Functional capacities and the adenylate energy charge in Escherichia coli under conditions of nutritional stress.

Functional capacities in Escherichia coli cells starved for glucose were examined by comparing protein synthesis, utilization of new substrates, and maintenance of viability with the adenylate energy charge of the culture. When growth ceased because of glucose exhaustion in an E. coli culture, the energy charge dropped from 0.90 to about 0.80. During this time, the viable-cell count and the capacity for protein synthesis and for induction of new enzymes were maintained only if other substrates were available in the medium. The culture could be maintained for many hours without growth or death if glucose was added slowly; the energy charge in this case stabilized at about 0.80. A consistent transient decrease in the energy charge to around 0.80, accompanied by a decrease in protein synthesis, was also observed during the adaptation from glucose to other substrates during diauxic growth on glucose and glycerol or lactose.     

The Response of Sugarcane (Saccharum officinarum) Cultured Cells to Anoxia and the Selection of a Tolerant Cell Line

The effect of anoxia on the sugarcane (Saccharum officinarum L.) cultured cells was studied in order to elaborate a technique for in vitro selection of cell lines, which would be tolerant to anaerobic stress. Inhibitory and lethal doses of anaerobic incubation were established from the state of the mitochondrial ultrastructure during the anaerobic incubation of cells either with or without exogenous glucose, as well as from the pattern of the post-anaerobic callus growth. An intact state of the mitochondrial ultrastructure and the viability of some cells in the presence of 3% glucose were shown to be maintained for at least 14 days of anaerobic incubation, while the index of post-anaerobic growth decreased by almost 50% even after 72-hour-long anaerobiosis. In the absence of exogenous glucose, a marked destruction of mitochondria and a twofold decrease in the callus growth index were observed as early as after six-hour-long anaerobic stress. A 48-hour-long incubation under these conditions resulted in the maintenance of the intact ultrastructure only in 7–10% of cells, while a 96-hour-long anaerobiosis brought about the complete degradation of the subcellular structure and cell death. A 48-hour-long anaerobiosis without exogenous glucose was chosen for selecting the anoxia-tolerant cell lines. After three cycles of selection, the anoxia tolerance of the selected cell line exceeded the respective index of the initial callus several-fold. In selected line, about 50% of cells retained viability and could resume growth even after 96-hour-long anaerobic incubation. The experimental results obtained were used to determine the possible causes of the heterogeneity of callus cells as regards their anoxia resistance.     

Evidence Relating Cessation of Respiration, Cell Envelope Changes, and Death in Ultraviolet-Irradiated Escherichia coli B/r Cells

Ionic and nonionic detergents have little effect on respiring bacteria, but in cultures poisoned with KCN rapid solubilization of the cell membrane, as indicated by turbidity losses, takes place. Ultraviolet radiations cause Escherichia coli cells grown in minimal medium with glycerol as a carbon source to cease respiring and growing about 1 h after irradiation. We tested the effect of the nonionic detergent Triton X-100 on growth and cell membrane dissolution (both measured by turbidity changes), respiration, and viability of unirradiated and irradiated E. coli B/r cells. When the detergent was added to cells immediately after irradiation, a decrease in turbidity occurred only when respiration was about to cease; when it was added after cessation of respiration, the turbidity loss was immediate. In both cases the turbidity loss was about 60%, and disintegration of the cell walls did not take place. 5-Fluorouracil (FU) and thermal (42 C) treatments cause respiration of irradiated cells to be maintained and also cause viability increases. Irradiated cells treated with FU and detergent show no turbidity loss just prior to the time respiration normally ceases, but a loss does occur in irradiated cells incubated with detergent at 42 C. We conclude that FU maintains respiration for all of the cells, but that thermal treatment maintains respiration for only part of the cells. In all cases the detergent had only a negligible effect on the respiration and viability of unirradiated and irradiated cells. We conclude that Triton X-100 causes solubilization of cell membranes of only nonrespiring cells that are not destined to survive.     

Aging of Escherichia coli

Clifton, C. E. (Stanford University, Stanford, Calif.). Aging of Escherichia coli. J. Bacteriol. 92:905-912. 1966.-The rates of endogenous and exogenous (glucose) respiration decreased much more rapidly than did the viable count during the first 24 hr of aging of washed, C(14)-labeled cells of Escherichia coli K-12 suspended in a basal salt medium devoid of ammonium salts. The rates of decrease of respiration and of death approached each other as the age of the cells increased, but death was not the only factor involved in decreased respiratory activity of the suspensions. The greatest decrease in cellular contents with aging was noted in the ribonucleic acid fraction, of which the ribose appeared to be oxidized, while uracil accumulated in the suspension medium. The viable count and respiratory activities remained higher in glucose-fed than in nonfed suspensions. Proline-labeled cells fed glucose tended to lose more of their proline and to convert more proline into C(14)O(2) than in unfed controls. On the other hand, uracil-labeled cells fed glucose retained more of the uracil than did nonfed cells, but glucose elicited some oxidation of uracil. An exogenous energy source such as glucose aided in the maintenance of a population, but it was not the only factor needed for such maintenance.     

Proton motive force is not obligatory for growth of Escherichia coli.

When 50 microM carbonyl cyanide-m-chlorophenyl hydrazone (CCCP), a protonophore, was added to growth medium containing glucose at pH 7.5, Escherichia coli TK1001 (trkD1 kdpABC5) started exponential growth after 30 min; the generation time was 70 min at 37 degrees C. Strain AS1 (acrA), another strain derived from E. coli K-12, also grew in the presence of 50 microM CCCP under the same conditions, except that the lag period was ca. 3 h. When this strain was grown in the presence of 50 microM CCCP and then transferred to fresh medium containing 50 microM CCCP, cells grew without any lag. Neither a membrane potential nor a pH gradient was detected in strain AS1 cells growing in the presence of CCCP. When either succinate or lactate was substituted for glucose, these strains did not grow in the presence of 50 microM CCCP. Thus, it is suggested that E. coli can grow in the absence of a proton motive force when glucose is used as an energy source at pH 7.5.     

Comparative growth analysis of the facultative anaerobes Bacillus subtilis,Bacillus licheniformis,and Escherichia coli

Bacillus subtilis anaerobic respiration and fermentative growth capabilities were compared to two other facultative anaerobes, Bacillus licheniformis and Escherichia coli. In glycerol defined medium, B. subtilis grew with nitrate, but not nitrite or fumarate, while B. licheniformis grew with nitrate or fumarate, but not nitrite. Growth of E. coli occurred in glycerol defined medium with either nitrate, nitrite, or fumarate. In order to grow by fermentation, B. subtilis required both glucose and pyruvate, while B. licheniformis and E. coli were capable of using either glucose or pyruvate.     

Relaxation of catabolite repression in streptomycin-dependent Escherichia coli

Acetohydroxy acid synthetase, which is sensitive to catabolite repression in wild-type Escherichia coli B, was relatively resistant to this control in a streptomycin-dependent mutant. The streptomycin-dependent mutant was found to be inducible for beta-galactosidase in the presence of glucose, although repression of beta-galactosidase by glucose occurred under experimental conditions where growth of the streptomycin-dependent mutant was limited. Additional glucose-sensitive enzymes of wild-type E. coli B (citrate synthase, fumarase, aconitase and isocitrate dehydrogenase) were found to be insensitive to the carbon source in streptomycin-dependent mutants: these enzymes were formed by streptomycin-dependent E. coli B in equivalent quantities when either glucose or glycerol was the carbon source. Two enzymes, glucokinase and glucose 6-phosphate dehydrogenase, that are glucose-insensitive in wild-type E. coli B were formed in equivalent quantity on glucose or glycerol in both streptomycin-sensitive and streptomycin-dependent E. coli B. The results indicate a general decrease or relaxation of catabolite repression in the streptomycin-dependent mutant. The yield of streptomycin-dependent cells from glucose was one-third less than that of the streptomycin-sensitive strain. We conclude that the decreased efficiency of glucose utilization in streptomycin-dependent E. coli B is responsible for the relaxation of catabolite repression in this mutant.     

Regulation of glucosamine utilization in Staphylococcus aureus and Escherichia coli.

Glucosamine- or N-acetylglucosamine-requiring mutants of Staphylococcus aureus 209P and Escherichia coli K12, which lack glucosamine-6-phosphate synthetase [2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase (amino-transferring); EC 5.3.1.19], were isolated. Growth of these mutants on glucosamine was inhibited by glucose, but growth on N-acetylglucosamine was not. Addition of glucose to mutant cultures growing exponentially on glucosamine inhibited growth and caused death of bacteria, though chloramphenicol prevented death. Uptake of glucosamine by S. aureus and E. coli mutants was severely inhibited by glucose whereas uptake of N-acetylglucosamine was only slightly inhibited. Uptake of glucose was not inhibited by either glucosamine or N-acetylglucosamine. In glucosamine auxotrophs, glucose causes glucosamine deficiency which interrupts cell wall synthesis and results in some loss of viability in the presence of continued protein synthesis.     

Properties of a Mutant of Escherichia coli with a Temperature-sensitive Fructose-1,6-Diphosphate Aldolase

B?ck, August (Purdue University, Lafayette, Ind.), and Frederick C. Neidhardt. Properties of a mutant of Escherichia coli with a temperature-sensitive fructose-1,6-diphosphate aldolase. J. Bacteriol. 92:470-476. 1966.-A mutant of Escherichia coli in which fructose-1,6-diphosphate aldolase functions at 30 C but not at 40 C was used to study the physiological effect of a specific block in the Embden-Meyerhof glycolytic pathway. Growth of the mutant at 40 C was found to be inhibited by the presence of glucose or certain related compounds in the medium. At 40 C, glucose was metabolized at 30 to 40% of the control rate and was abnormal in that glucose was converted into other six-carbon substances (probably gluconate, in large part) that were released into the culture medium. The inhibition was complete, but transient; its duration depended upon the initial amount of inhibitor added. The resumption of growth at 40 C was correlated with the further catabolism of the excreted compounds. When glycerol was used to grow the mutant at 40 C, the growth inhibition by glucose was accompanied by cessation of glycerol metabolism. Growth on alpha-glycerol phosphate was not inhibited under these conditions, implicating glycerol kinase as a possible site of inhibition; no inhibition of glycerol kinase by sugar phosphates, however, could be detected in vitro. The inhibitory effect of glucose on growth at 40 C is not caused by a deficit of intracellular adenosine triphosphate, but may be the result of a generalized poisoning of many cell processes by a greatly increased intracellular concentration of fructose-1,6-diphosphate, the substrate of the damaged enzyme.     

Isolation of a Mutant of Escherichia coli with a Temperature-sensitive Fructose-1,6-Diphosphate Aldolase Activity

B?ck, August (Purdue University, Lafayette, Ind.), and Frederick C. Neidhardt. Isolation of a mutant of Escherichia coli with a temperature-sensitive fructose-1,6-diphosphate aldolase activity. J. Bacteriol. 92:464-469. 1966.-A mutant of Escherichia coli was isolated which was able to grow in rich medium at 30 C but not at 40 C. Upon exposure to 40 C, the cells immediately stopped ribonucleic acid (RNA) and deoxyribonucleic acid synthesis, but protein synthesis continued at a diminished rate for a short time. Addition of chloramphenicol did not release RNA synthesis from inhibition at 40 C. Synthesis of beta-galactosidase could be induced at high temperature despite the presence of glucose in the medium, indicating a lesion in glucose catabolism. Of many catabolic enzymes tested in cell-free extracts, only fructose-1,6-diphosphate aldolase activity appeared to be altered in the mutant cells. No activity was demonstrable in extracts of mutant cells grown at either 30 or 40 C, but determination of glucose-oxidation patterns revealed that the enzyme is probably active in vivo at 30 C. Temperature-resistant secondary mutants were found to have partially or fully restored aldolase activity, and temperature-resistant recombinants had normal aldolase activity, indicating that the growth pattern and the altered aldolase had a common genetic basis. Linkage data permitted the assignment of an approximate map location for the mutated aldolase gene.     

环化腺苷酸对细菌生长的影响

用大肠杆菌(Escherichia coli AS 1.797)、北京棒状杆菌(Corynebacterium pekinense AS1.299)和巨大芽孢杆菌(Bacills megatertum AS 1.217)研究了细胞内环化腺苷酸(cAMP)浓度和外源cAMP对细胞生长的影响。结果表明,大肠杆菌在不同碳源中生长时,细胞的生长量随细胞内cAMF’浓度升高而降低。在以葡萄糖作碳源时,细胞内cAMP浓度低,外源cAMP。对生长有抑制作用,而cAMP的类似物5'-AMP则无抑制作用。在以乳糖、麦芽糖和甘油分别作碳源时,细胞内cAMP浓度高,外源cAMP对生长无影响。北京捧状杆菌以葡萄糖作碳源时,细胞生长也受外源cAMP的抑制,但cAMP的抑制作用不是专一的,它的作用可用类似物5’-AMP来代替。自身不合cAMP的巨大芽孢杆菌在不同碳原(包括葡萄糖)中生长时,生长不受外源cAMP抑制,也不受5’-Amt’的影响。因此认为,cAMP不是细菌生长的必需物,而是生长调节物,但这种调节物对巨大芽孢杆菌无效。     

Roles of glucose and acetate as carbon sources in<Emphasis Type="SmallCaps">l</Emphasis>-histidine production with<Emphasis Type="Italic">Brevibacterium flavum</Emphasis> FERM1564 revealed by metabolic flux analysis

The metabolic flux pattern forl-histidine production was analyzed when glucose and/or acetate were used as carbon sources. Totall-histidine production was enhanced when mixed substrate (glucose and acetate) was used, compared with that when either glucose or acetate was used as the sole carbon source. Theoretical maximum carbon fluxes through the main pathways forl-histidine production, cell growth, and ATP consumption for cell maintenance were obtained by the linear programming (LP) method. By comparison of the theoretical maximum carbon fluxes with actual ones, it was found that a large amount of glucose was actually used for maintenance of cell viability. On the other hand, acetate was used for cell growth. After depletion of acetate in the mixed substrate culture, the flux for glucose tol-histidine synthesis was markedly enhanced. A strategy for effectivel-histidine production using both carbon sources was proposed.     

Determination of deoxyribonucleic acid replication time in exponentially growing Escherichia coli B/r.

The time necessary to replicate the chromosome (C period) was measured in Escherichia coli B/r (ATCC 12407) and a low-thymine-requiring derivative of that strain. In the Thy- strain, C was measured as a function of growth rate and exogenous thymine concentration either from step-up or chloramphenicol experiments. In the Thy+ parental strain, C was measured only as a function of the growth rate and only by the chloramphenicol method. The C period was found to decrease with growth rate and, in the Thy- strain, the C period also decreased with increasing thymine concentration. It approached a value of approximately 37 min at high growth rates.     

Relation of cell growth and colicin tolerance to vitamin B12 uptake in Escherichia coli.

The uptake of vitamin B12 was measured in cells of Escherichia coli whose growth had been inhibited by any of a variety of treatments. In all cases, the secondary, energy-dependent phase of B12 uptake was depressed in proportion to the decrease in growth rate, but uptake was constant in cells growing logarithmically at different rates. The depression of B12 uptake activity was independent of the site of cell metabolism affected by the inhibitor or by its effect on cell viability, and was both more rapid and of greater degree than the effects on the uptake of any of the six amino acids tested. The decline was not affected by inhibitors of either cell division or proteolysis and was manifested without any apparent decrease in the surface B12 binding activity. Transport activity was rapidly regained upon reversal of the inhibition of protein synthesis. Prompted by this response, the uptake of B12 was contrasted to the apparent uptake of the E colicins, which share the same outer membrane receptor. Sensitivity to colicin E1, measured by its inhibition of proline uptake, was not affected by growth inhibition by antibiotic treatment. Finally, there was no specific depression of B12 uptake in cells rendered colicin tolerant either by mutation or as a consequence of phage f1 infection.     

Between feast and famine: endogenous inducer synthesis in the adaptation of Escherichia coli to growth with limiting carbohydrates.

Escherichia coli adapted to growth with low carbohydrate concentrations bypassed the requirement for exogenous inducer with at least three well-studied sugar regulons. Induction of mgl and gal genes became independent of added galactose in bacteria approaching stationary phase or during continuous culture with micromolar glucose in the medium. Bacteria became independent of exogenous induction because endogenous galactose and cyclic AMP (cAMP) pools were sufficient for high expression of mgl and gal genes under glucose limitation. Limitation-stimulated induction of mgl was dependent on a functional galETK operon for synthesis of the inducer galactose. Intracellular galactose levels were maximal not during starvation (or slow steady-state growth rates approaching starvation) but at fast growth rates with micromolar glucose. The extent of mgl/gal induction correlated better with inducer availability than with cAMP concentrations under all conditions tested. Endogenous inducer accumulation represents an adaptation to low-nutrient environments, leading to derepression of high-affinity transport systems like Mgl essential for bacterial competitiveness at low nutrient concentrations.     

Role of protein synthesis in the survival of carbon-starved Escherichia coli K-12.

In a typical Escherichia coli K-12 culture starved for glucose, 50% of the cells lose viability in ca. 6 days (Reeve et al., J. Bacteriol. 157:758-763, 1984). Inhibition of protein synthesis by chloramphenicol resulted in a more rapid loss of viability in glucose-starved E. coli K-12 cultures. The more chloramphenicol added (i.e., the more protein synthesis was inhibited) and the earlier during starvation it was added, the greater was its effect on culture viability. Chloramphenicol was found to have the same effect on a relA strain as on an isogenic relA+ strain of E. coli. Addition of the amino acid analogs S-2-aminoethylcysteine, 7-azatryptophan, and p-fluorophenylalanine to carbon-starved cultures to induce synthesis of abnormal proteins had an effect on viability similar to that observed when 50 micrograms of chloramphenicol per ml was added at zero time for starvation. Both chloramphenicol and the amino acid analogs had delayed effects on viability, compared with their effects on synthesis of normal proteins. The need for protein synthesis did not arise from cryptic growth, since no cryptic growth of the starving cells was observed under the conditions used. From these and previous results obtained from work with peptidase-deficient mutants of E. coli K-12 and Salmonella typhimurium LT2 (Reeve et al., J. Bacteriol. 157:758-763, 1984), we concluded that a number of survival-related proteins are synthesized by E. coli K-12 cells as a response to carbon starvation. These proteins are largely synthesized during the early hours of starvation, but their continued activity is required for long-term survival.     

Aerobic growth and respiration of a delta-aminolevulinic acid synthase (hemA) mutant of Bradyrhizobium japonicum.

Oxygen-dependent growth of the Bradyrhizobium japonicum hemA mutant MLG1 (M.L. Guerinot and B.K. Chelm, Proc. Natl. Acad. Sci. USA 83:1837-1841, 1986) was demonstrated in cultured cells in the absence of exogenous delta-aminolevulinic acid (ALA), but growth of analogous mutants of Rhizobium meliloti or of Escherichia coli was not observed unless ALA was added to the yeast extract-containing media. No heme could be detected in extracts of strain MLG1 cells as measured by the absorption or by the peroxidase activity of the heme moiety, but the rates of growth and endogenous respiration of the mutant were essentially identical to those found in the parent strain. A role for ALA in the viability of strain MLG1 could not be ruled out since the ALA analog levulinic acid inhibited growth, but neither ALA synthase nor glutamate-dependent ALA synthesis activity was found in the mutant. The data show that the cytochromes normally discerned in wild-type B. japonicum cultured cells by absorption spectroscopy are not essential for aerobic growth or respiration.     

The physiological effects and metabolic alterations caused by the expression of Rhizobium etli pyruvate carboxylase in Escherichia coli

Oxaloacetate (OAA) plays an important role in the tricarboxylic acid cycle and for the biosynthesis of a variety of cellular compounds. Some microorganisms, such as Rhizobium etli and Corynebacterium glutamicum, are able to synthesize OAA during growth on glucose via either of the enzymes pyruvate carboxylase (PYC) or phosphoenolpyruvate carboxylase (PPC). Other microorganisms, including Escherichia coli, synthesize OAA during growth on glucose only via PPC because they lack PYC. In this study we have examined the effect that the R. etli PYC has on the physiology of E. coli. The expressed R. etli PYC was biotinylated by the native biotin holoenzyme synthase of E. coli and displayed kinetic properties similar to those reported for alpha4 PYC enzymes from other sources. R. etli PYC was able to restore the growth of an E. coli ppc null mutant in minimal glucose medium, and PYC expression caused increased carbon flow towards OAA in wild-type E. coli cells without affecting the glucose uptake rate or the growth rate. During aerobic glucose metabolism, expression of PYC resulted in a 56% increase in biomass yield and a 43% decrease in acetate yield. During anaerobic glucose metabolism, expression of PYC caused a 2.7-fold increase in succinate concentration, making it the major product by mass. The increase in succinate came mainly at the expense of lactate formation. However, in a mutant lacking lactate dehydrogenase activity, expression of PYC resulted in only a 1.7-fold increase in succinate concentration. The decreased enhancement of succinate formation in the /dh mutant was hypothesized to be due to accumulation of pyruvate and NADH, metabolites that affect the interconversion of the active and inactive form of the enzyme pyruvate formate-lyase.     

Removal of inhibitors of bacterial growth by dialysis culture.

Dialysis culture was used to investigate the extent to which growth inhibition in bacterial cultures may be caused by accumulation of metabolites. Escherichia coli B was grown in a glucose/salts medium. A concentrated nutrient solution was pumped at a constant rate into the growing culture to ensure that growth was not limited by exhaustion of nutrients. In this way the only difference between growth conditions in dialysis and non-dialysis cultures was the transfer of dialysable metabolites from the culture vessel to the reservoir in the dialysis culture system. By adjusting the glucose concentration in the feed and maintaining a constant rate of feeding, glucose-limited growth could be achieved. Under these conditions, with oxygen in excess, bacterial yields of 140 to 150 g dry wt l-1 were obtained in dialysis culture compared with 30 to 40 g l-1 in non-dialysis culture. The high yields in dialysis culture depended on the removal of end-products of glucose metabolism. Growth inhibition was demonstrated to be the result of the combined influence of acetate, lactate, pyruvate, succinate, propionate and isobutyrate in concentrations found at the end of growth in non-dialysis cultures of Escherichia coli B.     

The glucose permease of the phosphotransferase system of Bacillus subtilis: evidence for IIGlc and IIIGlc domains

Glucose is taken up in Bacillus subtilis via the phosphoenolpyruvate:glucose phosphotransferase system (glucose PTS). Two genes, orfG and ptsX, have been implied in the glucose-specific part of this PTS, encoding an Enzyme IIGlc and an Enzyme IIIGlc, respectively. We now show that the glucose permease consists of a single, membrane-bound, polypeptide with an apparent molecular weight of 80,000, encoded by a single gene which will be designated ptsG. The glucose permease contains domains that are 40-50% identical to the IIGlc and IIIGlc proteins of Escherichia coli. The B. subtilis IIIGlc domain can replace IIIGlc in E. coli crr mutants in supporting growth on glucose and transport of methyl alpha-glucoside. Mutations in the IIGlc and IIIGlc domains of the B. subtilis ptsG gene impaired growth on glucose and in some cases on sucrose. ptsG mutants lost all methyl alpha-glucoside transport but retained part of the glucose-transport capacity. Residual growth on glucose and transport of glucose in these ptsG mutants suggested that yet another uptake system for glucose existed, which is either another PT system or regulated by the PTS. The glucose PTS did not seem to be involved in the regulation of the uptake or metabolism of non-PTS compounds like glycerol. In contrast to ptsl mutants in members of the Enterobacteriaceae, the defective growth of B. subtilis ptsl mutants on glycerol was not restored by an insertion in the ptsG gene which eliminated IIGlc. Growth of B. subtilis ptsG mutants, lacking IIGlc, was not impaired on glycerol. From this we concluded that neither non-phosphorylated nor phosphorylated IIGlc was acting as an inhibitor or an activator, respectively, of glycerol uptake and metabolism.