Identification of adaptive changes in an evolving population of Escherichia coli: the role of changes with regulatory and highly pleiotropic effects

A population of Escherichia coli initiated with a single clone developed extensive morphological and physiological polymorphism after being maintained for 773 generations in glucose-limited continuous culture. To understand the mechanisms of adaptation to this environment, total protein patterns of four adaptive clones and of the parent strains were examined by two-dimensional gel electrophoresis. Approximately 20% of the proteins (approximately 160 in absolute numbers) showed significantly different levels of expression in pairwise comparisons of parent and adapted clones. The extent of these changes points to the importance of mutations with regulatory and/or highly pleiotropic effects in the adaptive process. The four evolved clones all expressed fewer proteins than did the parent strain, supporting the hypothesis of energy conservation during evolutionary change. Forty-two proteins that could be assigned to known cellular functions were identified. The changes in some of them indicated that the evolved clones developed different adaptive mechanisms to glucose-limited environment. Changes were observed in the expression levels of proteins associated with translation, membrane composition, shock response, and active transport. A fraction of the changes could not be either explained or predicted from a consideration of the nature of the environment in which the clones evolved.     

Global regulatory mutations in csrA and rpoS cause severe central carbon stress in Escherichia coli in the presence of acetate

The csrA gene encodes a small RNA-binding protein, which acts as a global regulator in Escherichia coli and other bacteria (T. Romeo, Mol. Microbiol. 29:1321-1330, 1998). Its key regulatory role in central carbon metabolism, both as an activator of glycolysis and as a potent repressor of glycogen biosynthesis and gluconeogenesis, prompted us to examine the involvement of csrA in acetate metabolism and the tricarboxylic acid (TCA) cycle. We found that growth of csrA rpoS mutant strains was very poor on acetate as a sole carbon source. Surprisingly, growth also was inhibited specifically by the addition of modest amounts of acetate to rich media (e.g., tryptone broth). Cultures grown in the presence of >/=25 mM acetate consisted substantially of glycogen biosynthesis (glg) mutants, which were no longer inhibited by acetate. Several classes of glg mutations were mapped to known and novel loci. Several hypotheses were examined to provide further insight into the effects of acetate on growth and metabolism in these strains. We determined that csrA positively regulates acs (acetyl-coenzyme A synthetase; Acs) expression and isocitrate lyase activity without affecting key TCA cycle enzymes or phosphotransacetylase. TCA cycle intermediates or pyruvate, but not glucose, galactose, or glycerol, restored growth and prevented the glg mutations in the presence of acetate. Furthermore, amino acid uptake was inhibited by acetate specifically in the csrA rpoS strain. We conclude that central carbon flux imbalance, inhibition of amino acid uptake, and a deficiency in acetate metabolism apparently are combined to cause metabolic stress by depleting the TCA cycle.     

Functional analysis of macrophage hybridomas. I. Production and initial characterization

A series of macrophage hybridomas were generated by fusion of splenic adherent cells with P388D1 tumor cells. Forty-two cell lines were established, and each was cloned by limiting dilution. Six clones that exemplified the spectrum of macrophage heterogeneity were selected for further analysis. Qualitative and quantitative differences in phenotype and functional activity were noted. Some clones constitutively expressed Ia antigens, whereas others only expressed detectable levels of Ia after lymphokine activation. The level of antigen-presenting activity generally correlated with the level of Ia expression. Furthermore, interclonal differences were noted in the levels of receptor-mediated phagocytosis and IL 1 secretion. Generally, the hybridoma clones maintained stable phenotypic and functional properties during approximately 1 yr of continuous in vitro culture. These cloned hybridoma cell lines represent a useful resource to analyze macrophage biology and to dissect structure and function relationships.     

Mutation detection in rice waxy mutants by PCR-RF-SSCP

PCR-RF-SSCP (PRS), which combines cleaved amplified polymorphic sequence (CAPS) and single-strand conformation polymorphism (SSCP), is expected to be a useful technique for DNA polymorphism analysis. We evaluated the ability of PRS to detect single nucleotide polymorphism (SNP) using the Waxy gene, Wx, of rice, and subsequently were able to identify point mutations in wx mutant lines. The approximately 6-kb Wx gene was divided into five regions for PCR amplification. Two regions, in which most of the point mutations of the wx mutants have been identified, were amplified by PCR and cloned into a vector, and those clones containing SNPs produced as a result of the inherent inaccuracy of PCR were used for the evaluation of PRS. The efficiency of PRS in the detection of SNPs of these clones was over 70%. PRS analysis of the wx genes in 18 waxy mutants was carried out in the five regions using two different restriction endonucleases and two gel conditions, with and without glycerol. Of the 18 lines tested, 17 showed band patterns different from that of the wild type. Most of the mutations identified in this study were nucleotide changes in exons, which result in amino acid changes. One mutation generated an in-frame stop codon, and another was a frame shift mutation by one-base deletion. Two mutations found at a splice site were considered to inhibit normal splicing of mRNA. These results show that PRS is a useful technique for detecting point mutations in large plant genes.     

Oxidation of glycerol,lactate, and propionate by Propionibacterium freudenreichii in a poised-potential amperometric culture system

Growth of Propionibacterium freudenreichii was studied with glycerol, lactate, and propionate as energy sources and a three-electrode poised-potential amperometric electrode system with hexacyanoferrate (III) as mediator. In batch culture experiments with glycerol and lactate as substrates, hexacyanoferrate (III) was completely reduced. Growth yields increased and the fermentation patterns were shifted towards higher acetate formation with increasing hexacyanoferrate (III) concentrations (0.25–8.0 mM). In experiments with regulated electrodes, glycerol, lactate, and propionate were oxidized to acetate and CO₂, and the electrons were quantitatively transferred to the working electrode. Growth yields of 29.0, 13.4 and 14.2 g cell material per mol were calculated, respectively. The high cell yield obtained during propionate oxidation cannot be explained solely by substrate level phosphorylation indicating that additional energy was conserved via electron transport phosphorylation. Furthermore, this result indicated complete reversibility of the methyl-malonyl-CoA pathway in propionic acid bacteria.     

Differences in the Utilization of Glycerol and Glucose by Mycobacterium phlei

A basic difference was found in the kinetics of uptake and utilization of glucose and glycerol by washed suspensions of Mycobacterium phlei. With glucose, the rates of uptake, respiration, and assimilation were saturated at low external substrate concentration. With glycerol, these rates were found to increase with increasing substrate concentration and did not show saturation at any concentration tested. Qualitatively similar patterns were observed for cells grown on either glycerol or glucose. Above a saturation concentration, ratios of cell (14)C to CO(2) (14)C for uniformly labeled (14)C-glucose were constant at a value of 0.96. Glycerol-U-(14)C, on the other hand, yielded cell-(14)C/CO(2)-(14)C ratios which were highest at the lowest glycerol concentration tested, and decreased with increasing substrate concentration. The distribution of the glucose and glycerol carbons assimilated into M. phlei were qualitatively similar. Quantitatively, however, the uptake and assimilation of glycerol was far more rapid than that of glucose for all substrate concentrations employed. These quantitative differences in the utilization of glycerol and glucose could account for the increased content of nonessential lipid and polysaccharide found in glycerol-grown M. phlei.     

Movement of acetate across the cytoplasmic membrane of the unicellular cyanobacteria Synechococcus and Aphanocapsa

Mutants of Synechococcus and of Aphanocapsa which were unable to activate acetate have been used to demonstrate that acetate entered the cells rapidly in darkness, and to a greater extent in light. Total internal concentrations under different conditions can be explained if acetic acid equilibrates rapidly across the cell membrane while acetate ion is strongly hindered. Acetate as well as other weak acids such as 5,5-dimethyl-2,4-oxazolidenedione can therefore be used as a probe of internal pH in these mutants. The intracellular pH was maintained at about 7.1 in darkness and 7.6 in light when external pH was varied from 6.8–8.0 No carrier involved in acetic acid equilibration could be demonstrated. Of other organic acids investigated, only propionate distributed in accordance with pH differences between the cells and surrounding fluid. The low uptake rates of glycolate, pyruvate and leucine appeared limited by slow movement of molecules into the cells.Abbreviations TEA Tricthanolamine - DMO 5,5-dimethyl-2,4-oxazolidenedione     

Growth, enzyme activity, sugar transport, and hormone supplement responses in cells cloned from a pig kidney cell line LLC-PK1

Three clones of the pig kidney cell line LLC-PK1 were isolated and characterized with regard to morphology, growth, proximal tubule enzyme activity, sugar uptake capacity, and hormone and drug responsiveness in a defined medium. Clone N4 was similar in morphology to the wild type (WT), whereas clone F8 showed loose attachment to the substrate, formed large, sweeping domes, and had an elongated desmosome junction between cells. The third clone, F2, did not form domes and showed a marked reduction in growth rate. Cultures of WT, N4, and F8 had higher specific activities of the enzyme alkaline phosphatase and gamma-glutamyl transpeptidase at confluence relative to growing cells; however, there was no evidence of an increase in activity of either enzyme at confluence in F2. Phlorizin-sensitive alpha-methyl-D-glucoside uptake and cytochalasin B-sensitive 2-deoxy-D-glucose uptake were measured in confluent cultures grown on porous filter supports. None of the clones lacked either of the hexose transport systems, although quantitative differences were evident. N4 cells grown in a defined medium in 96-well culture plates were tested in situ for their enzyme responses to differentiation inducers, tumor promoters, and hormones. Alkaline phosphatase activity was significantly increased at confluence by serum, parathyroid hormone (PTH), and vasopressin (AVP), and was decreased by tetradecanoylphorbol acetate (TPA) and epinephrine (EPI). Glutamyl transpeptidase activity was decreased at confluence by serum, TPA, and EPI. Similar tests on alpha-methyl-D-glucoside uptake showed that serum, TPA, PTH, and AVP had no significant effect on phlorizin-sensitive uptake; however, calcitonin increased uptake by 84% (n = 18). It was concluded that LLC-PK1 clones maintained in a defined medium are useful models for studying renal cell function.     

Substrate utilization by rat stomach in vivo. Arteriovenous differences for glucose, lactate, ketone bodies, fatty acids and glycerol under control and acid-secreting conditions.

Arteriovenous differences for several potential metabolic substrates were measured across the fundic wall of the stomach of rats that had been starved overnight. There was an uptake of glucose and D-3-hydroxybutyrate, but no significant arteriovenous differences for acetoacetate, pyruvate, non-esterified fatty acids and glycerol were apparent. Lactate output represented a substantial fraction of glucose uptake when the arterial lactate concentration was within the resting physiological range, but when the arterial lactate concentration was above 1.3 mM, lactate was taken up by the stomach. Stimulation of acid secretion by pentagastrin did not affect the value of arteriovenous differences. Thus blood flow to the fundic mucosa and substrate metabolism may be similarly enhanced by pentagastrin. It is concluded that metabolism of glucose and D-3-hydroxybutyrate, and to a lesser extent of glutamine and branched-chain amino acids [Anderson & Hanson (1983) Biochem. J. 210, 451-455], could supply energy to power acid secretion.     

Uptake and turnover of acetate in hypersaline environments

Abstract: Acetate uptake and turnover rates were determined for the heterotrophic community in hypersaline environments (saltern crystallizer ponds, the Dead Sea) dominated by halpphilic Archaea. Acetate was formed from glycerol, which is potentially the major available carbon source for natural communities of halophilic Archaea. Values of [ K _t+ S _n] (the sum of the substrate affinity and the substrate concentration present in situ) for acetate measured in saltern crystallizer ponds were around 4.5–11.5 μM, while in the Dead Sea during a Dunaliella bloom values up to 12.8 μM were found. Maximal theoretical rates ( V _max) of acetate uptake in saltern crystallizer ponds were 12–56 nmol l⁻¹ h⁻¹, with estimated turnover times for acetate ( T _t) between 127–730 h at 35°C. V _max values measured in the Dead Sea were between 0.8 and 12.8 nmol l⁻¹ h⁻¹, with turnover times in the range of 320–2190 h. V _max values for acetate were much lower than those for glycerol. Comparisons with pure cultures of halophilic Archaea grown under different conditions showed that the natural communities were not adapted for preferential use of acetate. Both in natural brines and in pure cultures of halophilic Archaea, acetate incorporation rates rapidly decreased above the optimum pH value, probably since acetate enters the cell only in its unionized form. The low affinity for acetate, together with low potential utilization rates result in the long acetate turnover times, which explains the accumulation of acetate observed when low concentrations of glycerol are supplied as a nutrient to natural communities of halophilic Archaea.     

Some properties of hepatic glycerol kinase and their relation to the control of glycerol utilization

1. Glycerol kinase (EC 2.7.1.30) is shown to catalyse a non-equilibrium reaction in rat liver; and, as it is the first enzyme in the pathway metabolizing glycerol, its properties may be pertinent to the metabolic regulation of glycerol uptake and utilization by this tissue. 2. The properties of hepatic glycerol kinase were studied by using a radiochemical technique to measure the enzyme activity. When the concentration of ATP is low the activity of glycerol kinase is inhibited by high concentrations of glycerol; but when the concentration of ATP is high there is no inhibition and the double-reciprocal plot is linear, providing a K(m) for glycerol of 3.16x10(-6)m. Glycerol kinase is activated by high ATP concentrations provided that the concentration of the second substrate (glycerol) is high; at low concentrations of glycerol ATP does not activate the enzyme so that the double-reciprocal plot is linear, providing a K(m) for ATP of 5.8x10(-5)m. It is suggested that these kinetics may be explained by a model similar to that described by Ferdinand (1966) for phosphofructokinase. 3. Hepatic glycerol kinase is inhibited by ADP and AMP, and raising the Mg(2+) concentration increases the inhibition by these two compounds; this suggests that ADP-Mg(2+) and AMP-Mg(2+) complexes are the inhibitory species. The physiological significance of these inhibitions may be to prevent phosphorylation of glycerol when the hepatic ATP concentration is low. It is suggested that this inhibition may provide an approach to the problem of measurement of rates of lipolysis by glycerol release in tissues that contain glycerol kinase (e.g. liver, kidney, muscle, adipose tissue). 4. Hepatic glycerol kinase is inhibited by l-3-glycerophosphate competitively with respect to glycerol. The physiological significance of this inhibition may be that factors that change the intracellular concentration of l-3-glycerophosphate could change glycerol uptake by the tissue. Thus it is suggested that thyroxine treatment or feeding rats on a diet high in glycerol, which increase the activity of glycerophosphate oxidase in liver and kidney cortex respectively, lead to an increased glycerol uptake through a decrease in the concentration of glycerophosphate in these tissues. It is known that ethanol administration decreases glycerol uptake by liver, and this can be explained by the increased concentration of l-3-glycerophosphate causing inhibition of glycerol kinase.     

Role of cyclic adenosine 3',5'-monophosphate-dependent protein kinase in hormone-stimulated beta-endorphin secretion in AtT20 cells.

Secretion of beta-endorphin from mouse pituitary AtT20 cells is stimulated by a variety of compounds that raise intracellular cAMP and Ca2+. To investigate the role of cAMP-dependent protein kinases in secretion, AtT20 cells were transfected with an expression vector coding for a regulatory (R) subunit of cAMP-dependent protein kinase containing mutations in both cAMP-binding sites. Expression of the mutant regulatory subunit in stable transformants (RAB cells) results in a dominant inhibition of cAMP-dependent protein kinase activity. Isoproterenol (1 microM) or analogs of cAMP stimulated beta-endorphin secretion from AtT20 cells, but failed to stimulate secretion in RAB cells expressing the mutant R subunit. Secretion in response to CRF (100 nM) was inhibited by 80% in these mutant clones, whereas the secretory response to vasoactive intestinal peptide (VIP; 100 nM) or phorbol ester (100 nM phorbol myristate acetate) was not inhibited by the R subunit mutation. Intracellular cAMP was elevated in response to CRF (11- to 15-fold), isoproterenol (5- to 10-fold), and VIP (4- to 8-fold) in RAB cells. Similar concentrations of VIP were required to evoke beta-endorphin secretion in either RAB cells or AtT20 cells. As with most secretagogues, VIP-induced secretion was inhibited in the presence of either EGTA or a voltage-sensitive Ca2+ channel antagonist, PN200-110. The secretory response to VIP was unaffected by down-regulation of protein kinase-C. These results suggest that CRF and isoproterenol work via cAMP-dependent protein kinase to activate beta-endorphin secretion, whereas VIP can act by a different mechanism that does not involve cAMP-dependent protein kinase or protein kinase-C.     

Identification and characterization of coenzyme B12-dependent glycerol dehydratase- and diol dehydratase-encoding genes from metagenomic DNA libraries derived from enrichment cultures

To isolate genes encoding coenzyme B(12)-dependent glycerol and diol dehydratases, metagenomic libraries from three different environmental samples were constructed after allowing growth of the dehydratase-containing microorganisms present for 48 h with glycerol under anaerobic conditions. The libraries were searched for the targeted genes by an activity screen, which was based on complementation of a constructed dehydratase-negative Escherichia coli strain. In this way, two positive E. coli clones out of 560,000 tested clones were obtained. In addition, screening was performed by colony hybridization with dehydratase-specific DNA fragments as probes. The screening of 158,000 E. coli clones by this method yielded five positive clones. Two of the plasmids (pAK6 and pAK8) recovered from the seven positive clones contained genes identical to those encoding the glycerol dehydratase of Citrobacter freundii and were not studied further. The remaining five plasmids (pAK2 to -5 and pAK7) contained two complete and three incomplete dehydratase-encoding gene regions, which were similar to the corresponding regions of enteric bacteria. Three (pAK2, -3, and -7) coded for glycerol dehydratases and two (pAK4 and -5) coded for diol dehydratases. We were able to perform high-level production and purification of three of these dehydratases. The glycerol dehydratases purified from E. coli Bl21/pAK2.1 and E. coli Bl21/pAK7.1 and the complemented hybrid diol dehydratase purified from E. coli Bl21/pAK5.1 were subject to suicide inactivation by glycerol and were cross-reactivated by the reactivation factor (DhaFG) for the glycerol dehydratase of C. freundii. The activities of the three environmentally derived dehydratases and that of glycerol dehydratase of C. freundii with glycerol or 1,2-propanediol as the substrate were inhibited in the presence of the glycerol fermentation product 1,3-propanediol. Taking the catalytic efficiency, stability against inactivation by glycerol, and inhibition by 1,3-propanediol into account, the hybrid diol dehydratase produced by E. coli Bl21/pAK5.1 exhibited the best properties of all tested enzymes for application in the biotechnological production of 1,3-propanediol.     

Insulin affects glucose uptake by muscle and mammary tissues of lactating ewes

Effects of insulin on exchanges of glucose across skeletal muscle and mammary tissue were measured in short-term studies in lactating ewes. Insulin secretion was suppressed by a primed/continuous infusion of somatostatin, then insulin was administered by continuous intravenous infusion of doses that were increased, in a step-wise manner, from 0 to 2 U h-1. Plasma glucose was maintained essentially constant by frequent monitoring and intravenous administration of exogenous glucose. Somatostatin suppressed but did not completely inhibit insulin secretion as shown by maintenance of plasma concentration of C-peptide. As plasma insulin was increased, while arterial glucose was maintained stable, uptake of glucose by skeletal muscle increased and glucose uptake by the mammary gland decreased. These observations confirm the role of insulin in regulating glucose uptake by skeletal muscle and raise the possibility that insulin also regulates glucose uptake by the mammary gland.     

Potassium Fluxes on Hyperosmotic Shock and the Effect of Phenol and Bronopol (2-bromo-2-nitropropan-1,3-diol) on Deplasmolysis of Pseudomonas aeruginosa

Potassium fluxes and the effect of phenol and bronopol on deplasmolysis of Pseudomonas aeruginosa were followed in sucrose and glycerol plasmolysing systems.
In sucrose, K⁺ uptake related to the solute concentration. Proline increased the rate and overall K⁺ uptake, the latter by a factor of three. It was concluded that there was no rigid maximum in the accumulation of intracellular K⁺ as long as intracellular neutrality in electrical charges was maintained.
In glycerol, K⁺ uptake was parallel with glycerol penetration. The process was reversed, however, on equilibration of glycerol. This suggested that glycerol inhibited K⁺ retention against a concentration gradient rather than that K⁺ was excluded as a consequence of the osmotic established steady state. This view was enforced by the fact that the reversal of K⁺ uptake occurred in 20 and 30% glycerol but not in 10%.
Phenol and bronopol did not affect deplasmolysis in glycerol significantly, although some effect on K⁺ uptake and glycerol permeability could be seen. In the sucrose system, phenol acted according to its mode of action generally accepted, i.e. inhibiting deplasmolysis at low and allowing solute penetration at higher concentrations, whereas very high concentrations caused coagulation of the cytoplasm. Bronopol inhibited deplasmolysis, except at very low concentrations. Proline did not prevent the inhibition of deplasmolysis in either of the solute systems, except at the very low bronopol concentrations where the deplasmolysis rate only was affected.     

Isomeric lipid signatures reveal compartmentalized fatty acid metabolism in cancer

The cellular energy and biomass demands of cancer drive a complex dynamic between uptake of extracellular FAs and their de novo synthesis. Given that oxidation of de novo synthesized FAs for energy would result in net-energy loss, there is an implication that FAs from these two sources must have distinct metabolic fates; however, hitherto, all FAs have been considered part of a common pool. To probe potential metabolic partitioning of cellular FAs, cancer cells were supplemented with stable isotope-labeled FAs. Structural analysis of the resulting glycerophospholipids revealed that labeled FAs from uptake were largely incorporated to canonical (sn-) positions on the glycerol backbone. Surprisingly, labeled FA uptake also disrupted canonical isomer patterns of the unlabeled lipidome and induced repartitioning of n-3 and n-6 PUFAs into glycerophospholipid classes. These structural changes support the existence of differences in the metabolic fates of FAs derived from uptake or de novo sources and demonstrate unique signaling and remodeling behaviors usually hidden from conventional lipidomics.     

Genetic basis of growth adaptation of Escherichia coli after deletion of pgi, a major metabolic gene

Bacterial survival requires adaptation to different environmental perturbations such as exposure to antibiotics, changes in temperature or oxygen levels, DNA damage, and alternative nutrient sources. During adaptation, bacteria often develop beneficial mutations that confer increased fitness in the new environment. Adaptation to the loss of a major non-essential gene product that cripples growth, however, has not been studied at the whole-genome level. We investigated the ability of Escherichia coli K-12 MG1655 to overcome the loss of phosphoglucose isomerase (pgi) by adaptively evolving ten replicates of E. coli lacking pgi for 50 days in glucose M9 minimal medium and by characterizing endpoint clones through whole-genome re-sequencing and phenotype profiling. We found that 1) the growth rates for all ten endpoint clones increased approximately 3-fold over the 50-day period; 2) two to five mutations arose during adaptation, most frequently in the NADH/NADPH transhydrogenases udhA and pntAB and in the stress-associated sigma factor rpoS; and 3) despite similar growth rates, at least three distinct endpoint phenotypes developed as defined by different rates of acetate and formate secretion. These results demonstrate that E. coli can adapt to the loss of a major metabolic gene product with only a handful of mutations and that adaptation can result in multiple, alternative phenotypes.