Intracellular accumulation of potassium and glutamate specifically enhances survival of Escherichia coli in seawater.

The high resistance of Escherichia coli grown in saline media to seawater was suppressed by an osmotic down-shock. The shock released several molecules into the medium, including potassium, glutamate, and glycine betaine when cells were previously grown in the presence of this osmolyte. Incubation of such sensitized cells in a solution containing K+ (80 mM) and glutamate (50 mM) at pH 7.4 restored their resistance to seawater up to a level close to that observed initially. The protective effect was partly due to the rapid accumulation of K+; a significant exponential relationship between intracellular concentration of K+ and resistance to seawater was observed. Glutamate was accumulated more slowly and progressively completed the action of K+. These data emphasize the specific influence of potassium glutamate on osmotically stressed E. coli cells. They confirm that regulation of osmotic pressure and, probably, of intracellular pH strongly enhances survival of E. coli in seawater. Osmotic fluctuations in waters carrying enteric bacteria from intestines to seawater, together with variations in their K+ and amino acid contents, could modify the ability of cells to survive in marine environments. These results demonstrate the need to strictly control conditions (K+ content, temperature) used to wash cells before their transfer to seawater microcosms. They suggest that the K+ and glutamate contents of media in which E. coli cells are transported to the sea can influence their subsequent survival in marine environments.     

Influence of osmoregulation processes on starvation survival of Escherichia coli in seawater.

The adaptation of enteric bacteria in seawater has previously been described in terms of nutrient starvation. In the present paper, we bring experimental arguments suggesting that survival of these microorganisms could also depend on their ability to overcome the effects of osmotic stress. We analyzed the influence of osmoregulatory mechanisms (potassium transport, transport and accumulation of organic osmolytes) on the survival of Escherichia coli in seawater microcosms by using mutants lacking components of the osmotic stress response. Long-term protection was afforded to cells by growth in a medium whose osmotic pressure was increased by either NaCl, LiCl, or saccharose. Achievement of the protection state depended at least partly on osmoregulatory mechanisms, but differed when these were activated or induced during prior growth or in resting cells suspended in phosphate buffer or in seawater. When achieved during growth, K+ transport, glycine-betaine (GBT) synthesis or transport, and trehalose synthesis helped increase the ability to survive in seawater. Protection by GBT was also obtained with resting cells in a phosphate buffer at high osmotic pressure. However, when added only to the seawater, GBT did not change the survival ability of cells no matter what their osmoregulation potential. These results showed that the survival of E. coli cells in seawater depends, at least partly, on whether they possess certain genes which enable them to regulate osmotic pressure and whether they can be stimulated to express those genes before or after their release into the environment. This expression requires nutrients as the substrates from which the corresponding gene products are made.     

Intracellular Trp repressor levels in Escherichia coli.

A radioimmunoassay for the Trp repressor protein of Escherichia coli was developed with antisera raised against purified Trp repressor protein. This assay was used to directly measure the intracellular Trp repressor content in several E. coli K-12 and B/r strains. Repressor levels varied from 2.5- to 3-fold in response to L-tryptophan concentration in the growth medium (15 to 44 ng of repressor per mg of protein). Neither cell growth rate nor culture age had a significant effect on repressor concentrations within the cell. Addition of L-tryptophan to the growth medium resulted in lowered intracellular levels of Trp repressor. The absolute amounts of native Trp repressor molecules per cell varied between 120 and 375 dimers in the presence and absence of L-tryptophan in the culture medium, respectively. Assuming an intracellular volume of 7.3 microliters/10(10) E. coli cells, the Trp repressor concentration varied from 270 to 850 nM in response to extracellular tryptophan levels. These findings represent the first direct measurements of Trp repressor levels in E. coli and confirm the autoregulatory nature of the trpR gene.     

Intracellular activation of albomycin in Escherichia coli and Salmonella typhimurium.

The antibiotic albomycin is actively taken up by Escherichia coli via the transport system for the structurally similar iron complex ferrichrome. Albomycin is cleaved, and the antibiotically active moiety is released into the cytoplasm, whereas the iron carrier moiety appears in the medium. Besides transport-negative mutants, additional albomycin-resistant mutants were isolated. The mutations were mapped outside the transport genes close to the pyrD gene at 21 min. The mutants were devoid of peptidase N activity. The molecular weight, sensitivity to inhibitors, and cytoplasmic location of the enzyme hydrolyzing albomycin in vitro corresponded to the known properties of peptidase N. The aminoacyl thioribosyl pyrimidine moiety of albomycin apparently has to be cleaved off the iron chelate transport vehicle to inhibit growth. Peptidase N is the major hydrolyzing enzyme. In Salmonella typhimurium peptidase N and peptidase A were equally active in hydrolyzing and activating albomycin.     

Regulation of Intracellular Proteolysis in Escherichia coli

Individual nitrogenous metabolites have been examined as regulating agents for the breakdown of intracellular proteins in Escherichia coli. Generally, NH(4) (+) is the most effective regulator. Its depletion progressively increases the basal proteolytic rate to maximum in most strains when the doubling time is increased to 2 h. In E. coli 9723, the rate is further increased at longer doubling times. Amino acids have individual effects on intracellular proteolysis. The basal rate in amino acid-requiring auxotrophs of E. coli 9723 is stimulated weakly by starvation for histidine, tryptophan, or tyrosine, moderately by four other amino acid depletions, and more strongly by eight others. The degree of stimulation roughly correlates with the frequency of the amino acid in the cell proteins. Amino acid analogues that incorporate extensively into protein generally slightly inhibit intracellular proteolysis, except for selenomethionine, which is slightly stimulatory. Metabolic inhibitors were studied at graded concentrations. Chloramphenicol inhibits the basal level of intracellular proteolysis when protein synthesis is slightly or moderately inhibited, and stimulates proteolysis slightly at higher levels. Graded inhibition of ribonucleic acid synthesis with rifampin progressively stimulates intracellular proteolysis. Uracil depletion is also stimulatory. Inhibition of deoxyribonucleic acid synthesis with mitomycin C or by thymine starvation slightly inhibits intracellular proteolysis. Intracellular proteolysis is postulated to be regulated primarily by active ribosomal function. At 43 to 45 C, intracellular proteolysis becomes maximally induced and unresponsive to normal regulatory control by metabolites. Most regulation is directed towards the breakdown of the more stable cell proteins. Total proteolysis in all cell proteins is no more than doubled by the most effective conditions of starvation.     

Intracellular accumulation of monomer precursors of polyphosphates and polyhydroxyalkanoates from Acinetobacter calcoaceticus and Escherichia coli]

The formation of polyhydroxyalkanoates granules in anaerobically grown Escherichia coli cells was found to be preceded by the intracellular accumulation of carbonic acids (predominantly, acetic acid), amounting to 9% of the cytosol. The intracellular concentration of acidic metabolites increased after the lyophilization of the bacterial biomass and decreased after its long-term storage (3.5-13.5 years). The decrease in the concentration of acidic metabolites is likely due to the dehydration of dimeric carbonic acids in the viscoelastic cytosol of resting bacterial cells. The hydrophobic obligately aerobic cells of Acinetobacter calcoaceticus IEGM 549 are able to utilize a wide range of growth substrates (from acetate and citrate to hydrophobic hydrocarbons), which is considerably wider than the range of the growth substrates of E. coli (predominantly, carbohydrates). The minimal essential and optimal concentrations of orthophosphates in the growth medium of A. calcoaceticus were found to be tens of times lower than in the case of E. coli. The intracellular content of orthophosphates in A. calcoaceticus cells reached 35-77% of the total phosphorus content (Ptotal), providing for the intense synthesis of polyphosphates. The Ptotal of the A. calcoaceticus cells grown in media with different proportions between the concentrations of acetate and phosphorus varied from 0.7 to 3.3%, averaging 2%. This value of Ptotal is about two times higher than that observed for fermenting E. coli cells. Lowering the cultivation temperature of A. calcoaceticus from 37-32 to 4 degrees C augmented the accumulation of orthophosphates in the cytoplasm, presumably owing to a decreased requirement of growth processes for orthophosphate. In this case, if the concentration of phosphates in the cultivation medium was low, they were completely depleted.     

Molybdenum accumulation in chlD mutants of Escherichia coli.

The content of molybdenum in wild-type and chlD cells was measured under a variety of growth conditions to determine if cells with a defective chlD gene were able to accumulate molybdenum. The chlD cells accumulated less molybdenum than wild-type cells did but concentrated molybdenum to a level at least 20-fold higher than the concentration in the culture medium. Molybdenum was present within spheroplasts of chlD cells and was not dialyzable. The chlD cells accumulated as much molybdenum as wild-type cells did when grown in medium containing 0.1 mM molybdate; thus, the capability of incorporation of molybdenum into cellular component(s) was equivalent to that of the wild type under these conditions.     

Survival of Escherichia coli K12 in seawater

Abstract The fate of an auxotrophic Escherichia coli K12 strain (NF1830) in coastal water was investigated. The E. coli K12 were enumerated after incubation for varying times in seawater. Incubated in raw seawater at 15 and 20°C, the NF1830 decreased from 10⁶ cfu/ml to below detection within six days of incubation, but when incubated at 7°C it persisted longer. The NF1830 was capable of cell division in sterile seawater. Growth was also shown to occur in raw seawater in the presence of autoclaved sediment. The E. coli K12 decreased in number at a much lower rate when incubated in seawater treated with eukaryotic inhibitors. These findings suggest that the die-off of the auxotrophic E. coli K12 strain seen in the raw seawater was caused by grazing of bacterial predators in the seawater.     

Replacement of potassium chloride by potassium glutamate dramatically enhances protein-DNA interactions in vitro

Although protein-nucleic acid interactions exhibit dramatic dependences on both ion concentration and type in vitro, large variations in intracellular ion concentrations can occur in Escherichia coli and other organisms without apparent effects on gene expression in vivo. E. coli accumulates K+ and glutamate as cytoplasmic osmolytes. The cytoplasmic K+ concentration in E. coli varies from less than 0.2 to greater than 0.9 m as a function of external osmolarity; corresponding cytoplasmic glutamate concentrations range from less than 0.03 to greater than 0.25 m. Only low levels of chloride occur in the cytoplasm of E. coli at all osmotic conditions. Since most in vitro studies have been performed in chloride salts, whereas glutamate is the more relevant physiological anion, we have measured the effects of the substitution of potassium glutamate (KGlu) for KCl on the kinetics and equilibria of a variety of site-specific protein-DNA interactions in vitro. Both the interaction of E. coli RNA polymerase with two phage lambda promoters and the interactions of various restriction enzymes with their DNA cleavage sites are enhanced by this substitution. Using the abortive initiation assay, we find a greater than 30-fold increase in the second-order rate constant for open complex formation at the lambda PR promoter and a 10-fold increase at the lambda PR' promoter, when KGlu is substituted for KCl. Replacement of KCl by KGlu does not affect the strong salt dependences of these interactions; increasing either KCl or KGlu concentrations decreases both reaction rates and extents. Substitution of glutamate for chloride does, however, shift the range of salt concentrations over which these interactions are observable to higher K+ concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solar radiation induces sublethal injury in Escherichia coli in seawater.

Sublethal injury was noted in Escherichia coli after cells were exposed to solar radiation. Injury was detected by differential plate counts between complete and minimal media that were observed with sunlight-exposed cells but not with cells kept in the dark. Since addition of catalase or pyruvate to minimal medium overcame or repaired this injury, the catalase system appeared to be the site of injury.     

Intracellular location of catabolite activator protein of Escherichia coli.

The catabolite activator protein was assayed in extracts from the minicell-producing Escherichia coli strain P678-54. The level of catabolite activator protein was found to be the same in both parent cells and purified minicells, regardless of whether the bacteria were grown on glucose (which leads to low intracellular cyclic adenosine monophosphate levels) or on glycerol-yeast extract or LB broth (which lead to high cyclic adenosine monophosphate concentrations in the cell). Thus, at any given time most catabolite activator protein molecules are found in the cytoplasm. The implications of this for the mechanism of catabolite activator protein action at catabolite-sensitive operons are discussed.     

Stringent Control of Intracellular Proteolysis in Escherichia coli

Regulation of intracellular proteolysis has been compared during amino acid deficiencies in seven double auxotrophs of Escherichia coli 9723f with a common phenylalanine requirement. Individual deficiencies were either more effective than, less effective, or equal to phenylalanine deficiency in stimulating intracellular proteolysis. For each amino acid, the same relationship prevailed in inhibiting uracil incorporation into nucleic acids, a reaction series regulated by the rel gene for stringent control. The three amino acids least abundant in the cellular protein were the least effective regulators. These findings are interpreted as supportive evidence for stringent control of intracellular proteolysis by the rel gene.     

Transient accumulation of potassium glutamate and its replacement by trehalose during adaptation of growing cells of Escherichia coli K-12 to elevated sodium chloride concentrations

The sequence of events following the addition of 0.5 M NaCl to cells of Escherichia coli growing in a minimal mineral medium was investigated. Immediately after upshock the cells took up a large amount of K⁺ and synthesized approximately half the equivalent amount of glutamate concomitantly. After 30 min the cells started to synthesize trehalose, and after 2 h they had replaced most of their initial osmoprotectants by the carbohydrate. Cell trehalose was rapidly replaced by proline, taken up from the medium when added to the osmoadapting cells. The initial rate of this proline uptake was extremely rapid, and with rates observed of up to 0.6 mmolxmin^-1xg^-1 of cell protein it was approximately ten times faster than that reported in the literature for non-growing cells. These results indicate that for osmoadaptation of growing cells of E. coli the uptake of proline has priority over the synthesis of trehalose, which in its turn is preferred above K⁺ and glutamate as osmoprotectants. We observed that two mutants with unknown lesions, but which are known to be impaired in osmoadaptation, were inhibited in replacing K⁺ and glutamate by trehalose, indicating that this is the basis for their defect in osmoadaptation. Further experiments revealed that neither internal pH nor the membrane potential nor the transmembrane protonmotive force are likely to be involved in osmoadaptation in E. coli. However, during osmoadaptation a high internal potassium concentration appeared to stimulate the derepression of proline-uptake systems (mainly system ProP).Abbreviations TPP tetraphenylphosphonium - pmf transmembrane protonmotive force - membrane potential, pH, transmembrane pH difference These parameters are defined as the difference between the outside and the inside of the cells     

NarK enhances nitrate uptake and nitrite excretion in Escherichia coli.

narK mutants of Escherichia coli produce wild-type levels of nitrate reductase but, unlike the wild-type strain, do not accumulate nitrite when grown anaerobically on a glucose-nitrate medium. Comparison of the rates of nitrate and nitrite metabolism in cultures growing anaerobically on glucose-nitrate medium revealed that a narK mutant reduced nitrate at a rate only slightly slower than that in the NarK+ parental strain. Although the specific activities of nitrate reductase and nitrite reductase were similar in the two strains, the parental strain accumulated nitrite in the medium in almost stoichiometric amounts before it was further reduced, while the narK mutant did not accumulate nitrite in the medium but apparently reduced it as rapidly as it was formed. Under conditions in which nitrite reductase was not produced, the narK mutant excreted the nitrite formed from nitrate into the medium; however, the rate of reduction of nitrate to nitrite was significantly slower than that of the parental strain or that which occurred when nitrite reductase was present. These results demonstrate that E. coli is capable of taking up nitrate and excreting nitrite in the absence of a functional NarK protein; however, in growing cells, a functional NarK promotes a more rapid rate of anaerobic nitrate reduction and the continuous excretion of the nitrite formed. Based on the kinetics of nitrate reduction and of nitrite reduction and excretion in growing cultures and in washed cell suspensions, it is proposed that the narK gene encodes a nitrate/nitrite antiporter which facilitates anaerobic nitrate respiration by coupling the excretion of nitrite to nitrate uptake. The failure of nitrate to suppress the reduction of trimethylamine N-oxide in narK mutants was not due to a change in the level of trimethylamine N-oxide reductase but apparently resulted from a relative decrease in the rate of anaerobic nitrate reduction caused by the loss of the antiporter system.     

Rapid glutamate decarboxylase assay for detection of Escherichia coli.

A rapid test procedure for the enzyme glutamate decarboxylase was developed for detection of Escherichia coli. The assay procedure was able to confirm the presence of E. coli in enteric broth cultures with 95% specificity for both pure cultures and environmental samples. The procedure was capable of detecting survivors among chlorine-exposed cells.     

Cation Transport in Escherichia coli : II. Intracellular chloride concentration

The intracellular Cl concentration in E. coli has been studied as a function of the Cl concentration in the growth medium and the age of the bacterial culture. The ratio of extracellular to intracellular Cl concentration is shown to be 3.0 in the logarithmic phase and 1.13 in the stationary phase, both ratios being independent of the extracellular Cl concentration. If it may be assumed that Cl is passively distributed in this organism, these results are consistent with a transmembrane P.D. of 29 mv, interior negative, during the logarithmic phase, and 3 mv, interior negative, in the stationary phase.