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.     

Defective plasmid partition in ftsH mutants of Escherichia coli

FtsH is an ATP-dependent protease that is essential for cell viability in Escherichia coli. The essential function of FtsH is to maintain the proper balance of biosynthesis of major membrane components, lipopolysaccharide and phospholipids. F plasmid uses a partitioning system and is localized at specific cell positions, which may be related to the cell envelope, to ensure accurate partitioning. We have examined the effects of ftsH mutations on the maintenance of a mini-F plasmid, and have found that temperature-sensitive ftsH mutants are defective in mini-F plasmid partition, but not replication, at permissive temperature for cell growth. A significant fraction of replicated plasmid molecules tend to localize close together on one side of the cell, which may result in failure to pass the plasmid to one of the two daughter cells upon cell division. By contrast, an ftsH null mutant carrying the suppressor mutation sfhC did not affect partitioning of the plasmid. The sfhC mutation also suppressed defective maintenance in temperature-sensitive ftsH mutants. Using this new phenotype caused by ftsH mutations, we also isolated a new temperature-sensitive ftsH mutant. Mutations in ftsH cause an increase in the lipopolysaccharide/ phospholipid ratio due to stabilization of the lpxC gene product, which is involved in lipopolysaccharide synthesis and is a substrate for proteolysis by the FtsH protease. It is likely that altered membrane structure affects the localization or activity of a putative plasmid partitioning apparatus located at positions equivalent to 1/4 and 3/4 of the cell length.     

Defective transport in S-adenosylmethionine synthetase mutants of Escherichia coli

The transport of several metabolites is decreased in mutant strains of Escherichia coli (Met K, E4 and E40), which contain decreased levels of S-adenosylmethionine synthetase. The rates and extents of uptake for lysine, leucine, methionine, and α-methylglucoside in both whole cells and membrane vesicles isolated from these mutants are 2- to 10-fold lower than in corresponding preparations from wild-type cells, although proline uptake is normal. The addition of S-adenosylmethionine to cultures of strain E40 can partially restore the rate and extent of lysine uptake. Lysine transport is lower in mutant vesicles in the presence of either d-lactate, succinate, α-hydroxylbutyrate, or NADH even though these substrates are oxidized at rates comparable to those in wild-type vesicles. This suggests that the defect is not related to the ability of vesicles to oxidize electron donors, but is very likely related to the ability of mutant vesicles to couple respiration to lysine transport. In addition, temperature-induced efflux of α-methylglucoside phosphate and dinitrophenol-induced efflux of lysine are similar in both the mutant and wild-type membranes, indicating that the barrier properties of the membrane and the activity of the lysine carrier are normal.     

Physiological improvement to enhance Escherichia coli cell-surface display via reducing extracytoplasmic stress

Cell physiology was impaired when enhanced yellow fluorescence protein (EYFP) was displayed on the Escherichia coli cell surface, resulting in growth arrest and poor display performance. Coexpression of Skp, a periplasmic chaperone known to interact with several outer membrane proteins for their transport and insertion in the outer membrane, was demonstrated to be effective to restore cell physiology. When Skp was coexpressed with EYFP display, host cells became less sensitive to ethylenediaminetetraacetic acid and sodium dodecyl sulfate, implying that cell physiology was improved. Most importantly, the display performance was highly enhanced as a result of the increased specific fluorescence intensity without growth arrest. The results of transmission electron microscopy indicate that the density of surface-displayed EYFP was highly increased upon Skp coexpression. Cells with EYFP display experienced extracytoplasmic stress, as reflected by the induced promoter activities of three stress-responsive genes, degP, cpxP, and rpoH. The extracytoplasmic stress reflected by the degP promoter activity appears to be consistent with the cell physiology observed phenotypically under various culture conditions for cell-surface display. Therefore, the PdegP::lacZ allele was proposed to be a suitable "sensor" for monitoring the extracytoplasmic stress and cell physiology during the course of E. coli cell-surface display.     

Impairment of cell division in tolA mutants of Escherichia coli at low and high medium osmolarities.

Mutations in the tolA gene of Escherichia coli cause the cell to become sensitive to detergents and to some antibiotics, to release periplasmic enzymes and to be resistant to group A colicins; tolA mutations also lead to mucoid phenotype. TolA is a three-domain protein anchored in the inner membrane by its N-terminal domain. The second domain is proposed to span the periplasmic space and to interact with trimeric porins of the outer membrane. TolA proteins are considered to be located in the adhesion zones between inner and outer membranes. Our observations by confocal and electron microscopy have revealed that tolA mutants show modified morphology and produce DNA-free cells. Increasing or decreasing medium osmolarity amplifies these defects; mutants become essentially unable to locate the division site properly so that cells of highly unequal lengths are produced. Moreover, septation is impaired with asymmetric constrictions and oblique septa. These results suggest that TolA could play a role in positioning the division sites via the organisation of either the outer membrane or the possible adhesion zones.     

Loss of O-antigen increases cell shape abnormalities in penicillin-binding protein mutants of Escherichia coli

Escherichia coli mutants lacking multiple penicillin-binding proteins (PBPs) produce aberrantly shaped cells. However, most of these experiments have been performed in E. coli K12 strains, which do not attach a complete O-antigen to their outer membrane lipopolysaccharide. We constructed mutants in different genetic backgrounds and found that the frequency of morphological deformities was higher in strains lacking the O-antigen. Also, complementing O-negative mutants with a heterologous O-antigen from Klebsiella returned a substantial fraction of misshapen cells to a normal morphology. Thus, the O-antigen contributes to cell shape in E. coli, perhaps by reducing the number of ectopic poles, which may be the proximal cause of shape abnormalities.     

Cell response of Escherichia coli to cisplatin-induced stress

Cisplatin is undoubtedly one of the most common and successful anticancer drugs worldwide. Though its DNA-based mechanism of action is well established, the contribution of the proteome to this process remains unclear. The possible impact of particular Escherichia coli proteins on the cytostatic activity of cisplatin was the subject of this study. Our main focus was not only the "bottom-up" identification of novel cisplatin protein targets through LC/LC-MS/MS analysis, but also a label-free quantification of their regulation profile by spectral-counting. The regulation of two proteins, aconitate hydratase 2 and 60 kDa chaperonin 1, could be linked to a platinated amino acid in the protein sequence, whereas in the cases of 30S ribosomal protein S1 and enolase, it could be shown that cisplatin fragments are coordinated to an essential site for the functionality of the protein. Nucleoside triphosphate pyrophosphohydrolase (MazG) regulates the programmed cell death and was found to be platinated on the protein surface, which probably correlates with the established mode of action. A possible new chapter in the understanding of cisplatin's mechanism of action and its severe side effects is opened, since evidence is provided that platinated proteins are not only involved in cellular stress response but also in energy metabolism through glycolysis and catabolic processes, in gene regulatory mechanisms and protein synthesis.     

Structural studies of the Escherichia coli O-antigen 6

The structure of the Escherichia coli O-antigen 6 has been investigated using n.m.r. spectroscopy, methylation analysis, and various specific degradations. It is concluded that the O-antigen is composed of pentasaccharide repeating-units having the following structure. (Formula: see text)     

Protein expression in response to folate stress in Escherichia coli.

Interruption of folate metabolism by trimethoprim results in the elevated expression of folate stress proteins in Escherichia coli. E. coli grown in culture medium supplemented with the folate-dependent metabolites glycine, methionine, and the purine nucleoside inosine shows reduced expression of folate stress proteins. The folate stress proteins include the universal stress protein, the ferric uptake regulatory repressor, and possibly, lipoamide dehydrogenase, the L protein component of the glycine cleavage enzyme complex.     

A two-plasmid system for identification of promoters recognized by RNA polymerase containing extracytoplasmic stress response sigma(E) in Escherichia coli

We have previously established a two-plasmid system in Escherichia coli for identification of promoters recognized by RNA polymerase containing a heterologous sigma factor. Attempts to optimize this system for identification of promoters recognized by RNA polymerase containing E. coli extracytoplasmic stress response sigma(E) failed owing to high toxicity of the expressed rpoE. A new system for identification of sigma(E)-cognate promoters was established, and verified using the two known sigma(E)-dependent promoters, rpoEp2 and degPp. Expression of the sigma(E)-encoding rpoE gene was under the control of the AraC-dependent P(BAD) promoter. A low level of arabinose induced a non-toxic, however, sufficient level of sigma(E) to interact with the core enzyme of RNA polymerase. Such an RNA polymerase holoenzyme recognized both known sigma(E)-dependent promoters, rpoEp2 and degPp, which were cloned in the compatible promoter probe plasmid, upstream of a promoterless lacZ alpha reporter gene. This new system has proved to be useful for identification of E. coli sigma(E)-cognate promoters. Moreover, the system could be used for identification of ECF sigma-cognate promoters from other bacteria.     

Structural studies of the Escherichia coli O-antigen 25

The structure of the Escherichia coli O-antigen 25 has been investigated using n.m.r. spectroscopy, methylation analysis, and various specific degradations. It is concluded that the O-antigen is composed of pentasaccharide repeating-units having the following structure.     

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.     

Involvement of oxidative stress in hydroquinone-induced cytotoxicity in catalase-deficient Escherichia coli mutants

Hydroquinone is a benzene-derived metabolite. To clarify whether the reactive oxygen species (ROS) are involved in hydroquinone-induced cytotoxicity, we constructed transformants of Escherichia coli (E. coli) strains that express mammalian catalase gene derived from catalase mutant mice (Cs(b), Cs(c)) and the wild-type (Cs(a)) using a catalase-deficient E. coli UM255 as a recipient. Specific catalase activities of these tester strains were in order of Cs(a) > Cs(c) > Cs(b) > UM255, and their susceptibility to hydrogen peroxide (H2O2) showed UM255 > Cs(b) > Cs(c) > Cs(a). We found that hydroquinone exposure reduced the survival of catalase-deficient E. coli mutants in a dose-dependent manner significantly, especially in the strains with lower catalase activities. Hydroquinone toxicity was also confirmed using zone of inhibition test, in which UM255 was the most susceptible, showing the largest zone of growth inhibition, followed by Cs(b), Cs(c) and Cs(a). Furthermore, we found that hydroquinone-induced cell damage was inhibited by the pretreatment of catalase, ascorbic acid, dimethyl sulfoxide (DMSO), and ethylenediaminetetraacetic acid (EDTA), and augmented by superoxide dismutase (both CuZnSOD and MnSOD). The present results suggest that H2O2 is probably involved in hydroquinone-induced cytotoxicity in catalase-deficient E. coli mutants and catalase plays an important role in protection of the cells against hydroquinone toxicity.     

Involvement of oxidative stress in hydroquinone-induced cytotoxicity in catalase-deficient Escherichia coli mutants

Hydroquinone is a benzene-derived metabolite. To clarify whether the reactive oxygen species (ROS) are involved in hydroquinone-induced cytotoxicity, we constructed transformants of Escherichia coli (E. coli) strains that express mammalian catalase gene derived from catalase mutant mice (Cs^b, Cs^c) and the wild-type (Cs^a) using a catalase-deficient E. coli UM255 as a recipient. Specific catalase activities of these tester strains were in order of Cs^a > Cs^c > Cs^b > UM255, and their susceptibility to hydrogen peroxide (H₂O₂) showed UM255 > Cs^b > Cs^c > Cs^a. We found that hydroquinone exposure reduced the survival of catalase-deficient E. coli mutants in a dose-dependent manner significantly, especially in the strains with lower catalase activities. Hydroquinone toxicity was also confirmed using zone of inhibition test, in which UM255 was the most susceptible, showing the largest zone of growth inhibition, followed by Cs^b, Cs^c and Cs^a. Furthermore, we found that hydroquinone-induced cell damage was inhibited by the pretreatment of catalase, ascorbic acid, dimethyl sulfoxide (DMSO), and ethylenediaminetetraacetic acid (EDTA), and augmented by superoxide dismutase (both CuZnSOD and MnSOD). The present results suggest that H₂O₂ is probably involved in hydroquinone-induced cytotoxicity in catalase-deficient E. coli mutants and catalase plays an important role in protection of the cells against hydroquinone toxicity.     

Defective F pili and other characteristics of Flac and Hfr Escherichia coli mutants resistant to bacteriophage R17.

Mutants resistant to the donor-specific bacteriophage R17 were isolated from Hfr and Flac-containing strains of Escherichia coli K-12. Thirty-five mutants were examined for the presence of F pili by electron microscopy. The pilus morphology was studied, as were the abilities of the cells to retract their pili and to synthesize new pili. Measurements were made of the efficiency of the conjugal deoxyribonucleic acid transfer and of M13 and R17 phage infection. All mutants had noticeable defects in pilus production, structure, or function. Mutants were found which produced unusually long pili, displayed wide variations in the number of pili per cell, and were deficient in pilus retraction and synthesis. Evidence is presented that there may be two pathways of pilus retraction.     

Immunological detection of heterogeneous O-antigen containing lipopolysaccharides in Escherichia coli

The components of the cell envelopes of Escherichia coli O1:K1, O7:K1, O18:K1 and O83:K1 strains were separated on SDS-polyacrylamide gels. Longitudinal slices (50 microns thick) of the gel were incubated with typing sera for E. coli O1, O7, O18 and O83, followed by detection of the bound antibodies with 125I-labelled protein A and autoradiography. The antisera reacted with many cell envelope components of strains both with the homologous O-serotype and heterologous O-serotypes. With O-typing sera cross-reactions with heterologous cells and cells boiled for 2 h were found. Up to 40 serotype-specific bands at regular positions with molecular weights between 12000 and 100000 were demonstrated. Since these bands were also observed when purified lipopolysaccharide and unabsorbed homologous O-typing sera were used, it was concluded that these bands represented lipopolysaccharide molecules with increasing molecular weight, all of which contained O-antigen specific immunodeterminants. The band patterns were not influenced by the growth conditions of the cells or the various isolation procedures for the cell envelopes. Comparison of various strains serotyped as O18 revealed strain differences with respect to their lipopolysaccharide band patterns. In the case of O21- and O83-serotyped strains lipopolysaccharide cross-reactions, which were detected by agglutination, were analysed in detail using the gel immunoradioassay method. These cross-reactions appeared to be caused by the presence of common determinants on their lipopolysaccharides and polysaccharide-like material. The cross-reacting antibodies could be removed by cross-absorption. It is concluded that the immunological detection of lipopolysaccharides and other components of E. coli in gels is an important tool in (1) the control of the specificity of typing antisera, (2) the study of the nature of cross-reacting antigens and (3) the study of the nature and uniformity of the various O- and K-serotypes.     

Polyamine transport and role of potE in response to osmotic stress in Escherichia coli

When transport of polyamines in Escherichia coli was examined, putrescine excretion was observed under two different physiological conditions: (i) strictly correlated to growth and (ii) following a hyperosmotic shock. Spermidine was not excreted. Characterization of a deletion mutant showed that PotE is not involved in these transport processes.