Energy-limited Conditions Research Articles

Two classes of plant-like vacuolar-type H +-pyrophosphatases in malaria parasites

In plants, cytosolic inorganic pyrophosphate (PP i) is hydrolyzed by energy-conserving vacuolar-type H +-pyrophosphatases (V-PPases) that harness the free energy of PP i hydrolysis to establish transmembrane H + gradients. Here we describe the identification and cloning of two genes, PfVP1 and PfVP2, from the malaria parasite Plasmodium falciparum. Inferred to encode type I (K +-dependent) and type II (K +-independent) V-PPases, respectively, PfVP1 and PfVP2 appeared more sequence divergent from each other than from their type I and type II counterparts in plants. The steady state levels of PfVP1 mRNA were high in comparison to PfVP2 mRNA throughout the erythrocytic phases of infection. Western analyses of trophozoite membranes using generic V-PPase antibodies (PAB HK and PAB TK) demonstrated appreciable amounts of a Mr 67 000 polypeptide whose associated aminomethylenediphosphonate- (AMDP) inhibitable PPase activity was markedly stimulated by K +. Immunofluorescence microscopy of infected erythrocytes revealed PfVP antigen associated with both the parasite plasma membrane and punctate intracellular inclusions. Transient transfection of a PfVP1– GFP fusion further supported the localization of PfVP1 to the parasite plasma membrane. Based on these findings and the growth-retarding effects of AMDP, P. falciparum is concluded to possess both type I and type II V-PPases of which the former has the greatest potential for contributing to the establishment of H + gradients across the parasite plasma membrane under conditions of energy limitation.

Hierarchies of ATP-consuming processes: direct compared with indirect measurements, and comparative aspects

The original aim of the present study was to deal with two problems that had emerged from a study on hierarchies of ATP-consuming processes in cells [Buttgereit and Brand (1995) Biochem. J. 312, 163-167]. Firstly, we wanted to find out whether the results of that study had been influenced by the method used for the determination of process activity and, secondly, we wondered whether and to what extent the structure of the hierarchy established for cell suspensions under energy-limiting conditions might depend on the type of cell or on the lifestyle, ecology and phylogenetic status of the species from which the cells were derived. We confined our study to the two most prominent ATP consumers of cells: protein synthesis and the Na+/K+-ATPase, measuring their activity directly by [3H]leucine incorporation and Rb+-flux respectively. We found large differences in the sensitivity of protein synthesis to energy limitation between hepatocytes from an anoxia-tolerant fish species and an anoxia-sensitive fish species (goldfish and rainbow trout respectively). On the other hand, Na+/K+-ATPase activity was hardly affected by energy limitation in the hepatocytes from both fish species. We also studied the response of a human hepatoma cell line, HepG2, to energy limitation and found both protein synthesis and Na+/K+-ATPase activity to be equally sensitive to energy limitation, but more sensitive than the Na+/K+-ATPase of the two fish species. A comparison of the indirect and direct methods for measuring protein synthesis revealed the rate of oxygen consumption to be functionally related to the concentration of cycloheximide, the inhibitor used. It was found that at 15mM cycloheximide [three orders of magnitude higher than the concentration at which the incorporation of free amino acids (FAA) into protein is inhibited] total oxygen consumption was suppressed by 71-75%, whereas the measured rate of [3H]leucine incorporation into protein suggested that the cycloheximide-sensitive fraction should have amounted to not more than approx. 10% of the total oxygen consumption. On the other hand, the amount of oxygen consumption suppressed with the high concentration of cycloheximide corresponded almost exactly to the increase in oxygen consumption of cells incubated in an FAA-enriched medium compared with cells incubated in a standard, FAA-free medium. Our major conclusions are, firstly, that high concentrations of cycloheximide disrupt cellular metabolism, bringing to a standstill all those processes that can be stimulated by incubating starved cells in an FAA-enriched medium, secondly, that the attempt to estimate the metabolic cost of protein synthesis by inhibiting oxygen consumption with cycloheximide leads to spurious results, and, thirdly, that the structure of a ‘hierarchy’ of ATP-consumers may reflect the lifestyle and physiology of the species studied.

Cooperative organization of bacterial colonies: from genotype to morphotype.

In nature, bacteria must often cope with difficult environmental conditions. To do so they have developed sophisticated cooperative behavior and intricate communication pathways. Utilizing these elements, motile microbial colonies frequently develop complex patterns in response to adverse growth conditions on hard surfaces under conditions of energy limitation. We employ the term morphotype to refer to specific properties of colonial development. The morphologies we discuss include a tip-splitting (T) morphotype, chiral (C) morphotype, and vortex (V) morphotype. A generic modeling approach was developed by combining a detailed study of the cellular behavior and dynamics during colonial development and invoking concepts derived from the study of pattern formation in nonliving systems. Analysis of patterning behavior of the models suggests bacterial processes whereby communication leads to self-organization by using cooperative cellular interactions. New features emerging from the model include various models of cell-cell signaling, such as long-range chemorepulsion, short-range chemoattraction, and, in the case of the V morphotype, rotational chemotaxis. In this regard, pattern formation in microorganisms can be viewed as the result of the exchange of information between the micro-level (the individual cells) and the macro-level (the colony).

Anti-fungal properties of chitinolytic dune soil bacteria

Anti-fungal properties of chitinolytic soil bacteria may enable them to compete successfully for chitin with fungi. Additionally, the production of chitinase may be part of a lytic system that enables the bacteria to use living hyphae rather than chitin as the actual growth substrate, since chitin is an important constituent of most fungal cell walls. Lysis of living fungal hyphae by chitinolytic bacteria has been reported frequently; however, these reports nearly always deal with bacteria that had been selected because of their mycolytic properties. Our main objective was to get a better understanding of the relationship between chitinolytic and anti-fungal properties of bacteria that occur naturally in soils, i.e. without artificial selection. Three inner dune sites, two of which were lime-poor and one lime-rich, along the Dutch coast were selected for this study. Bacteria that were able to degrade colloidal chitin in water–agar comprised 0.2–5.7% of the total amount of culturable bacteria of these dune sites. Pseudomonas spp. were the most abundant culturable, chitin-degrading bacteria at the lime-poor sites, whereas Xanthomonas spp. and Cytophaga spp. were important at the lime-rich site. Chitinolytic actinomycetes were relatively abundant at all three sites. Chitinolytic and non-chitinolytic bacteria were randomly selected and tested for the possession of antagonistic activities against fungal dune strains [ Chaetomium globosum, Fusarium culmorum, F. oxysporum, Idriella (Microdochium) bolleyi, Mucor hiemalis, Phoma exigua, Ulocladium sp.]. The tests were done using water–agar to simulate the energy-limiting conditions that bacteria will encounter in dune soils. The percentage of bacterial isolates that were antagonistic against these fungi was considerably higher for chitinolytic strains than for non-chitinolytic ones. Therefore, the possible involvement of chitinase with respect to the inhibition of fungal growth was studied in more detail. It appeared that in many cases the inhibition of fungal growth was not accompanied by bacterial chitinase production. There was also no clear relationship between the activity of other cell wall degrading enzymes ( β-1,3-glucanase and protease) and antagonism. Chitinolytic bacteria had selective rather than general anti-fungal properties, which were not necessarily related to differences in general susceptibility of the fungi towards antagonism. These results may indicate that antibiotics were involved in the antagonistic activities of chitinolytic bacteria against fungi. Only growing fungi were antagonized by the chitinolytic bacteria; none of the chitinolytic bacteria were able to lyse existing mycelium of any of the fungi. The relevance of the results for the ecology of chitinolytic soil bacteria is discussed.

Induction of ammonia monooxygenase and hydroxylamine oxidoreductase mRNAs by ammonium in Nitrosomonas europaea.

In Nitrosomonas europaea, ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO) catalyse the oxidation of ammonia (NH3) to nitrite (NO2-). A transcript of 3500 bases hybridizes to probes for amoA and amoB (genes that code for AMO proteins). A transcript of 2100 bases hybridizes to probes for hao (the gene that codes for HAO). Induction of the mRNAs detected by amo and hao probes required the presence of ammonium (NH4+). To correlate new levels of mRNA with de novo activity, existent mRNA pools and AMO activity were depleted prior to induction by NH4+. The mRNAs of AMO and HAO were depleted by depriving the cells of energy for at least 8 h; AMO activity was inactivated with acetylene (C2H2) after mRNA depletion. In cells treated this way, levels of new AMO mRNA and de novo AMO enzyme activity were correlated with increased NH4+ concentrations up to 1 mM after 3 h of incubation. HAO mRNA also increased in the NH4(+)-treated cells. Other proteins and RNAs induced by NH4+ were detected in 14CO2-labelling experiments. The AMO and HAO mRNAs were preferentially synthesized during energy-limiting conditions.

Time and energy constraints and the evolution of sexual size dimorphism ? to eat or to mate?

We present an empirical test of the ‘Ghiselin—Reiss small-male hypothesis’ for the evolution of sexual size dimorphism (SSD). In mating systems dominated by scramble competition, where male reproductive success is a function of encounter rate with females, small males may be favoured when food is limiting because they require lower absolute amounts of food. Given a trade-off between time and energy devoted to foraging and to mate acquisition, small males should be able to devote more time to the latter. If at the same time larger females are favoured, this mechanism will contribute to the evolution of SSD and may be the major determinant of the female-biased SSDs that characterize most animal taxa. We tested this hypothesis using the water strider,Aquarius remigis (Heteroptera: Gerridae), a scramble competitor which mates many times over a prolonged mating season and which shows female-biased SSD. Laboratory experiments demonstrated that foraging success and giving up times (GUTs) are lower for males than for females during the reproductive season and that male water striders flexibly alter their time budgets under conditions of energy limitation. Controlled feeding experiments showed that male and female longevity, female fecundity and male mating success are positively related to food availability. As predicted, male body size is negatively correlated with several indices of male fitness (longevity, number of mating attempts and mating success), while female body size is positively correlated with longevity. These results are consistent with the hypothesis that scramble competition for mates favours small males in this species and provides empirical support for the Ghiselin—Reiss small-male hypothesis.

Arabinogalactan Utilization in Continuous Cultures of Bifidobacterium longum: Effect of Co-culture with Bacteroides thetaiotaomicron

Studies showed that the plant cell wall polysaccharide arabinogalactan supported growth of Bifidobacterium longum in batch culture. Galactose was also utilized, but not arabinose, the other major constituent sugar of the polymer. Enzymes required for hydrolysis of arabinogalactan ('arabinogalactanase', α-arabinopyranosidase, β-galactosidase) were inducible and cell-associated in B. longum, and their expression was repressed by glucose. Considerable amounts of α-arabinopyranosidase and β-galactosidase were synthesized during growth on arabinogalactan, but only low levels of arabinogalactanase were detected. B. longum only grew on arabinogalactan in continuous culture under putative carbon-excess conditions. In C-limited chemostats, the bifidobacterium could not establish unless Bacteroides thetaiotaomicron was present in co-culture. The relationship between the two organisms was not simply commensal; at low specific growth rates, bacteroides cell population densities were approximately 30% lower than those recorded in axenic culture, indicating the existence of competitive interactions with the bifidobacterium. In contrast, at high specific growth rates, a mutualistic association was observed, in that Bact. thetaiotaomicron was maintained in the chemostats at high dilution rates if bifidobacteria were also present. Measurements of residual carbohydrate in spent culture fluid from C-limited chemostats indicated that a large part of the arabinogalactan molecule could not be broken down by either B. longum or Bact. thetaiotaomicron alone, or in co-culture. Formate and acetate were the major fermentation products of B. longum cultured in the presence of high concentrations of arabinogalactan, confirming that these bacteria were growing under energy-limited conditions.

Metabolic uncoupling in Saccharomyces cerevisiae

Aerobic chemostat cultures of S. cerevisiae were performed at different diln. rates under energy (glucose) limitation or at various extents of energy excess imposed by a N limitation. Energy excess induced metabolic uncoupling under steady-state conditions. The sp. EtOH prodn. rate was always higher the lower the feed medium N concn. throughout the whole range of diln. rates tested. In addn., the respiratory rate also increased under N limitation, for at least as long as the sp. O2 consumption rate was below the max. capacity. These results imply that the ATP yield (YATP in g biomass per mol ATP) and/or the amt. of ATP produced, i.e. the P/O ratio, must be able to change. By assuming a const. YATP of 16 g/mol, a decrease in the P/O ratio from 1 to 1.5 at low diln. rates and glucose limitation down to only 0.1-0.2 at high diln. rates and N limitation could be calcd. If, instead, a fixed P/O ratio of 1.0 was assumed, the YATP decreased from .apprx.20 down to <10 g/mol during these different conditions. The heat yield values, i.e., the amt. of heat produced per amt. of biomass formed, increased dramatically when the cells were subjected to a N limitation, indicating less efficient growth in terms of conserving substrate energy as biomass under energy excess compared to energy-limiting conditions. [on SciFinder (R)]

Role of an Escherichia coli stress-response operon in stationary-phase survival.

The phage shock protein operon (pspABCE) of Escherichia coli is strongly expressed in response to stressful environmental conditions, such as heat shock, ethanol treatment, osmotic shock, and filamentous phage infection. We show that bacteria lacking the pspABC genes exhibit a substantial decrease in the ability to survive prolonged incubation in stationary phase under alkaline conditions (pH 9). The psp mutant bacteria grow approximately as well as wild-type strains in the alkaline medium, and stationary-phase survival of the psp mutants improves substantially at pH values closer to the optimal growth range (pH 6-8). In late stationary-phase (1- to 2-day-old) cells, the operon can be strongly induced under certain conditions, and PspA can become one of the most highly expressed bacterial proteins. The combination of stationary-phase starvation and alkaline pH is likely to place a severe strain on the maintenance of endogenous energy sources, and, consistent with these effects, we find that psp expression is also induced by uncouplers of oxidative phosphorylation and other agents that interfere with energy production. The death rate of psp mutants in stationary phase is accelerated by the presence of wild-type bacteria in the same culture, suggesting that the psp operon may play a significant role in enabling E. coli to compete for survival under nutrient- or energy-limited conditions.

Coupling of Energy Supply and Energy Demand in Isolated Goldfish Hepatocytes

The allocation of metabolic energy under fluctuating conditions of supply and demand was studied in isolated goldfish hepatocytes. Inhibition of glycolytic ATP production resulted in a decrease in the activity of one of the major ATP consuming components of cellular energy budgets, the sodium pump ((Na⁺, K⁺)-ATPase), because of the removal of substrates for the trichloroacetic acid (TCA) cycle. This was confirmed through restoration of activity to control levels by adding pyruvate and malate to the suspension. Ouabain-sensitive oxygen consumption constituted a fairly constant fraction of 15% to 25% of total oxygen consumption, whereas the relative load exerted by the pump on anaerobic energy metabolism (on the basis of measurements of Rb⁺ flux and lactate production) was much higher, ranging from 90% to 50% during the experimental period. Moreover, when pump activity was inhibited by the addition of ouabain, oxidative energy output decreased immediately, whereas under the restricted conditions of glycolytic energy supply a corresponding ouabain-sensitive component of lactate production was not observed. Such an apparent mismatch between supply and demand ofATP is interpreted as reflecting the flexibility of energy allocation under energy-limiting conditions. When ATP production was reduced by inhibiting either glycolysis or oxidative phosphorylation, the concentration of ATP in the hepatocytes dropped within 30 min from the control steady state to a new, lower, steady state that was maintained for at least 60 min.

Growth-phase-dependent induction of 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase in the cyanobacterium Synechoccus sp. PCC7942

In most cyanobacteria, the only known pathway for oxidation of stored carbohydrate in the dark or under energy-limiting conditions is the hexose monophosphate shunt. To determine whether the increased use of the shunt under these conditions derives from an increase in the activity level of the respective enzymes, we measured the effect of growth phase during the growth of batch cultures of Synechococcus sp. strain PCC7942 on the specific activity of 6-phosphogluconate dehydrogenase (6PGD) and glucose 6-phosphate dehydrogenase. The specific activities were constant during the exponential growth phase of the culture, but they increased about fivefold during the transition into stationary phase. As an approach to determining the level of expression at which the growth-phase-dependent regulation of 6PGD level is exerted, we constructed operon and gene fusions between the gnd gene, which encodes 6PGD, and the Escherichia coli lacZ gene, which encodes β-galactosidase (βGal). Strains harboring the fusions integrated into the cyanobacterial chromosome were prepared, and the growth-phase dependence of βGal level was determined. The specific activity of βGal in cultures of both types of fusion strains increased during the transition into stationary phase, indicating that the growth-phase-dependent regulation is on the gnd mRNA level. Characterization of the growth-phase-dependent induction of 6PGD in strains carrying differing amounts of DNA upstream from the gnd structural gene led to the localization of the promoter and the regulatory site on the restriction map of the gene, whose sequence has previously been determined. The operon and protein fusion vectors prepared for this work may be of general use in the study of cyanobacterial gene expression.

Regulation of protease production in Clostridium sporogenes

The physiological and nutritional factors that regulate protease synthesis in Clostridium sporogenes C25 were studied in batch and continuous cultures. Formation of extracellular proteases occurred at the end of active growth and during the stationary phase in batch cultures. Protease production was inversely related to growth rate in glucose-excess and glucose-limited chemostats over the range D = 0.05 to 0.70 h-1. In pulse experiments, glucose, ammonia, phosphate, and some amino acids (tryptophan, proline, tyrosine, and isoleucine) strongly repressed protease synthesis. This repression was not relieved by addition of 4 mM cyclic AMP, cyclic GMP, or dibutyryl cyclic AMP. Protease formation was markedly inhibited by 4 mM ATP and ADP, but GTP and GDP had little effect on the process. It is concluded that protease production by C. sporogenes is strongly influenced by the amount of energy available to the cells, with the highest levels of protease synthesis occurring under energy-limiting conditions.

Regulation of methanol metabolism in the facultative methylotroph Nocardia sp. 239 during growth on mixed substrates in batch- and continuous cultures

The regulation of methanol metabolism in Nocardia sp. 239 was investigated. Growth on mixtures of glucose or acetate plus methanol in batch cultures resulted in simultaneous utilization of the substrates. The presence of glucose, but not of acetate, repressed synthesis of the ribulose monophosphate (RuMP) cycle enzymes hexulose-6-phosphate synthase (HPS) and hexulose-6-phosphate isomerase (HPI), and methanol was used as an energy source only. Comparable results were obtained following addition of formaldehyde (fed-batch system) to a culture growing on glucose. The synthesis of the methanol dissimilatory and assimilatory enzymes in Nocardia sp. 239 thus appears to be controlled differently. Methanol and/or formaldehyde induce the synthesis of these enzymes, but under carbon-excess conditions their inducing effect on HPS and HPI synthesis is completely overruled by glucose, or metabolites derived from it. Repression of the synthesis of these RuMP cycle enzymes was of minor importance under carbon- and energy-limiting conditions in chemostat cultures. Addition of a pulse of glucose to a formaldehyde-limited (2.5 mmol l−1 h−1) fed-batch culture resulted in a decrease in the levels of several enzymes of methanol metabolism (including HPI), whereas the HPS levels remained relatively constant. Increasing HPS/HPI activity ratios were also observed with increasing growth rates in formaldehyde-limited chemostat cultures. The data indicate that additional mechanisms, the identity of which remains to be elucidated, are involved in controlling the levels of these C1-specific enzymes in Nocardia sp. 239.

Efficiency of bacterial protein synthesis during anaerobic degradation of cattle waste

The rate of [15N]ammonia (15NH3) uptake or incorporation into bacterial cells was studied, using stirred, 3-liter benchtop digestors fed on a semicontinuous basis with cattle waste. The fermentations were carried out at 40 and 60 degrees C and at four different loading rates (3, 6, 9, and 12 g of volatile solids per liter of reactor volume per day). The rate of NH3-N incorporation for the period 1 to 5 h after feeding at the four different loading rates was 0.49, 0.83, 1.05, and 1.08 mg/liter per h in the mesophilic digestor and 0.68, 1.07, 1.17, and 1.21 mg/liter per h in the thermophilic digestor. Values were lower 7 to 21 h after feeding in both digestors and were related to the rate of fermentation or CH4 production. In the mesophilic digestors, the rate of bacterial cell production ranged from 3.97 to 8.72 mg of dry cells per liter per h, 1 to 5 h after feeding at the different loading rates. Corresponding values for the thermophilic digestors ranged from 5.46 to 9.77 mg of dry cells per liter per h. Cell yield values ranged from 2.3 to 3.1 mg of dry cells per mol of CH4 produced in the mesophilic and thermophilic digestors at the two lower loading rates. The values were higher (2.8 to 3.4) in the mesophilic digestors at the two higher loading rates because of the accumulation of propionate and a consequent reduction in CH4 production. Low cell yields such as those measured in this study are characteristic of low-specific-growth rates under energy-limited conditions.

Energy linked modifications of the radiation response in a human cerebral glioma cell line

Effects of cellular energy metabolism on the radiation response of a cell line derived from a human cerebral glioma have been studied under conditions of energy limitation produced by the presence of inhibitors of respiratory metabolism (KCN) and glycolysis (glucose analogues such as 2-DG, 5-TG, and 3-0-MG). Radiation 60Co induced DNA repair (Unscheduled DNA Synthesis) and micronuclei formation were studied as measures of radiation response. Glycolysis (lactate production) and levels of adenine and related nucleotides (UTP, GTP, ATP etc.) were measured as parameters of energy metabolism. Two 2-DG (5 mM) inhibited DNA repair and increased micronuclei frequency both in the presence and absence of respiration (KCN, 2 mM). Under similar experimental conditions, the presence of 2-DG also significantly reduced the cellular energy status. Five-TG and 3-0-MG on the other hand, neither significantly altered the energy status (Σ XTP) nor influenced the radiation response under respiratory proficient conditions. The results can be explained on the basis of a model postulating differential energy linked modulations of the repair and fixation processes acting on DNA lesions. Implications of the present results for the radiotherapy of brain tumors are discussed.

Mixed substrate‐metabolism in an obligate methanol utilizer: Chemostat studies

In Pseudomonas C, an obligate RMP-methylotroph, externally added formic acid and multi-carbon intermediates are co-metabolized in the obligate presence of methanol. Cell yield and methanol-carbon conversion to cell mass are optimal in a chemostat culture growing on a mixture of energy sources and methanol as the sole limiting carbon source. Growth on methanol as the sole, obligate energy source and an additional carbon source (e.g. pyruvate) is less efficient. Under these energy limited conditions, the cyclic oxidation pathway is predominantly operated.

Simultaneous Nitrification and Denitrification in Aerobic Chemostat Cultures of Thiosphaera pantotropha

Thiosphaera pantotropha is capable of simultaneous heterotrophic nitrification and aerobic denitrification. Consequently, its nitrification potential could not be judged from nitrite accumulation, but was estimated from complete nitrogen balances. The maximum rate of nitrification obtained during these experiments was 93.9 nmol min mg of protein. The nitrification rate could be reduced by the provision of nitrate, nitrite, or thiosulfate to the culture medium. Both nitrification and denitrification increased as the dissolved oxygen concentration fell, until a critical level was reached at approximately 25% of air saturation. At this point, the rate of (aerobic) denitrification was equivalent to the anaerobic rate. At this dissolved oxygen concentration, the combined nitrification and denitrification was such that cultures receiving ammonium as their sole source of nitrogen appeared to become oxygen limited and the nitrification rate fell. It appeared that, under carbon-and energy-limited conditions, a high nitrification rate was correlated with a reduced biomass yield. To facilitate experimental design, a working hypothesis for the mechanism behind nitrification and denitrification by T. pantotropha was formulated. This involved the basic assumption that this species has a "bottleneck" in its cytochrome chain to oxygen and that denitrification and nitrification are used to overcome this. The nitrification potential of other heterotrophic nitrifiers has been reconsidered. Several species considered to be "poor" nitrifiers also simultaneously nitrify and denitrify, thus giving a falsely low nitrification potential.

Effects of Glucose Concentrations on Cadmium, Copper, Mercury, and Zinc Toxicity to a Klebsiella sp

The influence of glucose concentration on Cd, Cu, Hg, and Zn toxicity to a Klebsiella sp. was studied by following the degradation of C-labeled glucose at pH 6.0. Uptake of C into the cells was also determined. The carbon concentrations ranged from 0.01 to 40 mg liter, which are equivalent to soluble C concentrations in natural environments. The toxicity of Cu, Cd, and Zn to a Klebsiella sp. was affected considerably by the C concentration. Copper at 10 M was toxic when the carbon concentration was 10 or 40 mg liter, while at 0.01 to 1.0 mg liter no toxicity was observed. Cadmium and zinc were toxic at 10 M in media containing 0.01 to 1.0 mg of C liter. At C concentrations greater than 1.0 mg liter, the inhibition of glucose degradation and carbon assimilation was observed at 10 M Cd and Zn. The toxicity of mercury seemed to be independent of the C concentration. Results of this study showed that the nutritional state of an organism may have a profound effect on its sensitivity to metals. Metals taken up by an energy-driven transport system may be less toxic under conditions of C starvation. The C concentration should be taken into account when evaluating results from toxicity studies, especially as most microorganisms in nature live under energy-limited conditions.

Contrasting effects of sulfide and thiosulfate on symbiotic CO2-assimilation of Phallodrilus leukodermatus (Annelida)

Experiments were conducted in order to specify the reductants responsible for the carbon dioxide fixation of the symbiotic sulfur bacteria in the gutless marine oligochaete Phallodrilus leukodermatus (Annelida) from shallow calcareous sediments in Bermuda. Carbon dioxide-uptake rates were suppressed by S= and stimulated by S2O3 =. Individuals which hosted bacteria containing reserve energy substances maintained a high short-term CO2-uptake activity, while bacteria in worm homogenates and in worms treated with an antibiotic (Baypen) did not show any significant metabolic activity. Absolute uptake rates in P. leukodermatus were usually considerably higher than those reported for other animals harbouring prokaryotic sulfuroxidizing symbionts. Utilization of thiosulfate rather than sulfide is compatible with the preferred occurrence of the worms around the redox discontinuity layer and has been confirmed in other “thiobiotic” animals. Sulfur stored in the symbiotic bacteria appears to be oxidized to sulfate and be excreted when the worms are held under energy-limited conditions. The data emphasize the complexity of the possible metabolic pathways involved in the oxidation of reduced-sulfur compounds by bacterial symbionts in marine invertebrates.

Pantothenate limitation does not induce uncoupled growth of Zymomonas in chemostat culture

Growth coefficients ofZymomonas mobilis were compared in glucose-limited chemostat culture using a complex medium and a defined minimal growth medium at non inhibitory concentrations of ethanol. Under carbon and energy limited conditions in the complex medium containing yeast extract, the max. molar growth yield (YGmax) and maintenance energy coefficient (me) were 10.8 g cell/mol glucose and 8.3 mmol glu/g cell/hr, respectively. Glucose-limited growth in the minimal medium with NH4Cl as nitrogen source promoted slight energetic uncoupling, as reflected in the decrease in the maximum growth yield. The growth yield with respect to calcium pantothenate was calculated to be 1.4×104 g cell/g Ca-pantothenate. However, pantothenate-limited growth did not result in a decrease in growth yield nor an increase in the specific rate of glucose catabolism. Steady-state growth measurements failed to confirm the previously held view of Belaichet al. (1972) that pantothenate deficiency induces energetic uncoupling inZymomonas.