Change of extracellular cAMP concentration is a sensitive reporter for bacterial fitness in high-cell-density cultures of Escherichia coli

Guanosine-3',5'-tetraphosphate (ppGpp) and sigmaS, two regulators of the starvation response of Escherichia coli, have received increasing attention for monitoring cell physiological changes in production processes, although both are difficult to quantify. The kinetics of cAMP formation and degradation were not yet investigated in such processes, although the complex regulation of cAMP by synthesis, release, and degradation in connection with straightforward methods for analysis renders it a highly informative target. Therefore, we followed the cAMP concentration in various nonrecombinant and in four different recombinant glucose-limited fed-batch processes in different production scales. The intracellular cAMP concentration increases strongly at the end of the batch phase. Most cAMP is released to the cultivation medium. The rates of accumulation and degradation of extracellular cAMP are growth-rate-dependent and show a distinct maximum at a growth rate of about 0.35 h(-1). At very low growth rates, below 0.05 h(-1), extracellular cAMP is not produced but rather degraded, independent of whether this low growth rate is caused by glucose limitation or by the high metabolic load of recombinant protein production. In contrast to intracellular cAMP, which is highly unstable, analysis of extracellular cAMP is simpler and the kinetics of accumulation and degradation reflect well the physiological situation, including unlimited growth, limitation, and severe starvation of a production host.     

Factors influencing recombinant protein yields in an insect cell-bacuiovirus expression system: multiplicity of infection and intracellular protein degradation

The insect cell (Sf9)-baculovirus (AcNPV) expression system was employed for the synthesis of beta-galactosidase, a model heterologous protein. In the recombinant virus studied, the lacZ gene is fused to a portion of the polyhedrin structural gene and is under the control of the polyhedrin promoter. The effect of the multiplicity of infection (MOI) on product titer was determined by infecting cells with MOI values ranging from 0 to 100 and monitoring the production of beta-galactosidase with time. The relationship between final product titer and MOI was dependent on the growth phase of the cells prior to infection. The final product titer from cells infected in the early exponential phase was relatively independent of MOI. For cells infected in late-exponential phase there was a logarithmic relationship between the final beta-galactosidase titer and the MOI used, with the highest MOI studied resulting in greatest protein synthesis. The synthesis and degradation rates of beta-galactosidase were investigated by a pulse-chase technique using L-[(35)S]-methionine. At 24 h postinfection, the degradation rate is of the same order of magnitude as the synthesis rate. However, the synthesis rate of beta-galactosidase increases dramatically at 96 h postinfection. During this later period, the degradation rate is negligible. Although degradation of recombinant protein occurs in this system, degradation activity declines as infection proceeds and is insignificant late in intention when recombinant protein expression is intense.     

The effects of species pool,dispersal and competition on the diversity–productivity relationship

Aim The diversity–productivity relationship is a controversial issue in ecology. Diversity is sometimes seen to increase with productivity but a unimodal relationship has often been reported. Competitive exclusion was cited initially to account for the decrease of diversity at high productivity. Subsequently, the roles of evolutionary history (species pool size) and dispersal rate have been acknowledged. We explore how the effects of species pool, dispersal and competition combine to produce different diversity–productivity relationships. Methods We use a series of simulations with a spatially explicit, individual‐based model. Following empirical expectations, we used four scenarios to characterize species pool size along the productivity gradient (uniformly low and high, linear increase and unimodal). Similarly, the dispersal rate varied along the productivity gradient (uniformly low and high, and unimodal). We considered both neutral communities and communities with competitive exclusion. Results and main conclusions Our model predicts that competitive interactions will result in unimodal diversity–productivity relationships. The model often predicts unimodal patterns in neutral communities as well, although the decline in richness at high productivity is less than in competing communities. A positive diversity–productivity relationship is simulated for neutral communities when the species pool size increases with productivity and the dispersal rate is high. This scenario is probably more widespread in nature than the others since positive diversity–productivity relationships have been observed more frequently than previously expected, especially in the tropics and for woody species. Our simulated effects of species pool, dispersal and competition on diversity patterns can be linked to empirical observations to uncover mechanisms behind the diversity–productivity relationship.     

Excretion and degradation of exogenous adenosine 3',5'-monophosphate by isolated perfused rat kidney

To determine the role played by the kidney in the metabolism and excretion of plasma adenosine 3′,5′-monophosphate (cAMP) we have studied the fate of this nucleotide (0.01–1.0mM) when it is perfused in a recirculating medium through the isolated rat kidney. cAMP was rapidly taken up and degraded by the kidney, the rate of its disappearance from the perfusate being at least twice its rate of excretion in the urine. Nevertheless, the cAMP excretory rate exceeded the filtration rate by 1.5 to 2 fold, and thus net secretion (transtubular transport) was demonstrated. The rates of filtration, perfusate clearance, and degradation of cAMP were proportional to its perfusate concentration. Methyl xanthines (caffeine and aminophylline) at 10mM, and probenecid at 0.9mM abolished transtubular transport of cAMP and greatly retarded disappearance of the nucleotide from the perfusate. It is concluded that there is a ready penetration of cAMP into renal cells from peritubular capillaries. Depending on the perfusate concentration of cAMP, transtubular transport may or may not exceed the simultaneous intra-renal breakdown of the compound. A low rate of cAMP excretion in the urine may accompany a considerably higher rate of cAMP clearance from the perfusate by the kidney.     

Different cyclic adenosine 3',5'-monophosphate requirements for induction of beta-galactosidase and tryptophanase. Effect of osmotic pressure on intracellular cyclic adenosine 3,5-monophosphate concentrations.

In this study we have tried to answer the following questions: (1) is it possible for different catabolite-repressible genes, although submitted to the same control, to be expressed selectively depending upon the growth conditions, and (2) what is the effect of increasing the osmolarity of the medium on the intracellular level of cAMP? Two conditions were found to cause a continuous variation of intracellular cAMP levels during growth. With different strains, higher cAMP levels are required for induction of the tryptophanase gene than one required for induction of the lactose operon. cAMP has been provided externally in adenyl cyclase minus cells of a mutant that has been made permeable by EDTA treatment. Although external cAMP concentrations, 10 times higher than the usual intracellular levels, are required for induction of beta-galactosidase and tryptophanase, the difference of requirements of cAMP is maintained. An increase in the osmolarity of the medium by sucrose addition causes a fourfold decrease in the intracellular cAMP level. As a consequence this prevents the induction of tryptophanase whereas beta-galactosidase is still inducible. After pulse induction, a difference in the kinetics of expression of the tryptophanase and beta-galactosidase genes was found. Its relationship with the previous results is discussed.     

Effect of growth conditions on catabolite repression and cyclic AMP synthesis in Escherichia coli 3000A1

Catabolite repression of beta-galactosidase synthesis in E. coli 3000A1 (adenine-) was studied under a variety of growth conditions. The differential rate of induced beta-galactosidase synthesis was maximal at the growth rate of 0.75 division per h, irrespective of whether growth conditions were aerobic or anaerobic. The addition of cyclic AMP (cAMP) to the medium partly restored the repressed synthesis of beta-galactosidase under some growth conditions, but showed little or no effect on the enzyme synthesis under other conditions. Although growth rate and profile of beta-galactosidase synthesis in glucose-grown cells were similar to those in arabinose-grown cells, the acceleration of beta-galactosidase synthesis upon the addition of cAMP was found only in glucose-grown cells. The cells aerobically grown in the presence of glycerol, xylose, or arabinose showed a high synthetic rate of cAMP and were insensitive to exogenously supplied cAMP as regards beta-galactosidase synthesis. Although the cells grown with glucose showed similar rates of cAMP synthesis under aerobic and anaerobic conditions, the differential rate of beta-galactosidase synthesis was much higher in the anaerobic state than in the aerobic state. These findings support the idea that catabolite repression found in the strain is caused through two mechanisms, i.e., cAMP-mediated and cAMP-independent ones.     

Regulation of cyclic AMP synthesis in Escherichia coli K-12: effects of the rpoD800 sigma mutation, glucose, and chloramphenicol

An immediate 12-fold inhibition in the rate of beta-galactosidase synthesis occurs in Escherichia coli cells containing the mutant sigma allele rpoD800 after a shift to 42 degrees C. In the present study we characterize the nature of the inhibition. The severe inhibition of beta-galactosidase synthesis was partly relieved by cyclic AMP (cAMP). We inferred that the inhibition might be mediated by a decreased intracellular concentration of cAMP. Consistent with this inference, the rate of cAMP accumulation in mutant cells after a temperature upshift was depressed relative to that in wild-type cells. Glucose and chloramphenicol, two agents known to inhibit differentially beta-galactosidase mRNA synthesis, caused a similar inhibition in the rate of cAMP accumulation. Thus, three diverse stimuli, glucose, chloramphenicol, and a temperature-sensitive sigma mutation, appear to affect beta-galactosidase synthesis by regulating the synthesis of cAMP.     

Species pool size and rainfall account for the relationship between biodiversity and biomass production in natural forests of China

The strength of biodiversity–biomass production relationships increases with increasing environmental stress and time. However, we know little about the effects of abiotic (e.g., climate) and biotic (e.g., species pool and community composition) factors on this trend. Whether variation in biomass production is best explained by phylogenetic diversity metrics or traditional measures of species richness also remains elusive. We compiled estimates of community composition and biomass production for tree species in 111 permanent quadrats spanning three natural forests (tropical, subtropical, and temperate) in China. Based on ~10 years of data, we compared temperature, rainfall, species pool size, and community composition in each forest each year. We estimated species richness and phylogenetic diversity in each quadrat each year; the latter metric was based on the sum of branch lengths of a phylogeny that connects species in each quadrat each year. Using generalized linear mixed‐effect models, we found that top‐ranked models included the interaction between forest and biodiversity and the interaction between forest and year for both biodiversity metrics. Variation in biomass production was best explained by phylogenetic diversity; biomass production generally increased with phylogenetic diversity, and the relationship was stronger in subtropical and temperate forests. Increasing species pool size, temperature, and rainfall and decreasing inter‐quadrat dissimilarity range shifted the relationship between biomass production and phylogenetic diversity from positive to neutral. When considered alone, species pool size had the strongest influence on biomass production, while species pool size, rainfall, and their interaction with phylogenetic diversity constituted the top‐ranked model. Our study highlights the importance of species pool size and rainfall on the relationship between phylogenetic diversity and biomass production in natural forest ecosystems.     

Effect of phalloidin on liver actin distribution,content, and turnover

Phalloidin increases F-actin microfilament content and actin-directed immunofluorescence in hepatocytes in vivo and also increases actin polymerization and the stability of F-actin in vitro. We studied the sensitivity of immunofluorescent staining of actin to an actin depolymerizing factor (ADF) as well as actin content, degree of polymerization, and turnover in livers of in vivo phalloidin-treated rats. Pretreatment with ADF abolished anti-actin antibody (AAA) staining of normal liver but did not modify staining of livers from phalloidin-treated animals. Plani-metric analyses of SDS-polyacrylamide gels snowed the percent actin of total protein was increased by approximately 40% and the absolute amount of actin by approximately 43%, ten days after daily phalloidin treatment (50 μg/100 gm body weight). Similar but smaller changes could be seen after one day of treatment. Ultracentrifugational analyses of liver extracts indicated no change in the amount or proportion of G-actin but a 194% increase in the proportion of F-actin in ten-day treated animals, changes also apparent in one day animals. Neither the relative fractional rate of actin synthesis nor its synthesis as a percent of total protein synthesis was altered either at one-day or ten-day post-phalloidin treatment. Dual-isotope experiments indicated that the rate of actin degradation was decreased selectively in the one- to three-day period -following drug treatment. Thus, phalloidin appears to stabilize actin against the depolymerizing actions of ADF, increases the proportion of F-actin without altering the size of the G-actin pool, and causes accumulation of actin by decreasing its relative rate of degradation.     

Bioenergetics of growth of a cyprinid, Phoxinus phoxinus: the effect of ration, temperature and body size on food consumption, faecal production and nitrogenous excretion

Food consumption, faecal production and nitrogen excretion by minnows, Phoxinus phoxinus , weighing 1–5.5 g were studied at five rations ranging from starvation to ad libitum and four temperatures ranging from 5 to 15°C.
The maximum rate of food consumption (C_max) was related to body weight ( W ) and temperature ( T ) by the relationship: C _max= aW^b1T^b2 . There were significant daily variations in C_max, which tended to decline over time. Absorption efficiency increased with increasing ration size and decreasing temperature. Body weight had no significant effect on the faecal production. The equation F = a C^b1e^b2 T described the relationship between faecal production ( F ), food consumption ( C ) and temperature. Ammonia-N predominated over urea-N in the excreta of most experimental fish. The proportion of urea-N in the total nitrogen excreted was generally higher at lower rations than at higher rations. Rates of nitrogen excretion increased with increased ration size and were, to a lesser extent, influenced by temperature. Body weight had no significant effect on the nitrogen excretion by feeding minnows. The equation N = a+b_lT+b₂C described the effects of food consumption and temperature on nitrogen excretion ( N ) other than urea-N excretion. The relationship between urea-N excretion ( N_u ), food consumption and temperature was described by the equation N_u= ae^b1T ((C+1) ^b 2.
On the average, 11 % of food energy was lost in faecal production and nitrogen excretion by minnows feeding on whiteworms, Enchytraeus spp.     

Temperature and induction effects on the degradation rate of an abnormal beta-galactosidase in Escherichia coli.

Intracellular protein degradation was investigated using an unstable fragment of Escherichia coli beta-galactosidase, the CSH11 mutant, as a model protein. This abnormal protein was expressed from a single copy gene in the chromosome and is converted to a detectable degradable intermediate. The in vivo degradation rates of both beta-galactosidase fragments were measured using pulse-chase radioactive labeling techniques, and their intracellular concentrations were determined using alpha-complementation assays. In the physiological range of 30 to 37 degrees C, the apparent degradation rate constant for the CSH11 fragment follows Arrhenius behavior; while the intermediate's apparent degradation rate constant is nearly unchanged. However, above 37 degrees C the degradation rates of both fragments increase significantly. Analysis of the labeled intermediate's rate of change above 40 degrees C reveals that the CSH11 fragment is being degraded by a second pathway which does not produce the intermediate. When the induction level of the abnormal beta-galactosidase was varied the degradation rates of both fragments behaved similarly, but they unexpectedly decreased with increasing IPTG concentration. The two parallel degradation pathways for CSH11 apparently operated at only the lower IPTG levels. The measured degradation rates did not correlate directly with the intracellular concentration of abnormal proteins.     

Uptake and degradation of cytoplasmic RNA by hepatic lysosomes. Quantitative relationship to RNA turnover.

Past evidence has suggested that the lysosomal pathway is an important site of cytoplasmic RNA degradation in the hepatic parenchymal cell (Lardeux, B. R., Heydrick, S. J., and Mortimore, G. E. (1987) J. Biol. Chem. 262, 14507-14519). We now provide additional support for this notion by quantitating degradable RNA in lysosomes and correlating its pool size with hepatic RNA degradation. Rat livers, previously labeled with [6-14C]orotic acid, were perfused with graded levels of amino acids over the full range of induced autophagy; RNA degradation was determined from [14C]cytidine release. Close correspondence between the marker beta-acetylglucosaminidase and the breakdown of RNA to cytidine in subcellular fractions indicated that the lysosome was the main site of catabolism, a conclusion supported by the fact that degradation was enhanced when external pH was lowered from 7 to 6. Although [14C]cytidine was also released in homogenates by the action of natural ribonucleases on cytosolic RNA, this source was eliminated by unlabeled exogenous RNA. The size of the degradable RNA pool in lysosomes, determined from the total release of cytidine in homogenates, correlated directly with rates of hepatic RNA degradation over the full range of basal and induced degradation. A direct correlation was also seen between RNA degradation and cytidine pools within lysosomal particles. Because cytosolic cytidine was not taken up by lysosomes under these conditions, the pool could only have arisen from the breakdown of intralysosomal RNA. As determined by cytidine production, these findings support the view that the lysosomal-vacuolar system is the main, if not sole, site of induced and basal RNA degradation in liver.     

Why mammalian lineages respond differently to sexual selection: metabolic rate constrains the evolution of sperm size

The hypothesis that sperm competition should favour increases in sperm size, because it results in faster swimming speeds, has received support from studies on many taxa, but remains contentious for mammals. We suggest that this may be because mammalian lineages respond differently to sexual selection, owing to major differences in body size, which are associated with differences in mass-specific metabolic rate. Recent evidence suggests that cellular metabolic rate also scales with body size, so that small mammals have cells that process energy and resources from the environment at a faster rate. We develop the 'metabolic rate constraint hypothesis' which proposes that low mass-specific metabolic rate among large mammals may limit their ability to respond to sexual selection by increasing sperm size, while this constraint does not exist among small mammals. Here we show that among rodents, which have high mass-specific metabolic rates, sperm size increases under sperm competition, reaching the longest sperm sizes found in eutherian mammals. By contrast, mammalian lineages with large body sizes have small sperm, and while metabolic rate (corrected for body size) influences sperm size, sperm competition levels do not. When all eutherian mammals are analysed jointly, our results suggest that as mass-specific metabolic rate increases, so does maximum sperm size. In addition, species with low mass-specific metabolic rates produce uniformly small sperm, while species with high mass-specific metabolic rates produce a wide range of sperm sizes. These findings support the hypothesis that mass-specific metabolic rates determine the budget available for sperm production: at high levels, sperm size increases in response to sexual selection, while low levels constrain the ability to respond to sexual selection by increasing sperm size. Thus, adaptive and costly traits, such as sperm size, may only evolve under sexual selection when metabolic rate does not constrain cellular budgets.     

A metabolic theory of ecology applied to temperature and mass dependence of N and P excretion by common carp

Our study used a metabolic theory of ecology (MTE) to explore scaling of metabolic rates by body size and temperature, and to predict nutrient excretion by common carp (Cyprinus carpio). At high biomasses, common carp have negative impacts on water quality, and one mechanism is excretion of the nutrients N and P. We measured whole-body and mass-specific excretion rates during summer and winter for fish of different sizes (wet mass range 28–1,196 g) to produce an allometric scaling model capable of predicting excretion at different temperatures. We found positive relationships between both dissolved and total nutrient concentrations and fish wet mass in summer and winter, with greater excretion rates in summer (mean water temperature 24.2°C) than in winter (mean water temperature 9.2°C). Mass-specific excretion rates decreased with increasing fish size, consistent with the MTE, and the temperature-adjusted model explained more variation for N excretion than for P. The proportion of dissolved nutrients (NH₄ and PO₄) to total nutrients increased with increasing fish size. The significance of these models is that they can be used to predict population-based nutrient excretion by common carp when thermal history, fish density and size distribution in a water body are known.     

Effect of exercise on protein metabolism in humans as explored with stable isotopes

Exercising for 3.75 h on a treadmill at 50% VO2 max in the fed state induced an increased excretion of 71 mg nitrogen/kg over the 18 h after exercise. However, measurements of the time course of changes in 13CO2 excretion from ingested [1-13C]leucine indicated that all of this increased nitrogen production occurs during the exercise period. Because of the reduced renal clearance and slow turnover of the urea pool, urea excretion lags behind urea production. Measurements of nitrogen flux from the plateau labeling of urinary ammonia achieved by repeated oral doses of 15N-labeled glycine indicated that the nitrogen loss resulted from an increase in protein degradation and a decrease in protein synthesis. Further studies with [1-13C]leucine indicated that a 2-h treadmill exercise induced an increase in the nitrogen loss from 5.4 to 16 mg . kg-1 . h-1 measured with a primed constant infusion of [1-13C]leucine. This resulted from a fall in whole-body protein synthesis. Glucose given at the rate of 0.88 g . kg-1 . h-1 depressed the rate of whole-body protein degradation and appeared to suppress the exercise-induced increase in nitrogen excretion. When leucine oxidation rates were measured at increasing work rates, a linear relationship between percentage of VO2 max and leucine oxidation was observed up to 89% VO2 max when 54% of the flux of leucine was oxidized. These changes may involve nonmuscle as well as muscle tissue. Thus the source of the increased nitrogen losses is probably liver. In muscle, protein degradation is actually decreased judged by methylhistidine excretion, whereas in liver, protein degradation may be increased. Also the fall in whole-body protein synthesis may reflect changes in nonmuscle tissues because in running rats protein synthesis in muscle is maintained. As far as leucine metabolism is concerned, because the increase in leucine oxidation occurs when leucine and its keto acid concentration falls, exercise must specifically activate the 2-oxoacid dehydrogenase.     

Sensing of Nitrogen Limitation by Bacillus subtilis: Comparison to Enteric Bacteria

Previous studies showed that Salmonella typhimurium apparently senses external nitrogen limitation as a decrease in the concentration of the internal glutamine pool. To determine whether the inverse relationship observed between doubling time and the glutamine pool size in enteric bacteria was also seen in phylogenetically distant organisms, we studied this correlation in Bacillus subtilis, a gram-positive, sporulating bacterium. We measured the sizes of the glutamine and glutamate pools for cells grown in batch culture on different nitrogen sources that yielded a range of doubling times, for cells grown in ammonia-limited continuous culture, and for mutant strains (glnA) in which the catalytic activity of glutamine synthetase was lowered. Although the glutamine pool size of B. subtilis clearly decreased under certain conditions of nitrogen limitation, particularly in continuous culture, the inverse relationship seen between glutamine pool size and doubling time in enteric bacteria was far less obvious in B. subtilis. To rule out the possibility that differences were due to the fact that B. subtilis has only a single pathway for ammonia assimilation, we disrupted the gene (gdh) that encodes the biosynthetic glutamate dehydrogenase in Salmonella. Studies of the S. typhimurium gdh strain in ammonia-limited continuous culture and of gdh glnA double-mutant strains indicated that decreases in the glutamine pool remained profound in strains with a single pathway for ammonia assimilation. Simple working hypotheses to account for the results with B. subtilis are that this organism refills an initially low glutamine pool by diminishing the utilization of glutamine for biosynthetic reactions and/or replenishes the pool by means of macromolecular degradation.     

The relationship of cAMP levels and the rate of pool labelling of cAMP and related bases, nucleosides and nucleotides to the HeLa cell cycle.

Cellular cAMP levels as well as the rate of pool labelling of cAMP and related bases, nucleosides and nucleotides were determined in synchronized cultures of HeLa cells after pulse-labelling with [¹⁴C]adenine. The cAMP levels were found to be maximal in G 1 and minimal in G 2 and mitosis, as previously reported by others. The rate of labelling of the cAMP pools, however, was found to be maximal in G 2 and decreased to a minimum in G 1. This suggests that the rate of cAMP synthesis is highest when pool level is lowest and vice versa. A comparison of cAMP levels and the rate of 5′AMP pool labelling throughout the HeLa cell cycle indicated an inverse relationship. Such a relationship emphasizes the role of the cyclic 3′,5′-phosphodiesterase activity during the cell cycle. The kinetics of pool labelling of IMP, ATP, and hypoxanthine throughout the cell cycle suggested that the adenylate energy charge fluctuated as a function of the cell cycle. The apparent activation of the adenylate cyclase during G 2 and mitosis as reflected by the increased rate of cAMP pool labelling suggests that the super phosphorylation of H 1 histone during G 2-mitotic transition may be mediated by cAMP-dependent phosphokinases.