Role of CspC and CspE in regulation of expression of RpoS and UspA, the stress response proteins in Escherichia coli

Nine homologous proteins, CspA to CspI, constitute the CspA family of Escherichia coli. Recent studies are aimed at elucidating the individual cellular functions of these proteins. Two members of this family, CspC and CspE, are constitutively produced at 37 degrees C. In the present study, these two proteins were evaluated for their cellular role(s). The expression of three stress proteins, OsmY, Dps, and UspA, is significantly affected by the overexpression and deletion of CspC and CspE. RpoS is a regulatory element for osmY and dps. Further analysis showed a larger amount and greater stability of the rpoS mRNA as well as a higher level of RpoS itself with the overexpression of CspC and CspE. This suggests that CspC and CspE upregulate the expression of OsmY and Dps by regulating the expression of RpoS itself. Indeed, this upregulation is lost in the Delta rpoS strain. Other RpoS-controlled proteins such as ProP and KatG, are also upregulated by the overexpression of CspC. The present study suggests that CspC and CspE are the important elements involved in the regulation of the expression of RpoS, a global stress response regulator, and UspA, a protein responding to numerous stresses. In the light of these observations, it seems plausible that CspC and CspE function as regulatory elements for the expression of stress proteins in the complex stress response network of E. coli.     

Structural and functional characterization of osmotically inducible protein C (OsmC) from Thermococcus kodakaraensis KOD1

Osmotically inducible protein C (OsmC) is involved in the cellular defense mechanism against oxidative stress caused by exposure to hyperoxides or elevated osmolarity. OsmC was identified by two-dimensional electrophoresis (2DE) analysis as a protein that is overexpressed in response to osmotic stress, but not under heat and oxidative stress. Here, an OsmC gene from T. kodakaraensis KOD1 was cloned and expressed in Escherichia coli. TkOsmC showed a homotetrameric structure based on gel filtration and electron microscopic analyses. TkOsmC has a significant peroxidase activity toward both organic and inorganic peroxides in high, but not in low temperature.     

Antibiotic‐dependent perturbations of extended spectrum beta‐lactamase producing Klebsiella pneumoniae proteome

Extended spectrum beta‐lactamase producing Klebsiella pneumoniae (ESBL‐KP) causes life‐threatening infections in susceptible and immuno‐compromised individuals. Because of the emergence of multidrug resistance and tolerance, it is crucial to better understand the mechanisms by which ESBL‐KP can adapt to antibiotic stress. The aim of this study was to provide an overview of the global proteome changes occurring in ESBL‐KP in response to sub‐lethal concentrations of the antibiotics doxycycline (DC, bacteriostatic) and streptomycin (SM, bactericidal), which both impair ribosomal synthesis of bacterial proteins. These results represent the greatest experimental coverage of the ESBL‐KP proteome yet described. The 1538 proteins, representing 30% of the 5126 predicted KP gene products were identified from the combined experimental groups. Antibiotic stress resulted in significantly elevated levels of 42 proteins for DC and 55 for SM treatments, whereas 53 proteins were reduced for DC‐ and six for SM‐treated bacteria. Specifically, the ESBL‐KP response to DC was accompanied by the reduced levels of the porins LamB, CirA, FepA, and OmpC. In contrast to DC, the stress response to SM demonstrated a dramatic increase in the peroxidase detoxification pathway proteins PutA, KatG, KatE, and Dps, which prevent harmful hydroxyl radical formation. The results from this proteomic study are important for understanding adaptive responses to antibiotics, and may provide novel targets for the development of new therapeutic strategies.     

Characterization of a cytoplasmic trehalase of Escherichia coli.

Escherichia coli can synthesize trehalose in response to osmotic stress and is able to utilize trehalose as a carbon source. The pathway of trehalose utilization is different at low and high osmolarity. At high osmolarity, a periplasmic trehalase (TreA) is induced that hydrolyzes trehalose in the periplasm to glucose. Glucose is then taken up by the phosphotransferase system. At low osmolarity, trehalose is taken up by a trehalose-specific enzyme II of the phosphotransferase system as trehalose-6-phosphate and then is hydrolyzed to glucose and glucose-6-phosphate. Here we report a novel cytoplasmic trehalase that hydrolyzes trehalose to glucose. treF, the gene encoding this enzyme, was cloned under ara promoter control. The enzyme (TreF) was purified from extracts of an overexpressing strain and characterized biochemically. It is specific for trehalose exhibiting a Km of 1.9 mM and a Vmax of 54 micromol of trehalose hydrolyzed per min per mg of protein. The enzyme is monomeric, exhibits a broad pH optimum at 6.0, and shows no metal dependency. TreF has a molecular weight of 63,703 (549 amino acids) and is highly homologous to TreA. The nonidentical amino acids of TreF are more polar and more acidic than those of TreA. The expression of treF as studied by the expression of a chromosomal treF-lacZ fusion is weakly induced by high osmolarity of the medium and is partially dependent on RpoS, the stationary-phase sigma factor. Mutants producing 17-fold more TreF than does the wild type were isolated.     

Impact of nutritional factors on the proteome of intestinal Escherichia coli: induction of OxyR-dependent proteins AhpF and Dps by a lactose-rich diet

To study the impact of nutritional factors on protein expression of intestinal bacteria, gnotobiotic mice monoassociated with Escherichia coli K-12 were fed three different diets: a diet rich in starch, a diet rich in nondigestible lactose, and a diet rich in casein. Two-dimensional gel electrophoresis and electrospray-tandem mass spectrometry were used to identify differentially expressed proteins of bacteria recovered from small intestine and cecum. Oxidative stress response proteins such as AhpF, Dps, and Fur, all of which belong to the oxyR regulon, were upregulated in E. coli isolates from mice fed the lactose-rich diet. Luciferase reporter gene assays demonstrated that osmotic stress caused by carbohydrates led to the expression of ahpCF and dps, which was not observed in an E. coli ΔoxyR mutant. Growth of ahpCF and oxyR deletion mutants was strongly impaired when nondigestible sucrose was present in the medium. The wild-type phenotype could be restored by complementation of the deletions with plasmids containing the corresponding genes and promoters. The results indicate that some OxyR-dependent proteins play a major role in the adaptation of E. coli to osmotic stress. We conclude that there is an overlap of osmotic and oxidative stress responses. Mice fed the lactose-rich diet possibly had a higher intestinal osmolality, leading to the upregulation of OxyR-dependent proteins, which enable intestinal E. coli to better cope with diet-induced osmotic stress.     

Thylakoid proteome variation of Eutrema salsugineum in response to drought and salinity combined stress

It is well known that plant responses to stress involve different events occurring at different places of the cell/leaf and at different time scales in relation with the plant development. In fact, the organelles proteomes include a wide range of proteins that could include a wide range of proteins showing a considerable change in cellular functions and metabolism process. On this basis, a comparative proteomics analysis and fluorescence induction measurements were performed to investigate the photosynthetic performance and the relative thylakoid proteome variation in Eutrema salsugineum cultivated under salt stress (200 mM NaCl), water deficit stress (PEG) and combined treatment (PEG + NaCl) as a hyperosmotic stress. The obtained results showed a significant decrease of plant growth under drought stress conditions, with the appearance of some toxicity symptoms, especially in plants subjected to combined treatment. Application of salt or water stress alone showed no apparent change in the chlorophyll a fluorescence transients, primary photochemistry (fluorescence kinetics of the O-J phase), the PQ pool state (J-I phase changes), (Fv/Fm) and (Fk/Fj) ratios. However, a considerable decrease of all these parameters was observed under severe osmotic stress (PEG + NaCl). The thylakoid proteome analysis revealed 58 proteins showing a significant variation in their abundance between treatments (up or down regulation). The combined treatment (PEG + NaCl) induced a decrease in the expression of the whole PSII core subunit (D1, D2, CP43, CP47, PsbE and PsbH), whereas the OEC subunits proteins remained constant. An increase in the amount of PsaD, PsaE, PsaF, PsaH, PsaK and PsaN was detected under drought stress (PEG5%). No significant change in the accumulation of Cyt b6 and Cyt f was observed. Some regulated proteins involved in cellular redox homeostasis were detected (glutamine synthetase, phosphoglycerate kinase, transketolase), and showed a significant decrease under the combined treatment. Some oxidative stress related proteins were significantly up-regulated under salt or drought stress and could play a crucial role in the PSI photoprotection and the control of ROS production level.     

Sensitivity of Escherichia coli to proline analogues during osmotic stress and anaerobiosis

L.M. REESE, K.O. CUTLER AND C.E. DEUTCH. 1996. The sensitivity of wild-type Escherichia coli K-12 to a series of proline analogues was determined in cultures containing increasing concentrations of NaCl under both aerobic and anaerobic conditions. The bacteria were most sensitive to L-azetidine-2–carboxylate and L-thiazolidine-4–carboxylate. The minimum inhibitory concentrations for these compounds decreased progressively during osmotic stress, but the bacteria were much more sensitive to these proline analogues under aerobic conditions than during anaerobiosis. The reduced sensitivity under anaerobic conditions did not reflect degradation of the compounds in the culture medium. Since both urine and medullary renal tissue contain relatively low oxygen concentrations, these results raise doubts about the potential use of proline or glycine betaine analogues in treating urinary tract infections.     

The Tat System for Membrane Translocation of Folded Proteins Recruits the Membrane-stabilizing Psp Machinery in Escherichia coli

Tat systems transport folded proteins across energized membranes of bacteria, archaea, and plant plastids. In Escherichia coli, TatBC complexes recognize the transported proteins, and TatA complexes are recruited to facilitate transport. We achieved an abstraction of TatA from membranes without use of detergents and observed a co-purification of PspA, a membrane-stress response protein. The N-terminal transmembrane domain of TatA was required for the interaction. Electron microscopy displayed TatA complexes in direct contact with PspA. PspB and PspC were important for the TatA-PspA contact. The activator protein PspF was not involved in the PspA-TatA interaction, demonstrating that basal levels of PspA already interact with TatA. Elevated TatA levels caused membrane stress that induced a strictly PspBC- and PspF-dependent up-regulation of PspA. TatA complexes were found to destabilize membranes under these conditions. At native TatA levels, PspA deficiency clearly affected anaerobic TMAO respiratory growth, suggesting that energetic costs for transport of large Tat substrates such as TMAO reductase can become growth limiting in the absence of PspA. The physiological role of PspA recruitment to TatA may therefore be the control of membrane stress at active translocons.     

Proteome-based identification of fusion partner for high-level extracellular production of recombinant proteins in Escherichia coli

Extracellular production of recombinant proteins in Escherichia coli has several advantages over cytoplasmic or periplasmic production. However, nonpathogenic laboratory strains of E. coli generally excrete only trace amounts of proteins into the culture medium under normal growth conditions. Here we report a systematic proteome-based approach for developing a system for high-level extracellular production of recombinant proteins in E. coli. First, we analyzed the extracellular proteome of an E. coli B strain, BL21(DE3), to identify naturally excreted proteins, assuming that these proteins may serve as potential fusion partners for the production of recombinant proteins in the medium. Next, overexpression and excretion studies were performed for the 20 selected fusion partners with molecular weights below 40 kDa. Twelve of them were found to allow fused proteins to excrete into the medium at considerable levels. The most efficient excreting fusion partner, OsmY, was used as a carrier protein to excrete heterologous proteins into the medium. E. coli alkaline phosphatase, Bacillus subtilis alpha-amylase, and human leptin used as model proteins could all be excreted into the medium at concentrations ranging from 5 to 64 mg/L during the flask cultivation. When only the signal peptide or the mature part of OsmY was used as a fusion partner, no such excretion was observed; this confirmed that these proteins were truly excreted rather than released by outer membrane leakage. The recombinant protein of interest could be recovered by cleaving off the fusion partner by enterokinase as demonstrated for alkaline phosphatase as an example. High cell density cultivation allowed production of these proteins to the levels of 250-700 mg/L in the culture medium, suggesting the good potential of this approach for the excretory production of recombinant proteins.     

Escherichia coli phage-shock protein A (PspA) binds to membrane phospholipids and repairs proton leakage of the damaged membranes

Escherichia coli phage-shock protein A (PspA), a 25.3 kDa peripheral membrane protein, is induced under the membrane stress conditions and is assumed to help maintain membrane potential. Here, we report that purified PspA, existing as a large oligomer, is really able to suppress proton leakage of the membranes. This was demonstrated for membrane vesicles prepared from the PspA-lacking E. coli mutants, and for membrane vesicles damaged by ethanol and Triton X-100 prepared from the mutant and the wild-type cells. PspA also suppressed proton leakage of damaged liposomes made from E. coli total lipids. Furthermore, we found that PspA bound preferentially to liposomes containing phosphatidylserine and phosphatidylglycerol. All these effects were not observed for monomer PspA that was prepared by refolding of urea-denatured PspA. These results indicate that oligomers of PspA bind to membrane phospholipids and suppress proton leakage.     

Paired Bacillus anthracis Dps (mini-ferritin) have different reactivities with peroxide

Dps (DNA protection during starvation) proteins, mini-ferritins in the ferritin superfamily, catalyze Fe(2+)/H(2)O(2)/O(2) reactions and make minerals inside protein nanocages to minimize radical oxygen-chemistry (metal/osmotic/temperature/nutrient/oxidant) and sometimes to confer virulence. Paired Dps proteins in Bacillus, rare in other bacteria, have 60% sequence identity. To explore functional differences in paired Bacilli Dps protein, we measured ferroxidase activity and DNA protection (hydroxyl radical) for Dps protein dodecamers from Bacillus anthracis (Ba) since crystal structures and iron mineralization (iron-stain) were known. The self-assembled (200 kDa) Ba Dps1 (Dlp-1) and Ba Dps2 (Dlp-2) proteins had similar Fe(2+)/O(2) kinetics, with space for minerals of 500 iron atoms/protein, and protected DNA. The reactions with Fe(2+) were novel in several ways: 1) Ba Dps2 reactions (Fe(2+)/H(2)O(2)) proceeded via an A(650 nm) intermediate, with similar rates to maxi-ferritins (Fe(2+)/O(2)), indicating a new Dps protein reaction pathway, 2) Ba Dps2 reactions (Fe(2+)/O(2) versus Fe(2+)/O(2) + H(2)O(2)) differed 3-fold contrasting with Escherichia coli Dps reactions, with 100-fold differences, and 3) Ba Dps1, inert in Fe(2+)/H(2)O(2) catalysis, inhibited protein-independent Fe(2+)/H(2)O(2) reactions. Sequence similarities between Ba Dps1 and Bacillus subtilis DpsA (Dps1), which is regulated by general stress factor (SigmaB) and Fur, and between Ba Dps2 and B. subtilis MrgA, which is regulated by H(2)O(2) (PerR), suggest the function of Ba Dps1 is iron sequestration and the function of Ba Dps2 is H(2)O(2) destruction, important in host/pathogen interactions. Destruction of H(2)O(2) by Ba Dps2 proceeds via an unknown mechanism with an intermediate similar spectrally (A(650 nm)) and kinetically to the maxi-ferritin diferric peroxo complex.     

Diversion of the metabolic flux from pyruvate dehydrogenase to pyruvate oxidase decreases oxidative stress during glucose metabolism in nongrowing Escherichia coli cells incubated under aerobic, phosphate starvation conditions

Ongoing aerobic metabolism in nongrowing cells may generate oxidative stress. It is shown here that the levels of thiobarbituric acid-reactive substances (TBARSs), which measure fragmentation products of oxidized molecules, increased strongly at the onset of starvation for phosphate (P(i)). This increase in TBARS levels required the activity of the histone-like nucleoid-structuring (H-NS) protein. TBARS levels weakly increased further in DeltaahpCF mutants deficient in alkyl hydroperoxide reductase (AHP) activity during prolonged metabolism of glucose to acetate. Inactivation of pyruvate oxidase (PoxB) activity decreased the production of acetate by half and significantly increased the production of TBARS. Overall, these data suggest that during incubation under aerobic, P(i) starvation conditions, metabolic flux is diverted from the pyruvate dehydrogenase (PDH) complex (NAD dependent) to PoxB (NAD independent). This shift may decrease the production of NADH and in turn the adventitious production of H(2)O(2) by NADH dehydrogenase in the respiratory chain. The residual low levels of H(2)O(2) produced during prolonged incubation can be scavenged efficiently by AHP. However, high levels of H(2)O(2) may be reached transiently at the onset of stationary phase, primarily because H-NS may delay the metabolic shift from PDH to PoxB.     

Altered expression level of Escherichia coli proteins in response to treatment with the antifouling agent zosteric acid sodium salt

Zosteric acid sodium salt is a powerful antifouling agent. However, the mode of its antifouling action has not yet been fully elucidated. Whole cell proteome of Escherichia coli was analysed to study the different protein patterns expressed by the surface-exposed planktonic cells without and with sublethal concentrations of the zosteric acid sodium salt. Proteomic analysis revealed that at least 27 proteins showed a significant (19 upregulated and 8 downregulated, P?相似文献   

Induction and function of the phage shock protein extracytoplasmic stress response in Escherichia coli

The phage shock protein (Psp) F regulon response in Escherichia coli is thought to be induced by impaired inner membrane integrity and an associated decrease in proton motive force (pmf). Mechanisms by which the Psp system detects the stress signal and responds have so far remained undetermined. Here we demonstrate that PspA and PspG directly confront a variety of inducing stimuli by switching the cell to anaerobic respiration and fermentation and by down-regulating motility, thereby subtly adjusting and maintaining energy usage and pmf. Additionally, PspG controls iron usage. We show that the Psp-inducing protein IV secretin stress, in the absence of Psp proteins, decreases the pmf in an ArcB-dependent manner and that ArcB is required for amplifying and transducing the stress signal to the PspF regulon. The requirement of the ArcB signal transduction protein for induction of psp provides clear evidence for a direct link between the physiological redox state of the cell, the electron transport chain, and induction of the Psp response. Under normal growth conditions PspA and PspD control the level of activity of ArcB/ArcA system that senses the redox/metabolic state of the cell, whereas under stress conditions PspA, PspD, and PspG deliver their effector functions at least in part by activating ArcB/ArcA through positive feedback.     

Analysis of protein expression in response to osmotic stress in Escherichia coli

Two strains of Escherichia coli isogenic except for the cya (adenylate cyclase) allele were grown with [35S]methionine and cysteine in minimal defined glucose medium and in this medium with 600 mM NaCl to induce osmotic stress. Cells were grown for approximately two generations. The labeled proteins were separated by 2-dimensional electrophoresis and were quantified fluorographically. Of the 263 major proteins (proteins incorporating 0.10% or more of the total radioactivity) in the cya+ control culture, radioactivity in 41 proteins was at least ten times greater in cells grown with osmotic stress. Six of these individual proteins each accounted for 1.0% or more of the total radioactive label in the cells. Conversely, radioactivity in 31 major proteins appeared to decrease at least ten times when cells grew with osmotic stress. These data indicate that the response of the bacterium to osmotic stress involves induction of some proteins and repression of others. 61% of the proteins that appear to be stimulated by salt stress were found in both strains indicating there is no obligatory requirement for cAMP.     

Proteomic changes in Debaryomyces hansenii upon exposure to NaCl stress

The proteome of the highly NaCl-tolerant yeast Debaryomyces hansenii was investigated by two-dimensional polyacrylamide gel electrophoresis (2D PAGE), and 47 protein spots were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) followed by mass spectrometry (MS). The influence of NaCl on the D. hansenii proteome was investigated during the first 3 h of NaCl exposure. The rate of protein synthesis was strongly decreased by exposure to 8% and 12% (w/v) NaCl, as the average incorporation rates of l-[(35)S]methionine within the first 30 min after addition of NaCl were only 7% and 4% of the rate in medium without NaCl. In addition, the number of protein spots detected on 2D gels prepared from cells exposed to 8% and 12% (w/v) NaCl exceeded less than 28% of the number of protein spots detected on 2D gels prepared from cells without added NaCl. Several proteins were identified as being either induced or repressed upon NaCl exposure. The induced proteins were enzymes involved in glycerol synthesis/dissimilation and the upper part of glycolysis, whereas the repressed proteins were enzymes involved in the lower part of glycolysis, the route to the Krebs cycle, and the synthesis of amino acids. Furthermore, one heat shock protein (Ssa1p) was induced, whereas others (Ssb2p and Hsp60p) were repressed.