Immediate and transient inhibition of the respiration of Escherichia coli under hyperosmotic shock

Abstract A range of isolates of Serpula lacrymans show identical molecular profiles by Western Blotting or lectin staining. One isolate, BF-050, showed differences in silver stained protein profiles from old mycelium but profiles from young mycelium of BF-050 were identical to the type strain of S. lacrymans FPRL 12C. Both S. lacrymans and the related organism, Serpula himantoides , shared different profiles for old and young mycelium confirming that growth phase antigens/proteins are not limited to S. lacrymans . Lectin staining profiles could distinguish between S. lacrymans FPRL 12C, BF-015B, S. himantoides and all other basidiomycete organisms tested. Differences between strains of S. lacrymans including BF-050, were not apparent.     

Selective mRNA degradation by polynucleotide phosphorylase in cold shock adaptation in Escherichia coli

Upon cold shock, Escherichia coli cell growth transiently stops. During this acclimation phase, specific cold shock proteins (CSPs) are highly induced. At the end of the acclimation phase, their synthesis is reduced to new basal levels, while the non-cold shock protein synthesis is resumed, resulting in cell growth reinitiation. Here, we report that polynucleotide phosphorylase (PNPase) is required to repress CSP production at the end of the acclimation phase. A pnp mutant, upon cold shock, maintained a high level of CSPs even after 24 h. PNPase was found to be essential for selective degradation of CSP mRNAs at 15 degrees C. In a poly(A) polymerase mutant and a CsdA RNA helicase mutant, CSP expression upon cold shock was significantly prolonged, indicating that PNPase in concert with poly(A) polymerase and CsdA RNA helicase plays a critical role in cold shock adaptation.     

Caveolin internalization by heat shock or hyperosmotic shock

We investigated the cellular localization of caveolin, a landmark protein of caveolae, by indirect immunofluorescence after heat shock or hyperosmotic shock. Caveolin was internalized to the perinucleus by heat shock (43 degrees C) and relocalized in the plasma membrane after recovery of NIH3T3 cells at 37 degrees C for 4 h. The caveolin internalization was also observed after cells were exposed to hyperosmotic shock. Caveolin disappeared from detergent-insoluble complexes in the heat-shocked cells, but alkaline phosphatase was still there, suggesting that their responses to heat shock are quite different even though both of them were enriched in detergent-insoluble complexes of normal cells. Caveolin was internalized by the actin depolymerizer cytochalasin D, but not by the tubulin depolymerizer nocodazole. In addition, cellular exposure to hydrogen peroxide caused caveolin internalization along with disintegrated microfilaments and intact microtubules. Since cellular exposure to heat shock showed disintegrated microfilaments but intact microtubules, caveolin internalization might be due to depolymerized microfilaments. When cells were exposed to heat shock and allowed to recover for 4 h, actin depolymerization and caveolin internalization were not induced by a second heat shock, suggesting that some heat shock protein(s) might prevent actin depolymerization and caveolin internalization.     

Evolutionary adaptation to freeze-thaw-growth cycles in Escherichia coli

Fifteen populations of Escherichia coli were propagated for 150 freeze-thaw-growth (FTG) cycles in order to study the phenotypic and genetic changes that evolve under these stressful conditions. Here we present the phenotypic differences between the evolved lines and their progenitors as measured by competition experiments and growth curves. Three FTG lines evolved from an ancestral strain that was previously used to start a long-term evolution experiment, while the other 12 FTG lines are derived from clones that had previously evolved for 20,000 generations at constant 37 degrees C. Competition experiments indicate that the former FTG group improved their mean fitness under the FTG regime by about 90% relative to their progenitor, while the latter FTG group gained on average about 60% relative to their own progenitors. These increases in fitness result from both improved survival during freezing and thawing and more rapid recovery to initiate exponential growth after thawing. This shorter lag phase is specific to recovery after freezing and thawing. Future work will seek to identify the mutations responsible for evolutionary adaptation to the FTG environment and use them to explore the physiological mechanisms that allow increased survival and more rapid recovery.     

Role of heat shock protein DnaK in osmotic adaptation of Escherichia coli.

Escherichia coli can adapt and recover growth at high osmolarity. Adaptation requires the deplasmolysis of cells previously plasmolyzed by the fast efflux of water promoted by osmotic upshift. Deplasmolysis is essentially ensured by a net osmo-dependent influx of K+. The cellular content of the heat shock protein DnaK is increased in response to osmotic upshift and does not decrease as long as osmolarity is high. The dnaK756(Ts) mutant, which fails to deplasmolyze and recover growth, does not take up K+ at high osmolarity; DnaK protein is required directly or indirectly for the maintenance of K+ transport at high osmolarity. The temperature-sensitive mutations dnaJ259 and grpE280 do not affect the osmoadaptation of E. coli at 30 degrees C.     

Oxidation-reduction and production of molecular hydrogen by Escherichia coli in the hyperosmotic medium

It was shown that Escherichia coli is able to grow in anaerobic conditions in hyperosmotic media containing 0.5 M sodium chloride or equivalent amount of sucrose. However, in the presence of 0.5 M NaCl, bacterial growth rate and the intensity of oxidation-reduction processes decrease, and the production of molecular hydrogen is absent. Growth rate in the presence of 0.5 M NaCl is four times lower than that in the presence of sucrose. Under hyperosmotic stress by 0.5 M NaCl but not by equivalent amount of sucrose, the uptake of K+ with a high rate is observed. Proline is able to increase the growth rate and the intensity of oxidation-reduction processes and to restore the production of molecular hydrogen as well as to induce the uptake of K+ with a high rate under a hyperosmotic stress. Such effects are observed at pH 7.5 and are absent at pH 5.5. Proline also increases cell size independently of medium pH. It is likely that the effect of proline on oxidation-reduction processes and production of H2 is mediated through the accumulation of K+ in bacteria.     

Pervasive compensatory adaptation in Escherichia coli

To investigate compensatory adaptation (CA), we used genotypes of Escherichia coli which were identical except for one or two deleterious mutations. We compared CA for (i) deleterious mutations with large versus small effects, (ii) genotypes carrying one versus two mutations, and (iii) pairs of deleterious mutations which interact in a multiplicative versus synergistic fashion. In all, we studied 14 different genotypes, plus a control strain which was not mutated. Most genotypes showed CA during 200 generations of experimental evolution, where we define CA as a fitness increase which is disproportionately large relative to that in evolving control lines, coupled with retention of the original deleterious mutation(s). We observed greater CA for mutations of large effect than for those of small effect, which can be explained by the greater benefit to recovery in severely handicapped genotypes given the dynamics of selection. The rates of CA were similar for double and single mutants whose initial fitnesses were approximately equal. CA was faster for synergistic than for multiplicative pairs, presumably because the marginal gain which results from CA for one of the component mutations is greater in that case. The most surprising result in our view, is that compensation should be so readily achieved in an organism which is haploid and has little genetic redundancy This finding suggests a degree of versatility in the E. coil genome which demands further study from both genetic and physiological perspectives.     

Characterization of Escherichia coli nucleoids released by osmotic shock

Nucleoids were isolated by osmotic shock from Escherichia coli spheroplasts at relatively low salt concentrations and in the absence of detergents. Sucrose-protected cells, made osmotically sensitive by growth in the presence of ampicillin or by digestion with low lysozyme concentrations (50-5 μg/ml), were shocked by 100-fold dilution of the sucrose buffer. Liberated nucleoids stained with 4',6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI), the dimeric cyanine dye TOTO-1, or fluorescent DNA-binding protein appeared as cloud-like structures, in the absence of phase contrast. Because UV-irradiation disrupted the DAPI-stained nucleoids within 5-10 s, they were imaged by time-lapse microscopy with exposure times less than 2 s. The volume of nucleoids isolated from ampicillin- or low-lysozyme spheroplasts and minimally exposed to UV (<2 s) was on average ～42 μm(3). Lysozyme at concentrations above 1 μg/ml in the lysate compacted the nucleoids. Treatment with protease E or K (20-200 μg/ml) and sodium dodecyl sulfate (SDS; 0.001-0.01%) caused a twofold volume increase and showed a granular nucleoid at the earliest UV-exposure; the expansion could be reversed with 50 μM ethidium bromide, but not with chloroquine. While DNase (1 μg/ml) caused a rapid disruption of the nucleoids, RNase (0.1-400 μg/ml) had no effect. DAPI-stained nucleoids treated with protease, SDS or DNase consisted of granular substructures at the earliest exposure similar to UV-disrupted nucleoids obtained after prolonged (>4 s) UV irradiation. We interpret the measured volume in terms of a physical model of the nucleoid viewed as a branched DNA supercoil crosslinked by adhering proteins into a homogeneous network.     

Metabolic adaptation of Escherichia coli to long-term exposure to salt stress

The aim of this work was to understand the relevance of central carbon metabolism in salt stress adaptation of Escherichia coli. The cells were grown anaerobically in batch and chemostat reactors at different NaCl concentrations using glycerol as a carbon source. Enzyme activities of the main metabolic pathways, external metabolites, ATP level, NADH/NAD⁺ ratio, l-carnitine production and the expression level of the main genes related to stress response were used to characterize the metabolic state under the osmotic stress. The results provided the first experimental evidence of the important role played by central metabolism adaptation and cell survival after long-term exposure to salt stress. Increased glycolytic fluxes and higher production of fermentation products indicated the importance of energy metabolism. Carbon fluxes under stress conditions were controlled by the decrease in the isocitrate dehydrogenase/isocitrate lyase ratio and the phosphoenolpyruvate carboxykinase/phosphoenolpyruvate carboxylase ratio, and the increase in the phosphotransferase/acetyl-CoA synthetase ratio. Altogether, the results demonstrate that, under salt stress, E. coli enhances energy production by substrate-level phosphorylation (Pta–Ack pathway) and the anaplerotic function of the TCA cycle, in order to provide precursors for biosynthesis. The results are discussed in relation with the general stress response and metabolic adaptation of E. coli.     

Cold shock response in Escherichia coli

Sensing a sudden change of the growth temperature, all living organisms produce heat shock proteins or cold shock proteins to adapt to a given temperature. In a heat shock response, the heat shock sigma factor plays a major role in the induction of heat shock proteins including molecular chaperones and proteases, which are well-conserved from bacteria to human. In contrast, no such a sigma factor has been identified for the cold shock response. Instead, RNAs and RNA-binding proteins play a major role in cold shock response. This review describes what happens in the cell upon cold shock, how E. coli responds to cold shock, how the expression of cold shock proteins is regulated, and what their functions are.     

Acid shock proteins of Escherichia coli

Synthesis of total cellular proteins of Escherichia coli was studied after transfer of cultures from pH 6.9 to pH 4.3. Proteins induced by such an external pH shift down were identified by mono- and bi-dimensional electrophoresis. 30 to 45 min after an acid shift, a group of at least sixteen polypeptides was markedly induced. Four of these polypeptides corresponded to the well known heat shock proteins GroEL, DnaK, HtpG and HtpM. Their pH induction was RpoH-dependent. Three other pH-induced proteins were previously identified as stress proteins induced either by osmolarity or aerobiosis or low temperature (proteins 32 (defined in this paper), C70.0 and C62.7). Seven other proteins were specifically induced after an acid shift and were called acid shock proteins (ASP). The induction of one of these proteins was RpoH-dependent, whereas that of others was RpoH-independent.     

Evolutionary adaptation to environmental pH in experimental lineages of Escherichia coli

This study uses the enteric bacterium Escherichia coli as an experimental system to examine evolutionary responses of bacteria to an environmental acidic-alkaline range between pH 5.3 and 7.8 (15-5000 nM [H(+)]). Our goal was both to test general hypotheses about adaptation to abiotic variables and to provide insights into how coliform organisms might respond to changing conditions inside and outside of hosts. Six replicate lines of E. coli evolved for 2000 generations at one of four different constant pH conditions: pH 5.3, 6.3, 7.0, or 7.8. Direct adaptation to the evolutionary environment, as well as correlated changes in other environments, was measured as a change in fitness relative to the ancestor in direct competition experiments. The pH 5.3 group had the highest fitness gains, with a highly significant increase of 20%. The pH 7.8 group had far less significant gains and much higher variance among its lines. Analysis of individual lines within these two groups revealed complex patterns of adaptation: all of the pH 5.3 lines exhibited trade-offs (reduced fitness in another environment), but only 33% of the pH 7.8 lines showed such trade-offs and one of the pH 7.8 lines demonstrated exaptation by improving fitness in the pH 5.3 environment. Although there was also prevalent exaptation in other groups to the acidic environment, there were no such cases of exaptation to alkalinity. Comparison across the entire experimental pH range revealed that the most acidic lines, the pH 5.3 group, were all specialists, in contrast to the pH 6.3 lines, which were almost all generalists. That is, although none of the pH 5.3 lines showed any correlated fitness gains, all of the pH 6.3 lines did.     

Escherichia coli physiology and metabolism dictates adaptation to diverse host microenvironments

Bacterial growth in the host is required for pathogenesis. To successfully grow in vivo, pathogens have adapted their metabolism to replicate in specific host microenvironments. These adaptations reflect the nutritional composition of their host niches, inter-bacterial competition for carbon and energy sources, and survival in the face of bactericidal defense mechanisms. A subgroup of Escherichia coli, which cause urinary tract infection, bacteremia, sepsis, and meningitis, have adapted to grow as a harmless commensal in the nutrient-replete, carbon-rich human intestine but rapidly transition to pathogenic lifestyle in the nutritionally poorer, nitrogen-rich urinary tract. We discuss bacterial adaptations that allow extraintestinal pathogenic E. coli to establish both commensal associations and virulence as the bacterium transits between disparate microenvironments within the same individual.     

Behavioral responses of Escherichia coli to changes in temperature caused by electric shock.

The behavioral response of Escherichia coli to electric shock in 10(-2) M potassium phosphate plus 10(-4) M potassium EDTA was studied. When presented with a 150-V/cm electric shock that lasted 250 ms, the bacteria at first exclusively ran, then exclusively tumbled, and finally returned to their original running and tumbling. This response is due to increased temperature caused by the electric shock, i.e., to thermotaxis, and it is mediated by the chemotaxis machinery. A more severe electric shock, 150 V/cm for 550 ms, caused cells to tumble immediately, and then they went back to their original running and tumbling. The mechanism of that response is unknown since, unlike known thermotaxis, it does not require the chemotaxis machinery.     

Alcohols protect Escherichia coli against cold shock

Alcohols protect Escherichia coli against cold shock, and the concentration of alcohol which provided optimal protection declined with increasing hydrophobicity of the alcohol. The rate of loss of viability after the chilling transition was decreased by n-octanol, even when it was added after that chilling transition. Cold-shocked cells exhibited a sensitivity toward dioxygen, seen as greater enumeration on anaerobic, rather than on aerobic, trypticase-yeast extract agar plates, and addition of catalase or antioxidants, such as alpha-tocopherol or probucol, to the agar plates did not lessen this dioxygen sensitivity. Respiratory capacity was diminished by cold shock, and cyanide-sensitive respiration was more affected than was cyanide-resistant respiration. Discharging the proton gradient, with the uncoupler carbonyl cyanide trifluoromethoxy-phenylhydrazone, did not change sensitivity to cold shock. There was no evidence for minimal medium recovery after cold shock. The data presented, as well as that already in the literature, are explained on the basis of membrane damage caused by patches of ordering transitions in one membrane leaflet, unmatched by comparable transitions in the mating leaflet.     

Osmotic adaptation of T4 infected Escherichia coli

In contrast to enzymatic adaptation, osmotic adaption is possible with T4-infected Escherichia coli B cells. After an osmotic shift from 220 mOsM to 690 mOsM the intracellular content of potassium rises in infected cells as well as in uninfected cells. After osmotic shock the involved TrKA transport system shows an increased discrimination against rubidium (Rb⁺) and for potassium (K⁺).