Arginine metabolism inLactobacillus leichmannii

Lactobacillus leichmannii ATCC 4797 metabolizes arginine via the arginine dihydrolase pathway producing ornithine, ammonia, carbon dioxide, and ATP. The specific activities of arginine deiminase and ornithine transcarbamylase were two-or threefold lower (stationary growth phase) in galactose-grown cells. The addition of arginine increased the specific activities of these two enzymes with all growth sugars. When glucose was virtually exhausted from the medium, maximum activities of both enzymes were achieved. The formation of two first enzymes of the arginine dihydrolase pathway inL. leichmannii ATCC 4797 seems to be under the control of two processes: induction by arginine and repression of the induced synthesis by glucose.Dedicated to Dr. Luis F. Leloir on the occasion of his 80th birthday, 6 September 1986.     

The inducible microsomal fatty alcohol oxidase of Candida (Torulopsis) apicola

In the transition phase of Candida apicola IMET 43747 from logarithmic to stationary growth a pyridine-nucleotide-independent alcohol oxidase was induced coinciding with the beginning of sophorose lipid production. This enzyme was not repressed by glucose and was measurable in stationary cells grown on glucose or on a mixture of n-hexadecane and glucose. An NAD⁺-dependent aldehyde dehydrogenase behaved in the same way. Both enzymes were localized in the microsomal fraction. The alcohol oxidase accepted long-chain (fatty) aliphatic alcohols (C₈ to at least C₁₆) and diols starting from decanediol. Trace activities were found with -hydroxy fatty acids. Aromatic, secondary and tertiary alcohols were not oxidized. In the stationary growth phase the substrate specificity of the alcohol oxidase tends to be changed to more hydrophobic substrates. The physiological role of both enzymes, the alcohol oxidase and aldehyde dehydrogenase, is discussed including their possible involvement in the synthesis of sophorose lipid. Correspondence to: R. K. Hommel     

Arginine metabolism in lactic streptococci.

Streptococcus lactis metabolizes arginine via the arginine deiminase pathway producing ornithine, ammonia, carbon dioxide, and ATP. In the four strains of S. lactis examined, the specific activities of arginine deiminase and ornithine transcarbamylase were 5- to 10-fold higher in galactose-grown cells compared with glucose- or lactose-grown cells. The addition of arginine increased the specific activities of these two enzymes with all growth sugars. The specific activity of the third enzyme involved in arginine metabolism (carbamate kinase) was not altered by the composition of the growth medium. In continuous cultures arginine deiminase was not induced, and arginine was not metabolized, until glucose limitation occurred. In batch cultures the metabolism of glucose and arginine was sequential, whereas galactose and arginine were metabolized concurrently, and the energy derived from arginine metabolism was efficiently coupled to growth. No arginine deiminase activity was detected in the nine Streptococcus cremoris strains examined, thus accounting for their inability to metabolize arginine. All nine strains of S. cremoris had specific activities of carbamate kinase similar to those found in S. lactis, but only five S. cremoris strains had ornithine transcarbamylase activity.     

Isolation and characteristics ofTreponema zuelzerae nov. spec., an anaerobic,free-living spirochete

Summary An anaerobic, free-living spirochete was isolated from mud. The organism can be cultivated in ordinary nutrient media, e.g. yeast extract-glucose. End products of glucose fermentation are: lactic, acetic, and succinic acids, CO₂, and H₂. In cultures of this organism spheroid bodies are formed, especially during the stationary growth phase. Studies of slide cultures showed that these bodies, when inoculated in fresh medium, do not give rise to spiral cells whereas a rapid multiplication of normal cells, also present in the inoculum, was observed. Since the organism is serologically related toTreponema pallidum, it has been assigned to the genusTreponema, and is here described asTreponema zuelzerae nov. spec. Part of this work was carried out at the Hopkins Marine Station of Stanford University, Pacific Grove, U.S.A., under a Rockefeller Foundation fellowship. Present address: Laboratorium voor Microbiologie, Wageningen, the Netherlands.     

Adaptation of methane formation and enzyme contents during growth of Methanobacterium thermoautotrophicum (strain ΔH) in a fed-batch fermentor

During growth of Methanobacterium thermoautotrophicum in a fed-batch fermentor, the cells are confronted with a steady decrease in the concentration of the hydrogen energy supply. In order to investigate how the organism responds to these changes, cells collected during different growth phases were examined for their methanogenic properties. Cellular levels of the various methanogenic isoenzymes and functionally equivalent enzymes were also determined. Cells were found to maintain the rates of methanogenesis by lowering their affinity for hydrogen: the apparent K _m ^H2 decreased in going from the exponential to the stationary phase. Simultaneously, the maximal specific methane production rate changed. Levels of H₂-dependent methenyl-tetrahydromethanopterin dehydrogenase (H₂-MDH) and methyl coenzyme M reductase isoenzyme II (MCR II) decreased upon entry of the stationary phase. Cells grown under conditions that favored MCR II expression had higher levels of MCR II and H₂-MDH, whereas in cells grown under conditions favoring MCR I, levels of MCR II were much lower and the cells had an increased affinity for hydrogen throughout the growth cycle. The use of thiosulfate as a medium reductant was found to have a negative effect on levels of MCR II and H₂-MDH. From these results it was concluded that M. thermoautotrophicum responds to variations in hydrogen availability and other environmental conditions (pH, growth temperature, medium reductant) by altering its physiology. The adaptation includes, among others, the differential expression of the MDH and MCR isoenzymes.     

Properties of the Invertases of Cultured Sycamore Cells and Changes in Their Activity during Culture Growth

When cultured sycamore cells are homogenised in a phosphate-citrate buffer at pH 7.0 and the homogenate centrifuged two fractions are obtained both of which show the presence of an acid (opt. pH 4.0–4.5) and a neutral (opt. pH 7.0–7.4) invertase. The activity of the insoluble pellet appears to be located in its cell wall fragments. The acid and neutral invertases of the soluble fraction can be separated by fractional precipitation with (NH₄SO₄. The activities of these enzymes are low in stationary phase cells but they increase following subculture to reach peaks of activity towards the end of the period of most active cell growth and division and then decline again as the cells begin to enter stationary phase. The activities of both enzymes are higher in the cell wall than in the soluble fraction and the acid invertase reaches higher levels of activity than the neutral enzyme in both fractions. When cells are subcultured there occurs within a few hours an increase in the acid invertase and a decline in the neutral invertase activity in the cell wall fraction and a decline in the acid invertase of the soluble fraction prior to the large net increases in the activities of both enzymes in both locations which occurs as the cells embark upon cell division. The pattern of changes in the invertase activities through the growth cycle of batch propagated cultures is similar whether the cells are grown in sucrose, or glucose, or sucrose plus glucose; the highest levels of activities were recorded in the glucose-grown cells. The total yield of invertase activities and the distribution of activities between the soluble and cell wall fractions of the homogenates are affected by the pH of the extraction medium (within the range pH 4.0–8.0). It has not proved possible to completely remove the invertases from the cell wall fraction; upwards of 50 % of the acid invertase was recovered from this fraction by treatment with Triton-X followed by urea, but these treatments inactivated a high proportion of the neutral enzyme. These findings are compared with other studies on the activity and intra-cellular distribution of plant invertases and the possible roles of these enzymes discussed.     

Catalase Activities of Hydrocarbon-utilizing Candida Yeasts

The catalase activities of the Candida cells grown on hydrocarbons were generally much higher than those of the cells grown on Iauryl alcohol, glucose or ethanol. Km values for hydrogen peroxide of the enzymes from the glucose- and the hydrocarbon-grown cells of Candida tropicalis were the same level. The enzyme activities of the yeasts were higher at the exponential growth phase, especially of the hydrocarbon-grown cells, than at the stationary phase. Profuse appearance of microbodies having homogeneous matrix surrounded by a single-layer membrane has also been observed electronmicroscopically in the hydrocarbon- grown cells of several Candida yeasts. Cytochemical studies using 3,3′-diaminobenzidine (DAB) revealed that the catalase activity was located in microbodies. These facts suggest that the catalase activities would be related to the hydrocarbon metabolism in the yeasts.     

Effects of silicate resupply to silicate‐deprived Thalassiosira weissflogii (Bacillariophyceae) in stationary or senescent phase: short‐term patterns of growth and cell death

The ability of nutrient‐deprived phytoplankton to recover in the short term when nutrients are resupplied has been studied for nitrogen and phosphorus, but the case for silicate (Si) is poorly understood. Si‐limited Thalassiosira weissflogii (Grunow) Fryxell et Hasle (grown in batch culture) was harvested in stationary phase (when cell numbers stopped increasing ~2 d after Si depletion) and senescence (when cell numbers declined ~4 d after Si depletion) and Si was resupplied at different concentrations (from 0 to 100 μM). Cell numbers, proportion of dead cells, variable fluorescence emissions (F_v/F_m), and activities of proteases were measured during Si depletion and for 24 h after Si resupply. As Si was depleted, the specific growth rate declined, dead cells increased from ~2% in log phase, to ~25% in stationary phase to over 35% in senescence, and activities of proteases associated with cell death increased several‐fold. Concentration‐dependent recovery of growth rate was seen after 24 h for cultures resupplied with Si in stationary phase but not in senescence. However, resupply of Si at 100 μM to stationary phase cultures alone increased protease activity to nearly the levels seen in senescence. Differences in the responses to Si resupply suggest that the ability and time to recover from Si depletion depend not only on the growth phase but also on the concentration resupplied.     

Cytochrome P-450 in the sophorose-lipid-producing yeast Candida (Torulopsis) apicola

The appearance of cytochrome P-450 and of cytochrome oxidase aa₃ were determined in the sophorose lipid producing yeast Candida (Torulopsis) apicola IMET 43 747 grown on a mixture of glucose and n-hexadecane. Cytochrome P-450, detectable in both the logarithmic and the stationary growth phase was not repressed by glucose. At the end of the logarithmic growth phase the content of cytochrome P-450 was three- to fivefold increased, which was connected with initiation of sophorose lipid biosynthesis. After that it dropped to the basal level, which remained constant during sophorose lipid biosynthesis. Cytochrome P-450 from logarithmic cells was cross-reactive with an antibody derived against cytochrome P-450alk from C. tropicalis. With microsomal proteins of stationary cells no cross-reactivity was obtained. The microsomal hydroxylase system of stationary cells seem to be regulated by the carbohydrate used as carbon source. Correspondence to: R. K. Hommel     

Ornithine cycle in Nostoc PCC 73102. Arginase,OCT and arginine deiminase,and the effects of addition of external arginine,ornithine, or citrulline

Arginase, ornithine carbamoyl transferase (OCT) and arginine deiminase activities were found in cell-free extracts of Nostoc PCC 73102, a free-living cyanobacterium originally isolated from the cycad Macrozamia. Addition of either arginine, ornithine or citrulline to the growth medium induced significant changes in their in vitro activities. Moreover, growth in darkness, compared to in light, induced higher in vitro activities. The in vitro activities of arginase and arginine deiminase, two catabolic enzymes primarily involved in the breakdown of arginine, increased substantially by a combination of growth in darkness and addition of either arginine, or ornithine, to the growth medium. The most significant effects on the in vitro OCT activities where observed in cells grown with the addition of ornithine. Cells grown in darkness exhibited about 6% of the in vivo nitrogenase activity observed in cells grown in light. However, addition of external carbon (glucose and fructose) to cells grown in darkness resulted in in vivo nitrogenase activity levels similar to, or even higher than, cells grown in light. Growth with high in vivo nitrogenase activity or in darkness with the addition of external carbon, resulted in repressed levels of in vitro arginase and arginine deiminase activities. It is suggested that nitrogen starvation induces a mobilization of the stored nitrogen, internal release of the amino compound arginine, and an induction of two catabolic enzymes arginase and arginine deiminase. A similar and even more pronunced induction can be observed by addition of external arginine to the growth medium.     

Pathogenicity ofPseudomonas aeruginosa and its relationship to the choline metabolism through the action of cholinesterase,acid phosphatase,and phospholipase C

The increase of cholinesterase (ChE), acid phosphatase (Ac.Pase), and phospholipase C (PLC) activities byPseudomonas aeruginosa was associated with the choline consumption in growth media of varied composition (high or low P_i concentrations, presence or absence of ammonium ion, amino acids, polyamines, peptone, or tricarboxylic acid cycle intermediates). The highest production of the three enzymes occurred in the late stationary growth phase. The simultaneous presence of alkaline phosphatase (Alk.Pase) and the above enzymes was noted when the bacteria were grown in low P_i medium plus choline, in the absence of a preferred carbon source. The importance of choline in the production of ChE, Ac.Pase, and PLC was observed in either clinical isolates or collection strains ofP. aeruginosa. These enzymes catalyze the hydrolysis of acetylcholine, phosphorylcholine, and phosphatidylcholine. Through their action the bacteria may break down various compounds (e.g., acetylcholine, from the corneal epithelium; lung surfactant dipalmitoylphosphatidylcholine; phosphorylcholine, a product of the PLC action) or cell membranes through the coordinated action of PLC and Ac.Pase or Alk.Pase. The final consequence of the action of these enzymes is an increase of the free choline concentration. Extrapolated to an in vivo situation, if the stationary growth phase resembles the conditions thatP. aeruginosa encounters in its natural environments, then it is possible to include choline among the factors promoting the pathogenicity of this bacterium.     

Two δ1-pyrroline-5-carboxylate dehydrogenase isoforms are expressed in cultured Nicotiana plumbaginifolia cells and are differentially modulated during the culture growth cycle

δ¹-Pyrroline-5-carboxylate (P5C) dehydrogenase (EC 1.5.1.12) activity was measured in extracts from cultured tobacco (Nicotiana plumbaginifolia Viviani) cells. Two putative isozymes were resolved by anion-exchange fast protein liquid chromatography. These enzyme forms showed different patterns of expression during the culture growth cycle: activity-I increased in exponentially growing cells and declined rapidly in late logarithmic phase, while activity-II was found at substantial level only in cells which were entering the stationary phase. Both P5C dehydrogenases were partially purified and characterized with respect to kinetic and biochemical properties. They showed similar molecular masses as judged from retention patterns upon gel-filtration chromatography. The in vitro activity of both enzymes had a broad maximum around pH 7.4, and was progressively inhibited by Cl⁻ at concentrations ranging from 0.1 to 1 M. A pronounced difference was found between their apparent K _m values for the two substrates, P5C and NAD⁺, the higher affinities being shown by activity-I. Regulation of P5C dehydrogenase during salt-stress-induced proline accumulation was investigated. Following the addition of 175 mM NaCl to the culture medium the level of activity-I was substantially unaffected, while the specific activity of the other isozyme failed to increase even after the onset of the stationary phase of growth. Possible roles for P5C dehydrogenase isozymes in proline and arginine metabolism are discussed. Received: 23 May 1996 / Accepted: 18 December 1996     

Regulation of exoprotease production by temperature and oxygen in Vibrio alginolyticus

The production of an extracellular collagenase and an alkaline protease by Vibrio alginolyticus during stationary phase was inhibited by a temperature shift from 30 to 37°C and by a lack of oxygen. The stability of the exoproteases was unaffected by incubation at 37°C and aeration. The optimum growth temperature for the V. alginolyticus strain was 33.5°C Aeration enhanced the rate of growth of exponential phase cells. Temperature and oxygen did not affect the growth of stationary phase cells when the exoproteases were being produced. Macromolecular synthesis in stationary phase cells was not affected by temperature. There was no rapid release of the exoproteases after temperature shift down and chloramphenicol inhibited the production of the enzymes when added at time of temperature shift down from 37 to 30°C. The regulation of exoprotease production by temperature and oxygen was specific and has implications regarding the ecology of V. alginolyticus. Cerulenin, quinacrine and O-phenanthroline inhibited the production of the exoproteases.     

Studies of fungal growth and intermediary carbon metabolism under steady and non-steady state conditions

The physiology of Aspergillus nidulans strain 224 has been studied under conditions of batch- and glucose-limited chemostat-culture and the effect of different steady state growth rates and dissolved oxygen tensions (DOT) examined. Measurements of the specific activities of selected glucose enzymes, the extent of oxygen uptake inhibition by glycolytic inhibitors, and radiorespirometric analyses were made in order to follow the variations in glucose catabolism, which occurred under these conditions. Greatly increased activity of the hexosemonophosphate (HMP) pathway was found during: (i) exponential growth of batch cultures; (ii) at near maximum specific growth rates (μ = 0.072 hr^?1) (DOT = 156 mm Hg); and (iii) at low DOT levels (<30 mm Hg) (μ = 0.050 hr^?1) in chemostat cultures. These changes in glucose eatabolism have been discussed in terms of the biosynthetic demands of the fungus under the influence of changing growth pressures. Preliminary studies also have been made of transition state behavior following stepwise alteration of the DOT. A new steady state was established after 4–5 culture doublings during which period an “overshoot” in HMP pathway activity occurred; these kinetics are indicative of a derepression of certain glucose enzymes. Low molecular weight phenols are synthesized during the exponential phase in batch cultures and these are further metabliized to a major secondary metabolite, melanin, at the onset of stationary phase conditions. The kinetics of tyrosinase production in steady state chemostats differs from those that might be predicted for an enzyme associated solely with secondary metabolism. A primary physiological role for this oxidase in Aspergillus nidulans has been postulated.     

Effect of α-Naphthalene- and α-Naphthoxyacetic Acid on the Activity of Certain Enzymes of Atropa belladonna cv. lutea Cells in Suspension Culture

The growth of Atropa belladonna cv. lutea Döll cells and the activities of several enzymes were studied as a function of time in suspension cultures without added auxin, with α-naphthaleneacedc acid (NAA) and with α-naphthoxyacetic acid (NOA). The growth curves were rather similar and therefore easy to compare. The enzyme activities (per mg dry weight of culture) were distinctly higher in cultures grown with NAA than in the other two treatments, in which root formation was common. In some cases the enzyme activities were lower in cultures with NOA than in the control series but usually the curves were quite parallel. Promotion of enzyme activities by NAA (aldolase, aminopeptidase, gluta-mate: oxaloacetate transaminase) was clearest in the beginning of the phase of rapid cell division (6-day-old culture). These three enzymes reach their maxima at this stage of growth in the other series as well. Ribonuclease activity increased in the stationary phase cells (13 and 19 days) with NAA but with the other treatments the level of this activity was relatively stable. Peroxidase and phosphatase did not show any characteristic maxima, glutamate: pyruvate transaminase activity was low and no amylase activity could be demonstrated. The results indicate that synthetic plant growth substances can regulate the physiological state of a tissue by causing alterations in enzyme activities, but their action is clearly specific.     

Derepression and partial insensitivity to carbon catabolite repression of the methanol dissimilating enzymes inHansenula polymorpha

Summary The regulation of the synthesis of alcohol oxidase, catalase, formaldehyde dehydrogenase, and formate dehydrogenase was investigated in the methanol-utilizing yeastHansenula polymorpha during growth on different carbon and energy sources. When cells were grown on glucose, the enzymes of the dissimilatory methanol metabolism were not detected during the exponential phase of growth, but were formed in the late stationary phase without addition of methanol. Moreover, the enzymes were synthesized during growth on sorbitol, glycerol, ribose, and xylose. It was shown that the carbon catabolite insensitivity of the synthesis of methanol-specific enzymes is not limited to substrates that are slowly metabolized.     

Activity changes of three nucleolytic enzymes during the life cycle of Saccharomyces cerevisiae

Conditions were established for the assay of three nucleolytic enzymes: a Mg2+-independent endoribonuclease, a Mg2+-dependent endonuclease, and a Mg2+-dependent 5'-exonuclease in Saccharomyces cerevisiae cell extracts. The changes in the activities of these enzymes were determined throughout the life cycle of the organism. As the cells progressed from the exponential to the stationary growth phase, the specific activities of the Mg2+-independent endoribonuclease and of the Mg2+-dependent 5'-exonuclease increased, whereas the Mg2+-dependent endonuclease decreased. During sporulation the Mg2+-independent endoribonuclease and the Mg2+-dependent 5'-exonuclease increased several-fold over the first 10 h, but, since a similar increase was seen in nonsporulating control cells, the increases did not appear to be related to sporulation. However, the specific activity of the Mg2+-dependent endonuclease showed a sporulation-related increase during the first 3 h of sporulation, with a subsequent decline to very low levels. The specific activity of this enzyme increased again during germination to the levels seen in exponential phase cells. The Mg2+-independent endoribonuclease and the Mg2+-dependent 5'-exonuclease showed little change during germination of the ascospores. The high specific activity of the Mg2+-independent endoribonuclease during periods of nutrient deprivation is in agreement with the proposed role for this enzyme in the degradation of rRNA under these conditions.     

Glyoxalase-I activity and cell cycle regulation in yeast

The status of glyoxalase-I was explored in exponentially growing and G₁ arrested temperature sensitive (ts) cell division cycle (cdc) mutants of Saccharomyces cerevisiae. It was observed that the specific activity of this enzyme was correlated with overall growth status. The activity was high in actively growing cells and was low in G₁ arrested cells. Specific activities of glyoxalase-I were also low in G₁ arrested prolonged stationary phase (PSP) cells of S. cerevisiae and Candida albicans. The activity of glyoxalase-I recovered when G₁ arrested S. cerevisiae (ts) cells were allowed to regrow under permissive conditions. Results demonstrate that although glyoxalase-I activity is a good indicator of cell growth status, it is not involved in cell cycle regulation of this eukaryotic organism.     

Formation of polysaccharide depolymerase and glycoside hydrolase enzymes byBacteroides ruminicola subsp.ruminicola grown in batch and continuous culture

The activity of ten polysaccharide depolymerase and glycoside hydrolase enzymes was monitored inBacteroides ruminicola subsp.ruminicola throughout the growth cycle and over a range of dilution rates in carbon-limited continuous (chemostat) culture. The enzymes were principally cell associated, and the specific activities increased throughout the growth cycle to reach maximum levels in the late exponential and stationary growth phases. In chemostat-grown cells the activities were growth rate dependent and were highest at the lowest dilution rates examined (0.025/h), but remained constant over a wide range of growth rates (D=0.05–0.15/h). The specific activities were lower in cells with a generation time of 3 h (D=0.225/h). The major metabolites formed from xylose, in batch and continuous culture, were lactic, acetic, and succinic acids, with traces (1%–2% of total acid production) of branched and straight-chain C₃–C₅ volatile fatty acids. The proportions of the metabolites produced varied with the stage and rate of growth.     

Regulation ofEscherichia coli catalases by anaerobiosis and catabolite repression

Escherichia coli has two forms of catalases, HPI and HPII. Both enzymes, but mainly HPII, are induced in cells reaching the stationary growth phase or under anaerobic conditions and are repressed in the presence of glucose. The induction at the stationary phase is dependent onfnr, a gene that regulates the expression of anaerobically induced proteins. The inhibition by glucose is not affected by cyclic AMP (cAMP) but is reduced in acrp mutant. The results show that HPII belongs to the group of genes controlled by the Fnr protein and is catabolically repressed in a manner that is independent of cAMP.