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Enzyme reprofiling in bacteria during adaptation from one environmental condition to another may be regulated by both transcription and translation. However, little is known about the contribution of translational regulation. Recently, we have developed a pulse labeling method using the methionine analog azidohomoalanine to determine the relative amounts of proteins synthesized by Escherichia coli in a brief time frame upon a change in environmental conditions. Here we present an extension of our analytical strategy, which entails measuring changes in total protein levels on the same time scale as new protein synthesis. This allows identification of stable and labile proteins and demonstrates that altered levels of most newly synthesized proteins are the result of a change in translation rate rather than degradation rate. With this extended strategy, average relative translation rates for 10 min immediately after a switch from aerobiosis to anaerobiosis were determined. The majority of proteins with increased synthesis rates upon an anaerobic switch are involved in glycolysis and pathways aimed at preventing glycolysis grinding to a halt by a cellular redox imbalance. Our method can be used to compare relative translation rates with relative mRNA levels at the same time. Discrepancies between these parameters may reveal genes whose expression is regulated by translation rather than by transcription. This may help unravel molecular mechanism underlying changes in translation rates, e.g. mediated by small regulatory RNAs.

Enzyme reprofiling in bacteria during adaptation from one environmental condition to another may be regulated by both transcription and translation. However, little is known about the contribution of translational regulation. Recently, we have developed a pulse labeling method using the methionine analog azidohomoalanine to determine the relative amounts of proteins synthesized by Escherichia coliin a brief time frame upon a change in environmental conditions. Here we present an extension of our analytical strategy, which entails measuring changes in total protein levels on the same time scale as new protein synthesis. This allows identification of stable and labile proteins and demonstrates that altered levels of most newly synthesized proteins are the result of a change in translation rate rather than degradation rate. With this extended strategy, average relative translation rates for 10 min immediately after a switch from aerobiosis to anaerobiosis were determined. The majority of proteins with increased synthesis rates upon an anaerobic switch are involved in glycolysis and pathways aimed at preventing glycolysis grinding to a halt by a cellular redox imbalance. Our method can be used to compare relative translation rates with relative mRNA levels at the same time. Discrepancies between these parameters may reveal genes whose expression is regulated by translation rather than by transcription. This may help unravel molecular mechanism underlying changes in translation rates, e.g.mediated by small regulatory RNAs.

Two-component regulation systems (TCRSs) are the dominant type of signal transduction system in prokaryotes that are used to inform the cellular trancriptional machinery (and additional targets for regulation, like the motility apparatus) about actual changes in the extracellular physico-chemical conditions. We now review their molecular structure and enzymatic characteristics, their mutual interactions and its implications, and their role in cellular physiology. Specific emphasis is placed on the ArcB/A system, a representative of the phosphorelay type of TCRS, and a key player in the adjustment of the cellular make-up of enterobacteria in response to alterations in the oxygen availability. Also some applied aspects of the TCRSs are discussed, i.e. their role as a target to develop new anti-bacterials and their application in biotechnology (or: ‘synthetic biology’).

14-3-3 proteins form a family of highly conserved eukaryotic proteins involved in a wide variety of cellular processes, including signalling, apoptosis, cell-cycle control and transcriptional regulation. More than 150 binding partners have been found for these proteins. The yeast Saccharomyces cerevisiae has two genes encoding 14-3-3 proteins, BMH1 and BMH2. A bmh1 bmh2 double mutant is unviable in most laboratory strains. Previously, we constructed a temperature-sensitive bmh2 mutant and showed that mutations in RTG3 and SIN4, both encoding transcriptional regulators, can suppress the temperature-sensitive phenotype of this mutant, suggesting an inhibitory role of the 14-3-3 proteins in Rtg3-dependent transcription [van Heusden and Steensma (2001) Yeast 18, 1479–1491]. In the present paper, we report a genome-wide transcription analysis of a temperature-sensitive bmh2 mutant. Steady-state mRNA levels of 60 open reading frames were increased more than 2.0-fold in the bmh2 mutant, whereas those of 78 open reading frames were decreased more than 2.0-fold. In agreement with our genetic experiments, six genes known to be regulated by Rtg3 showed elevated mRNA levels in the mutant. In addition, several genes with other cellular functions, including those involved in gluconeogenesis, ergosterol biosynthesis and stress response, had altered mRNA levels in the mutant. Our data show that the yeast 14-3-3 proteins negatively regulate Rtg3-dependent transcription, stimulate the transcription of genes involved in ergosterol metabolism and in stress response and are involved in transcription regulation of multiple other genes.

A study has been initiated to integrate molecular and physiological responses of Saccharomyces cerevisiaeto heat stress conditions. We focus our research on a quantification of the energetics of the stress response. A series of continuous heat stresses was applied to exponentially growing cells of the strain X2180-1A at 28°C, by increasing the growth temperature to 37, 39, 40, 41, 42, or 43°C. Here, the results on cell growth and viability, as well as on anabolic and catabolic rates are presented. We observed a surprisingly ‘thin line’ for the cells between growing, surviving, and dying, with regard to growth temperature. The heat stress showed a dual effect on catabolism: immediately after the temperature increase a strong peak was seen, after which a new, steady level was reached. In addition, the yield on glucose decreased with increasing temperature. Our results indicate that life at elevated temperatures is energetically unfavourable and a non-lethal heat stress invokes a redistribution of catabolic and anabolic fluxes.

An integrated approach is used to develop a rapid sampling strategy for the quantitative analysis of in vivo kinetic behavior based on measured concentrations of intracellular metabolites in Saccharomyces cerevisiae. Emphasis is laid on small sample sizes during sampling and analysis. Subsecond residence times are accomplished by minimizing the dead volume of the sterile sampling system and by maximizing flow rates through application of vacuum to the sampling tubes in addition to the overpressure in the fermenter. A specially designed sample tube adapter facilitates sampling intervals of 4 to 5 s for various test tube types. Statistical analysis of the results obtained from enzymatic and liquid chromatography mass spectrometry (LC-MSMS) analysis of the metabolite concentrations was used to optimize the sampling protocol. The most notable improvement is reached through the introduction of vacuum drying of the cell extract. The presented system is capable of reliably dealing with fermenter samples as small as 1-g with a variation of less than 3%, and is thus ideally suited for intracellular measurements on small, lab-scale fermenters. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 406–415, 2001.

Manufacturers of polytetraflouroethylene (PTFE) grafts used for chronic hemodialysis access describe specific advantages for their respective grafts, which presumably result in greater graft patency rates, reduced complications and decreased overall costs. There are few data available in the literature to support or contradict these alleged benefits. Therefore, this prospective study was undertaken to evaluate and compare patency rates, complications and costs between two of the leading brands of PTFE that are currently being marketed for use as hemodialysis access grafts. Totals of 190 primary PTFE grafts (100 Gore-tex (W. L. Gore and Associates, Flagstaff, AZ) and 90 Impra (C. R. Bard Inc., Tempe, AZ)) were implanted in 168 consecutive patients with end-stage renal disease. A policy of non-interventions was employed for patent grafts, as no attempt was made to assist primary patency. Grafts that occluded during follow-up underwent secondary revision to maintain patency. There was no difference in primary and secondary patency by life-table analysis between Gore-tex and Impra grafts at 2 years (P> 0.53 and P> 0.13, respectively). There was also no significant difference between Gore-tex and Impra in the number of days before the first thrombectomy or in the number of thrombectomies or revisions per graft (P> 0.50). Likewise, the incidence of complications was similar between the two grafts. The cost of graft implantation and maintenance of patency was not significantly different between Gore-tex and Impra grafts. It is concluded that either graft can be used for hemodialysis access with similar expected outcomes for at least 2 years following implantation.

The kinetics of glucose transport and the transcription of all 20 members of the HXT hexose transporter gene family were studied in relation to the steady state in situ carbon metabolism of Saccharomyces cerevisiae CEN.PK113-7D grown in chemostat cultures. Cells were cultivated at a dilution rate of 0.10 h-1 under various nutrient-limited conditions (anaerobically glucose- or nitrogen-limited or aerobically glucose-, galactose-, fructose-, ethanol-, or nitrogen-limited), or at dilution rates ranging between 0.05 and 0.38 h-1 in aerobic glucose-limited cultures. Transcription of HXT1-HXT7 was correlated with the extracellular glucose concentration in the cultures. Transcription of GAL2, encoding the galactose transporter, was only detected in galactose-limited cultures. SNF3 and RGT2, two members of the HXT family that encode glucose sensors, were transcribed at low levels. HXT8-HXT17 transcripts were detected at very low levels. A consistent relationship was observed between the expression of individual HXT genes and the glucose transport kinetics determined from zero-trans influx of 14C-glucose during 5 s. This relationship was in broad agreement with the transport kinetics of Hxt1-Hxt7 and Gal2 deduced in previous studies on single-HXT strains. At lower dilution rates the glucose transport capacity estimated from zero-trans influx experiments and the residual glucose concentration exceeded the measured in situ glucose consumption rate. At high dilution rates, however, the estimated glucose transport capacity was too low to account for the in situ glucose consumption rate.

The kinetics of glucose transport and the transcription of all 20 members of the HXThexose transporter gene family were studied in relation to the steady statein situcarbon metabolism of Saccharomyces cerevisiaeCEN.PK113-7D grown in chemostat cultures. Cells were cultivated at a dilution rate of 0.10 h−1under various nutrient-limited conditions (anaerobically glucose- or nitrogen-limited or aerobically glucose-, galactose-, fructose-, ethanol-, or nitrogen-limited), or at dilution rates ranging between 0.05 and 0.38 h−1in aerobic glucose-limited cultures. Transcription ofHXT1–HXT7was correlated with the extracellular glucose concentration in the cultures. Transcription of GAL2, encoding the galactose transporter, was only detected in galactose-limited cultures. SNF3and RGT2, two members of the HXTfamily that encode glucose sensors, were transcribed at low levels. HXT8–HXT17transcripts were detected at very low levels. A consistent relationship was observed between the expression of individual HXTgenes and the glucose transport kinetics determined from zero-transinflux of 14C-glucose during 5 s. This relationship was in broad agreement with the transport kinetics of Hxt1–Hxt7 and Gal2 deduced in previous studies on single-HXTstrains. At lower dilution rates the glucose transport capacity estimated from zero-transinflux experiments and the residual glucose concentration exceeded the measured in situglucose consumption rate. At high dilution rates, however, the estimated glucose transport capacity was too low to account for the in situglucose consumption rate.

Clostridium butyricum was grown in a glucose-limited chemostat culture at a dilution rate of 0.1 h-1 at pH 6.0. With 0.9% w/v input glucose in the medium the cells were found to grow in suspension and glucose was fermented completely to acetate and butyrate. An increase in the input concentration of glucose resulted in increased concentrations of end-products, but not all extra glucose was consumed. It could be demonstrated that this was due to a lowering of the maximal growth rate by elevated levels of butyric acid. However, prolonged growth in the presence of high glucose concentrations led to an increase in biomass. This was caused by the selection of a variant that was less sensitive to butyrate. This variant was able to form aggregates in an anaerobic gas-lift reactor at high dilution rates. Inoculation of these aggregates in a conventional chemostat culture with high glucose input resulted in an aggregated culture that remained stable for at least 6 months, and in which all glucose was consumed. Whether the organisms grew in suspension or in aggregates was found to be determined by the concentration of butyrate. The isolation of aggregate-forming variants from chemostat cultures leads to a very simple and new type of immobilization technique.

The mechanism of granule formation by Selenomonas ruminantium was investigated. A basic protein has been isolated from the lysate of S. ruminantium which triggers cluster formation (small aggregates of 20–100 cells) of suspended cells. Evidence is presented that these basic proteins were of ribosomal origin. It is suggested that ribosomes are released into the culture broth by lysis and that the associated basic proteins are subsequently dissociated by high monovalent cation concentrations. It was found that these positively charged basic proteins interact with the negatively charged lipopolysaccharide of the organism to form the clusters. Adding lysate to suspended cells, followed by lowering of the pH from 5.8 to 4.5 also induced clustering. At dilution rates exceeding the maximum growth rate clusters were retained in anaerobic gas-lift reactors and grew into granules (1–3 mm). It is postulated that granules evolve from clusters. Within the clusters, lysis and a low pH are induced due to diffusion limitations. As a consequence dividing cells are entrapped within the clusters, resulting in growth.

When an aggregate-forming variant of Clostridium butyricum was grown in continuous culture under glucose-limited conditions (pH 5.2, dilution rate = 0.1 h-1, 30° C), glucose was mainly fermented to isopropanol and butanol. Production of solvents was pH dependent and initiated above a threshold concentration of acetic and butyric acid, which probably means that it was triggered by the undissociated concentration of these acids. This was confirmed by studies with a second reactor. Solvent productivity by this variant was found to be stable and the highest ever reported for Clostridium sp. in argued that, due to the aggregated growth mode, this variant is able to reach the threshold concentration of acids at which solventogenesis is initiated at high dilution rates.

Enterococcus faecalis NCTC 775 was grown anaerobically in chemostat culture with pyruvate as the energy source. At low culture pH values, high in vivo and in vitro activities were found for both pyruvate dehydrogenase and lactate dehydrogenase. At high culture pH values the carbon flux was shifted towards pyruvate formate lyase. Some mechanisms possibly involved in this metabolic switch are discussed. In particular attention is paid to the NADH/NAD ratio (redox potential) and the fructose-1,6-bisphosphate-dependent lactate dehydrogenase activity as possible regulatory factors.

Magnesium-limited chemostat cultures of Klebsiella pneumoniae NCTC 418 with 20 µM CaCl2 in the medium showed a low rate of gluconate plus 2-ketogluconate production relative to potassium- or phosphate-limited cultures. However, when the medium concentration of CaCl2 was increased to 1 mM, the glucose dehydrogenase (GDH) activities also increased and became similar to those observed in potassium- or phosphate limited cultures. It is concluded that this is due to Mg2+ and Ca2+ ions being involved in the binding of pyrroloquinoline quinone (PQQ) to the GDH apoenzyme. There seems to be an absolute requirement of divalent cations for proper enzyme functioning and in this respect Ca2+ ions could replace Mg2+ ions. The high GDH activity which has been found in cells grown under Mg2--limited conditions in the presence of higher concentrations of Ca2+ ions, is compatible with the earlier proposal that GDH functions as an auxiliary energy generating system involved in the maintenance of high transmembrane ion gradients.

The biomass concentration extant in potassiumlimited cultures of either Klebsiella pneumoniae or Bacillus stearothermophilus (when growing at a fixed temperature and dilution rate in a glucose/ammonium salts medium) increased progressively as the medium pH value was raised step-wise from 7.0 to 8.5. Because the macromolecular composition of the organisms did not vary significantly, this increase in biomass could not be attributed to an accumulation of storage-type polymers but appeared to reflect a pH-dependent decrease in the cells' minimum K+ requirement. Significantly, this effect of pH was not eviden with cultures in which no ammonium salts were present and in which either glutamate or nitrate was added as the sole nitrogen source; however, it was again manifest when various concentrations of NH4Cl were added to the glutamate-containing medium. This suggested a functional replacement of K+ by NH4+, a proposition consistent with the close similarity of the ionic radii of the potassium ion (1.33 Å) and the ammonium ion (1.43 Å). At pH 8.0, and with a medium containing both glutamate (30 mM) and NH4Cl (100 mM), cultures of B. stearothermophilus would grow without added potassium at a maximum rate of 0.7 h-1. Under these conditions the cells contained maximally 0.1% (w/w) potassium (derived from contaminating amounts of this element in the medium constituents), a value which should be compared with one of 1.4% (w/w) for cells growing in a potassiumlimited medium containing initially 0.5 mM K+. Qualitatively similar findings were made with cultures of K. pneumoniae; and whereas one may not conclude that NH4+can totally replace K+ in the growth of these bacteria, it can clearly do so very extensively.

Among the signal transfer systems in bacteria two types predominate: two-component regulatory systems and quorum sensing systems. Both types of system can mediate signal transfer across the bacterial cell envelope; however, the signalling molecule typically is not taken up into the cells in the former type of system, whereas it usually is in the latter. The Two-component systems include the recently described (eukaryotic) phosphorelay systems; quorum sensing systems can be based upon autoinducers of the N-acylated homoserine lactones, and on autoinducers of a peptidic nature. A single bacterial cell contains many signalling modules that primarily operate in parallel. This may give rise to neural-network behaviour. Recently, however, for both types of basic signal transfer modules, it has been demonstrated that they also can be organised in series (i.e. in a hierarchical order). Besides their hierarchical position in the signal transduction network of the cell, the spatial distribution of individual signalling modules may also be an important factor in their efficiency in signal transfer. Many challenges lie hidden in future work to understand these signal transfer processes in more detail. These are discussed here, with emphasis on the mutual interactions between different signal transfer processes. Successful contributions to this work will require rigorous mathematical modelling of the performance of signal transduction components, and -networks, as well as studies on light-sensing signal transduction systems, because of the unsurpassed time resolution obtainable in those latter systems, the opportunity to apply repeated reproducible stimuli, etc. The increased understanding of bacterial behaviour that already has resulted – and may further result – from these studies, can be used to fine-tune the beneficial activities of bacteria and/or more efficiently inhibit their deleterious ones.

Progressively increasing the input concentration of growth-limiting nutrient (glucose, ammonia, K+) to anaerobic chemostat cultures ofKlebsiella aerogenes(D=0.38 h−1; 35°C; pH 6.8) led to a non-linear increase in bacterial cell concentration. At modest population densities, residual growth-limiting substrate levels increased substantially, with increasing input concentration, and the culture bacterial dry weight tended to a constant value. With the glucose-limited culture, increasing the glucose input concentration above 20 g·1−1led to accumulation of unused glucose and a change in the fermentation pattern. There was a concomitant lowering of the yield value with respect to glucose consumption, and the calculated YATPvalue similarly declined. Addition of extra essential (non-limiting) nutrients to the culture was without effect. Similarly, addition of individual fermentation products (acetate, ethanol,d-lactate, 2,3-butanediol, succinate) to the feed medium, in varying concentrations and in different combinations, failed to influence the fermentation pattern or the energetics of cell synthesis. However, a clear correlation was observed between the yield values (of both glucose- and K+-limited cultures) and the steady state concentration of CO2in the effluent gas. Increasing the concentration CO2either by increasing the population density or lowering the sparging rate of nitrogen gas through the culture, effected a lowering of the yield values. It is suggested that dissolved CO2exerts an effect on both metabolism and the energetics of cell synthesis. A possible mechanism of energy dissipation (i.e., a futile cycle) involving carboxylation and decarboxylation reactions is proposed.

Streptomyces coelicolor was grown in variously limited chemostat cultures and the specific rate of extracellular actinorhodin production (qactinorhodin) was measured. The highest qactinorhodin values were observed in glucose- or ammonia-limited cultures, whereas almost no actinorhodin was produced in sulfate-, phosphate-, potassium-, or magnesium-limited cultures. The effect of the dilution rate on actinorhodin production was studied in glucose-limited cultures. It was found that qactinorhodin was highest at D = 0.06h⁻¹, which was well below the maximal D value tested (0.14 h⁻¹). This explains why, in batch cultures, actinorhodin production starts at the onset of the stationary phase. It was also found that the use of nitrilotriacetate instead of citrate as a chelating agent had a negative effect on actinorhodin production. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 577–582, 1997.

The aerobic respiratory chain of Escherichia coli can function with either of two different membrane-bound NADH dehydrogenases (NDH-1 and NDH-2) and with either of two ubiquinol oxidases (bd-type and bo-type). The amounts of each of these enzymes present in the E. coli membrane depend on growth conditions in general and particularly on the dissolved oxygen concentration. Previous in vitro studies have established that NDH-1 and NDH-2 differ in the extent to which they are coupled to the generation of an energy-conserving proton motive force. The same is true for the two ubiquinol oxidases. Hence, the bioenergetic efficiency of the aerobic respiratory chain must depend on the electron flux through each of the specific enzyme components which are being utilized. In this work, the specific rates of oxygen consumption for cells growing under glucose-limited conditions are reported for a series of isogenic strains in which one or more respiratory components are genetically eliminated. The results are compatible with the proton translocation values of the various components reported from in vitro measurements. The data show that (i) the bd-type oxidase is less efficient than is the bo-type oxidase, but the former is still a coupling site in the respiratory chain; and (ii) under the conditions employed, the wild-type strain uses both the NDH-1 and NDH-2 NADH dehydrogenases to a significant degree, but most of the electron flux is directed through the bo-type oxidase.

The pyruvate dehydrogenase complex of Lactococcus lactis subsp. lactis bv. diacetylactis has a specific activity of 6.6 U/mg and a Km of 1 mM for pyruvate. The specific activities of E2 and E3 in the complex are 30 and 0.36 U/mg, respectively. The complex is very sensitive to NADH inhibition and consists of four subunits: E1 alpha (44 kDa), E1 beta (35 kDa), E2 (73 kDa), and E3 (60 kDa). The L. lactis alpha-acetolactate synthase has a specific activity of 103 U/mg and a Km of 50 mM for pyruvate. Thiamine pyrophosphate (Km = 3.2 microM) and divalent cations are essential for activity. The native enzyme measures 172 kDa and consists of 62-kDa monomers. The role of both enzymes in product formation is discussed in view of NADH inhibition and competition for pyruvate.

Klebsiella aerogenes NCTC 418 was grown anaerobically in chemostat culture with glycerol as source of carbon and energy. Glycerol-limited cultures did not ferment the carbon source with maximal efficiency but produced considerable amounts of 1,3-propanediol. The fraction of glycerol converted to this product depended on the growth rate and on the limitation: faster growing cells produced relatively more of this compound. Under glycerol excess conditions the energetic efficiency of fermentation was decreased due to the high 1,3-propanediol excretion rate. Evidence is presented that 1,3-propanediol accumulation exerts a profound effect on the cells' metabolic behaviour.

Klebsiella aerogenes NCTC 418 was grown in chemostat culture under carbon limitation, with fructose, mannitol, gluconate or pyruvate as the growth-limiting substrates, respectively. It was found that under these conditions the carbon sources were fermented with maximal stoichiometry of ATP generation. The YATP values (g cells per mol ATP generated net) were similar for mannitol- and fructose-limited cultures, but gluconate-limited cultures expressed a value that was 20% lower. From these data it was concluded that gluconate-limited cells invest 0.5 ATP in the uptake of 1 gluconate.

Description of the morphology of yeast and mycelial forms ofParacoccidioides brasiliensis observed in the electron scanning microscope.

The stability of several pBR322-derived recombinant plasmids, carrying thethr operon fromEscherichia coli, was investigated in sulfur-limited chemostat cultures ofE. coli GT123. A marked increase in the segregational stability of one of these plasmids was observed. It is concluded that the increased stability was due to the spontaneous insertion of Tn1000 from the chromosome of the host into the plasmid.

Klebsiella aerogenes NCTC 418 was cultured anaerobically in chemostat cultures (pH 6.8; 35° C) under carbon, phosphate-, ammonia-, sulphate- and potassium-limited conditions with glucose as the sole carbon- and energy source. The rates of uptake of glucose and excretion of fermentation products were quantitatively determined and carbon, hydrogen- and oxygen balances were constructed with recoveries better than 90%.

A study was undertaken of the microbial composition of aggregates from an acidifying anaerobic gas-lift reactor. For this purpose a simple 100 ml anaerobic gas lift reactor was developed. It was found that the predominant organism in the aggregates was Selenomonas ruminantium. Both, microscopical observations and a newly developed enumeration technique led to the conclusion that the mixed granules consisted mainly of this organism. Grown in pure culture, S. ruminantium was capable of forming aggregates. These aggregates resembled the mixed aggregates both macro- as well as microscopically. Furthermore the fermentation pattern of this pure aggregated culture was similar to that of a mixed aggregated culture.

The presence of Paracoccidioides brasiliensis was determined in sputum samples from 50 patients with paracoccidioidomycosis using four different techniques: (a) cell-block preparations stained with silver methenamine, (b) direct microbiologie examination, (c) smears stained with Shorr, and (d) smears stained with silver methenamine. Overall, cell-block preparations and smears stained with silver methenamine proved to be the most sensitive techniques, followed by smears stained with Shorr and direct microbiologic examination in decreasing order of sensitivity. Sputum cytology tended to be less positive in patients with interstitial pulmonary lesions as determined by chest X-ray than in patients with alveolar lesions. In addition to its high sensitivity, cell-block preparation technique allows storage of blocks and slides for further studies.

The metabolism of gluconate by Klebsiella pneumoniae NCTC 418 was studied in continuous culture. Under all gluconate-excess conditions at low culture pH values (pH 4.5–5.5) the majority (70–90%) of the gluconate metabolized was converted to 2-oxogluconate via gluconate dehydrogenase (GADH), although specific 2-oxogluconate production rates under potassium-limited conditions were significantly lower than under other gluconate-excess conditions. At high culture pH values, metabolism shifted towards production of acetate. Levels of GADH were highest at low culture pH values and synthesis was stimulated by the presence of (high concentrations of) gluconate. An increase in activity of the tricarboxylic acid cycle was accompanied by a decrease in GADH activity in vivo and in vitro, suggesting that the GADH serves a role as an alternative energy-generating system. Anaerobic 2-oxogluconate production was found to be possible in the presence of nitrate as electron acceptor. Levels of gluconate kinase were highest when K. pneumoniae was grown under gluconate-limited conditions. Under carbon-excess conditions, levels of this enzyme correlated with the intracellular catabolic flux.

Escherichia coli Frag1 was grown under various nutrient limitations in chemostat culture at a fixed temperature, dilution rate and pH both in the presence and the absence of a high concentration of ammonium ions by using either ammonium chloride or dl-alanine as the sole nitrogen source. The presence of high concentrations of ammonium ions in the extracellular fluids of potassium-limited cultures of E. coli Frag1 caused an increase of the specific rate of oxygen consumption of these cultures. In contrast, under phosphate-, sulphate- or magnesium-limited growth conditions no such increase could be observed. The presence of high concentrations of ammonium ions in potassium-limited cultures of E. coli Frag5, a mutant strain of E. coli Frag1 which lacks the high affinity potassium uptake system (Kdp), did not increase the specific rate of oxygen consumption.

Klebsiella aerogenes NCTC418 was cultured anaerobically under glucose-limited conditions in chemostat cultures at various growth rates, ranging from 0.13 h-1 to 0.82 h-1. It was found that the specific uptake rate of glucose varied linearly with the growth rate and that under these conditions glucose was fermented solely to acetate and ethanol plus CO2+H2 and formate.

This study evaluated the influence of dry matter (DM) or energy intake on in vivo embryo production and quality. Nonlactating Nelore cows (n= 32, 4 to 10 y old) weighing 489.5 ± 11.3 kg and with BCS of 3.3 (1 to 5) were used. After 15 d on a maintenance diet [1.2% of DM per kg of body weight (BW)], cows were randomly divided into four groups. Maintenance (M), 0.7M, and 1.5M received the equivalent of 70, 100, and 150% of the M diet, respectively. The fourth group (Energy; E) received a diet with DM similar to the M group, but with an energy level equivalent to the 1.5M group. Cows were fed individually and offered all diets in a Latin-square arrangement every 42 d. Cows were submitted to a conventional superovulation protocol. Superovulatory response was assessed by ultrasonography and embryo quality was evaluated according to the IETS guidelines, as well as by pregnancy per fixed-time transfer (P/ET) of 274 vitrified embryos to Nelore recipients. Pre-prandial blood plasma insulin was performed by RIA at the onset of superovulation. Data were analyzed by PROC GLIMMIX of SAS (P< 0.05). Circulating insulin was greater in the E group (8.7 ± 0.9 µIU/mL) compared with groups 0.7 M (4.6 ± 0.9) and M (5.3 ± 0.85), not differing from group 1.5M (6.6 ± 0.9). Superovulation (CL number) was lower in donors fed high energy (9.7 ± 1.2) compared with cows receiving the standard diet [0.7M (13.0 ± 1.3), M (14.2 ± 1.2) or 1.5M (13.9 ± 1.2)] due to the negative correlation between circulating insulin and CL number (r= -0.32). Nevertheless, there was no difference between groups for number of ova/embryos (∼6), viable (∼3), or freezable embryos (∼2.7). Regardless of treatment, circulating insulin was negatively correlated with the number of viable embryos (r= -0.22). There was lower P/ET at 60 d in the 97 cows receiving embryos from donors fed high energy (E; 29.4%) compared with the 177 cows receiving embryos from donors fed the standard diet (0.7M, M or 1.5M; 43.3%). Moreover, probability of P/ET at 60 d evaluated by logistic regression decreased as circulating insulin of donor cows increased from 0.64 to 25.0 µIU/mL. In conclusion, there was effect of diet on the superovulatory response and P/ET. Additionally, high circulating insulin of the donors was associated with lower superovulatory response, less viable embryos and less P/ET at 60 d. Financial support from FAPESP and CNPq. We also thank InVitro Brasil and Hildergard Pritzelwitz Experimental Station.

ABSTRACTDue to the spread of resistance, antibiotic exposure receives increasing attention. Ecological consequences for the different niches of individual microbiomes are, however, largely ignored. Here, we report the effects of widely used antibiotics (clindamycin, ciprofloxacin, amoxicillin, and minocycline) with different modes of action on the ecology of both the gut and the oral microbiomes in 66 healthy adults from the United Kingdom and Sweden in a two-center randomized placebo-controlled clinical trial. Feces and saliva were collected at baseline, immediately after exposure, and 1, 2, 4, and 12 months after administration of antibiotics or placebo. Sequences of 16S rRNA gene amplicons from all samples and metagenomic shotgun sequences from selected baseline and post-antibiotic-treatment sample pairs were analyzed. Additionally, metagenomic predictions based on 16S rRNA gene amplicon data were performed using PICRUSt. The salivary microbiome was found to be significantly more robust, whereas the antibiotics negatively affected the fecal microbiome: in particular, health-associated butyrate-producing species became strongly underrepresented. Additionally, exposure to different antibiotics enriched genes associated with antibiotic resistance. In conclusion, healthy individuals, exposed to a single antibiotic treatment, undergo considerable microbial shifts and enrichment in antibiotic resistance in their feces, while their salivary microbiome composition remains unexpectedly stable. The health-related consequences for the gut microbiome should increase the awareness of the individual risks involved with antibiotic use, especially in a (diseased) population with an already dysregulated microbiome. On the other hand, understanding the mechanisms behind the resilience of the oral microbiome toward ecological collapse might prove useful in combating microbial dysbiosis elsewhere in the body.IMPORTANCEMany health care professionals use antibiotic prophylaxis strategies to prevent infection after surgery. This practice is under debate since it enhances the spread of antibiotic resistance. Another important reason to avoid nonessential use of antibiotics, the impact on our microbiome, has hardly received attention. In this study, we assessed the impact of antibiotics on the human microbial ecology at two niches. We followed the oral and gut microbiomes in 66 individuals from before, immediately after, and up to 12 months after exposure to different antibiotic classes. The salivary microbiome recovered quickly and was surprisingly robust toward antibiotic-induced disturbance. The fecal microbiome was severely affected by most antibiotics: for months, health-associated butyrate-producing species became strongly underrepresented. Additionally, there was an enrichment of genes associated with antibiotic resistance. Clearly, even a single antibiotic treatment in healthy individuals contributes to the risk of resistance development and leads to long-lasting detrimental shifts in the gut microbiome.

The cyanobacterium Microcystis PCC7806 was found to possess an NAD-dependent lactate dehydrogenase (EC 1.1.1.27) which catalyzes the reduction of pyruvate to l-lactate. The enzyme required fructose 1,6-bisphosphate for activity and displayed positive cooperativity towards pyruvate. Lactate was not formed during fermentation by cell suspensions, possibly due to low intracellular concentrations of fructose 1,6-bisphosphate and/or pyruvate.

The tendency of Saccharomyces cerevisiae to favor alcoholic fermentation over respiration is a complication in aerobic, biomass-directed applications of this yeast. Overproduction of Hap4p, a positive transcriptional regulator of genes involved in respiratory metabolism, has been reported to positively affect the balance between respiration and fermentation in aerobic glucose-grown batch cultures. In this study, the effects of HAP4 overexpression have been quantified in the prototrophic S. cerevisiae strain CEN. PK 113-7D under a variety of growth conditions. In aerobic glucose-limited chemostat cultures, overexpression of HAP4 increased the specific growth rate at which aerobic fermentation set in by about 10% relative to the isogenic wild-type. Upon relief of glucose-limited conditions, the HAP4-overexpressing strain produced slightly less ethanol than the wild-type strain. The effect of Hap4p overproduction was most drastic in aerobic, glucose-grown chemostat cultures in which ammonium was limiting. In such cultures, the biomass yield on glucose was double that of the wild-type.

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Potassium-limited cultures of Candida utilis grown with ammonium chloride as the sole nitrogen source attained a higher dry weight than similar cultures grown with sodium nitrate as the sole nitrogen source. This increase depended on the extracellular ammonia concentration and was not due to accumulation of storage polymers. We conclude that in this yeast ammonium ions can fulfill to some extent the physiological role of potassium ions and that the intracellular concentration of ammonium ions is predominantly determined by the ammonia concentration in the culture medium.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

The effect of NADH on the activity of the purified pyruvate dehydrogenase complexes (PDHc) of Enterococcus (Ec.) faecalis, Lactococcus lactis, Azotobacter vinelandii and Escherichia coli was determined in vitro. It was found that the PDHc of E. coli and L. lactis was active only at relatively low NADH/NAD ratios, whereas the PDHc of Ec. faecalis was inhibited only at high NADH/NAD ratios. The PDHc of Azotobacter vinelandii showed an intermediate sensitivity. The organisms were grown in chemostat culture under conditions that led to different intracellular NADH/NAD ratios and the PDHc activities in vivo could be calculated from the specific rates of product formation. Under anaerobic growth conditions, only Ec. faecelis expressed PDHc activity in vivo. The activities in vivo of the complexes of the different organisms were in good agreement with their properties determined in vitro. The physiological consequences of these results are discussed.

When grown in carbon source-limited chemostat cultures with lactate or glucose as the carbon and energy source and xylose as an additional source of reducing equivalents, Pseudomonas putida NCTC 10936 oxidized xylose to xylonolactone and xylonate. No other products were formed from this pentose, nor was it incorporated into biomass. The presence of xylose in these cultures resulted in higher Yglucose and Ylactate values as compared to cultures without xylose indicating that biologically useful energy was conserved during the periplasmic oxidation of xylose. As the Y0 values for growth on glucose or on lactate alone were equal to the Y0 values with xylose as co-substrate, it is concluded that for flucose- or lactate-limited growth energy conservation by PQQH2 oxidation is as efficient as by NADH2 oxidation.