as the carbon source showed high GDH plus GaDH activities in contrast to nitrogen-and sulphate-limited cultures. However, when fructose, which is not degraded by GDH, was the carbon source, specific oxygen consumption rates under these four conditions were essentially the same. The latter observation suggests that high transmembrane phosphate gradients which are supposedly present under phosphate-limited conditions do not cause high energetic demands due to futile cycling of phosphate ions. In addition, dissipation of the transmembrane phosphate gradient of phosphate-limited cells immediately increased the rate of intracellular glucose degradation. It is concluded that under phosphate-limited conditions (i) extensive futile cycling of phosphate ions is absent and (ii) low concentrations of phosphate ions limit intracellular degradation of glucose. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) activities of cell-free extracts of glucose-grown cells harvested from aerobic chemostat cultures limited in various nutrients showed that at least a tenfold overcapacity in GAPDH activity was present under phosphate-limited conditions with respect to the steady-state carbon fluxes through this enzyme. The physiological significance of this adaptation and the possible role of GDH and GaDH are discussed.

The parasitic protist Trichomonas vaginalis was cultured in chemostats with glucose or maltose as carbon and energy source. The maximum growth rate was about six divisions per day independent of the substrate, and the apparent K m for glucose was 0.375 mM. While growing on maltose, the growth rate depended linearly on the maltose concentration, indicating that in contrast to glucose metabolism a diffusion step is rate-limiting to maltose metabolism. Cultures were examined over a wide range of growth rates under four conditions: utilizing glucose or maltose as carbon and energy source, with the carbon source rate-limiting or present in excess. Cell density, cellular protein and carbohydrate content as well as residual substrate concentration in the culture fluid were measured at each steady state. The protein content was constant at 100 μg protein per cell except when T. vaginalis was cultured under glucose limitation; in the latter case, slow-growing cells had less protein than cells grown at high rates. When growing under glucose limitation T. vaginalis metabolism changed to become more energy efficient at growth rates exceeding about half the maximum rate. The maintenance energy at the low growth rates accounted for approximately half of the total carbon consumption, which is high in comparison to other micro-organisms. At low growth rates the yield on maltose exceeded that on glucose, when expressed in terms of carbon equivalents. The yields on maltose and glucose were equal, but much lower, when the carbon source was not rate-limiting. A comparison of the data of this study with similar studies on other organisms suggests that the high maintenance energy of T. vaginalis may be used primarily for maintaining homeostasis of the internal conditions to enable growth and survival in the vagina.

The anaerobic parasitic protist Trichomonas vaginalis was adapted in chemostats to eight different conditions defined by different growth rates and carbon regimes. Glucose or maltose was used as carbon and energy source. Cells cultured under well-defined steady states were tested in short-term experiments. The kinetics of glucose and maltose uptake were determined and their glucokinase and α-glucosidase activities were measured. Uptake in 20 min was measured with radiolabeled glucose and maltose, rather than analogues, using the silicone oil centrifugation technique. Hence, the accumulated label represents both transport and metabolic activity. The total uptake of glucose was highest in organisms that had been starved for glucose during growth. The kinetics of glucose uptake can be understood by assuming rate-limitation by transport across the plasma membrane at low external concentrations and by the subsequent metabolism at concentrations exceeding a cross-over value. The specific glucokinase activity correlated in only four out of eight cases with the saturation uptake. The kinetics of maltose uptake indicated rate-limitation at low maltose concentrations by a diffusion-limited step and at higher levels by metabolic steps. The uptake of maltose was primarily affected by the growth rate during culture, the highest growth rates resulting in most uptake. Maltose uptake was determined only partially by the cellular α-glucosidase activity. The activities of both transport and metabolic enzymes changed due to the culture conditions suggesting that the control over glucose and maltose metabolism is shared by several steps in the pathway.