Characterisation of cisplatin coordination sites in cellular <Emphasis Type="Italic">Escherichia coli</Emphasis> DNA-binding proteins by combined biphasic liquid chromatography and ESI tandem mass spectrometry

Combined multidimensional liquid chromatography and electrospray ionisation tandem mass spectrometry was employed to analyse platinated tryptic peptides from Escherichia coli cells treated with the anticancer drug cis-[PtCl₂(NH₃)₂] at pH 7.0. Prerequisites for the LC/LC/MS/MS analysis of protein targets that are fulfilled by cisplatin are (a) that the original protein binding sites have a high kinetic stability over the range 2.3 < pH < 8.5, and (b) that the metal fragment remains coordinated to a significant number of b⁺ and y⁺ peptide ions under MS/MS fragmentation conditions. Matching the MS/MS spectra of the platinated tryptic peptides to sequences of proteins in the E. coli database enabled the identification of 31 protein targets for cisplatin. Whereas six of these are high-abundance enzymes and ribosomal proteins in E. coli cells, five low-abundance DNA-binding proteins were also identified as specific targets. These include the DNA mismatch repair protein mutS, the DNA helicase II (uvrD) and topoisomerase I (top1). Two efflux proteins (acrD, mdtA), the redox regulator thioredoxin 1 (thiO) and the external filament-like type-1 fimbrial protein A chain (fimA1) were also characterised as specific cisplatin-binding proteins. Kinetically favoured carboxylate (D, E) and hydroxy (S, T, Y) O atoms were identified as the Pt coordination sites in 18 proteins and methionyl S atoms in 9 proteins.     

The Escherichia coli O157:H7 cattle immunoproteome includes outer membrane protein A (OmpA), a modulator of adherence to bovine rectoanal junction squamous epithelial (RSE) cells

Building on previous studies, we defined the repertoire of proteins comprising the immunoproteome (IP) of Escherichia coli O157:H7 (O157) cultured in DMEM supplemented with norepinephrine (O157 IP), a β‐adrenergic hormone that regulates E. coli O157 gene expression in the gastrointestinal tract, using a variation of a novel proteomics‐based platform proteome mining tool for antigen discovery, called “proteomics‐based expression library screening” (PELS; Kudva et al., 2006). The E. coli O157 IP (O157‐IP) comprised 91 proteins, and included those identified previously using proteomics‐based expression library screening, and also proteins comprising DMEM and bovine rumen fluid proteomes. Outer membrane protein A (OmpA), a common component of the above proteomes, and reportedly a contributor to E. coli O157 adherence to cultured HEp‐2 epithelial cells, was interestingly found to be a modulator rather than a contributor to E. coli O157 adherence to bovine rectoanal junction squamous epithelial cells. Our results point to a role for yet to be identified members of the O157‐IP in E. coli O157 adherence to rectoanal junction squamous epithelial cells, and additionally implicate a possible role for the outer membrane protein A regulator, TdcA, in the expression of such adhesins. Our observations have implications for the development of efficacious vaccines for preventing E. coli O157 colonization of the bovine gastrointestinal tract.     

Challenges Associated with Heterologous Expression of <Emphasis Type="Italic">Flavobacterium psychrophilum</Emphasis> Proteins in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A two-parameter statistical model was used to predict the solubility of 96 putative virulence-associated proteins of Flavobacterium psychrophilum (CSF259-93) upon over expression in Escherichia coli. This analysis indicated that 88.5% of the F. psychrophilum proteins would be expressed as insoluble aggregates (inclusion bodies). These solubility predictions were verified experimentally by colony filtration blot for six different F. psychrophilum proteins. A comprehensive analysis of codon usage identified over a dozen codons that are used frequently in F. psychrophilum, but that are rarely used in E. coli. Expression of F. psychrophilum proteins in E. coli was often associated with production of minor molecular weight products, presumably because of the codon usage bias between these two organisms. Expression of recombinant protein in the presence of rare tRNA genes resulted in marginal improvements in the expressed products. Consequently, Vibrio parahaemolyticus was developed as an alternative expression host because its codon usage is similar to F. psychrophilum. A full-length recombinant F. psychrophilum hemolysin was successfully expressed and purified from V. parahaemolyticus in soluble form, whereas this protein was insoluble upon expression in E. coli. We show that V. parahaemolyticus can be used as an alternate heterologous expression system that can remedy challenges associated with expression and production of F. psychrophilum recombinant proteins.     

ATP-binding cassette transporters in Escherichia coli

ATP-binding cassette (ABC) transporters are integral membrane proteins that actively transport molecules across cell membranes. In Escherichia coli they consist primarily of import systems that involve in addition to the ABC transporter itself a substrate binding protein and outer membrane receptors or porins, and a number of transporters with varied functions. Recent crystal structures of a number of ATPase domains, substrate binding proteins, and full-length transporters have given new insight in the molecular basis of transport. Bioinformatics approaches allow an approximate identification of all ABC transporters in E. coli and their relation to other known transporters. Computational approaches involving modeling and simulation are beginning to yield insight into the dynamics of the transporters. We summarize the function of the known ABC transporters in E. coli and mechanistic insights from structural and computational studies.     

Differential effect of YidC depletion on the membrane proteome of Escherichia coli under aerobic and anaerobic growth conditions

YidC of Escherichia coli belongs to the evolutionarily conserved Oxa1/Alb3/YidC family. Members of the family have all been implicated in membrane protein biogenesis of respiratory and energy transducing proteins. The number of proteins identified thus far to require YidC for their membrane biogenesis remains limited and the identification of new substrates may allow the elucidation of properties that define the YidC specificity. To this end we investigated changes in the membrane proteome of E. coli upon YidC depletion using metabolic labeling of proteins with ¹⁵N/¹⁴N combined with a MS‐centered proteomics approach and compared the effects of YidC depletion under aerobic and anaerobic growth conditions. We found that YidC depletion resulted in protein aggregation/misfolding in the cytoplasm as well as in the inner membrane of E. coli. A dramatic increase was observed in the chaperone‐mediated stress response upon YidC depletion and this response was limited to aerobically grown cells. A number of transporter proteins were identified as possible candidates for the YidC‐dependent insertion and/or folding pathway. These included the small metal ion transporter CorA, numerous ABC transporters, as well as the MFS transporters KgtP and ProP, providing a new subset of proteins potentially requiring YidC for membrane biogenesis.     

Why does ethanol induce cellular heat-shock response?

At the time of induction of the periplasmic protein alkaline phosphatase (AP) in Escherichia coli, the presence of ethanol (10% v/v) in the growth medium did not allow the induced AP to be translocated out to the periplasm. The nontransported AP was stored in the cytoplasm as the unfolded precursor form (AP with its amino-terminal signal sequence), which had no enzymatic activity. The presence of 10% v/v ethanol in the growth medium also induced the heat-shock response in E. coli, which was evident from the enhanced syntheses of several heat-shock proteins (HSPs) over their cellular basal levels. These results, in conjunction with our earlier findings on the occurrence of heat-shock response in an AP-signal sequence mutant of E. coli due to the export deficiency of AP precursor, suggest that the membrane protein precursors, stored in the cytoplasm due to the ethanol-mediated inhibition of translocation, behaved to the cells as abnormal proteins, which ultimately triggered the signal for the induction of heat-shock response in E. coli.     

Cellular Responses and Proteomic Analysis of <Emphasis Type="Italic">Escherichia coli</Emphasis> Exposed to Green Tea Polyphenols

The purpose of this study was to characterize the cellular response and proteomic analysis of Escherichia coli exposed to tea polyphenols (TPP) extracted from Korean green tea (Camellia sinensis L). TPP showed a dose-dependent bactericidal effect on E. coli. Analysis of cell-membrane fatty acids of E. coli cultures treated with TPP identified unique changes in saturated and unsaturated fatty acids, whereas scanning electron microscopic analysis demonstrated the presence of perforations and irregular rod forms with wrinkled surfaces in cells treated with TPP. Two-dimensional polyacrylamide gel electrophoresis of soluble protein fractions from E. coli cultures exposed to TPP showed 17 protein spots increased or decreased by TPP. Nine upregulated proteins were identified (including GroEL and proteins involved in cellular defense, such as GyrA, RpoS, SodC, and EmrK), whereas the expression of eight proteins was downregulated by exposure to TPP (including proteins involved in carbon and energy metabolism, such as Eno, SdhA, and UgpQ, as well as those involved in amino-acid biosynthesis, such as GltK and TyrB). These results provide clues for understanding the mechanism of TPP-induced stress and cytotoxicity on E. coli.     

Molecular analysis of the Escherichia coli hns gene encoding a DNA-binding protein, which preferentially recognizes curved DNA sequences.

Summary We previously demonstrated that the E. coli protein, H-NS (or Hla), encoded by the gene hns (or osmZ or bglY preferentially recognizes curved DNA sequences in vitro. In order to gain further insight into the complex function of H-NS and the significance of DNA curvature, we constructed a structurally defined hns deletion mutant on the E. coli chromosome. The hns deletion mutant thus obtained showed a variety of phenotypes previously for other lesions in hns. It was further demonstrated that, in this hns deletion background, numerous E. coli cellular proteins were either strongly expressed or remarkably repressed, as compared to their expression levels in wild-type cells.     

Escherichia coli as an expression system for K(+) transport systems from plants

The value of theEscherichia coli expression system has long been establishedbecause of its effectiveness in characterizing the structure andfunction of exogenously expressed proteins. When eukaryotic membraneproteins are functionally expressed in E. coli, thisorganism can serve as an alternative to eukaryotic host cells. A fewexamples have been reported of functional expression of animal andplant membrane proteins in E. coli. This mini-review describes the following findings: 1) homologousK⁺ transporters exist in prokaryotic cells and ineukaryotic cells; 2) plant K⁺ transporters canfunctionally complement mutant K⁺ transporter genes inE. coli; and 3) membrane structures of plant K⁺ transporters can be elucidated in an E. colisystem. These experimental findings suggest the possibility ofutilizing the E. coli bacterium as an expression system forother eukaryotic membrane transport proteins.

     

Isolation of oxidative stress response mutants in Escherichia coli for their use as a genetic screen to identify new anti-mycobacterials as functional analogues of isoniazid

Summary Tuberculosis is a leading killer disease of the world with increasing mortality due to HIV-infected individuals becoming highly prone to this infection. An attempt has been made in the present work to identify novel plant-derived compounds active against Mycobacterium tuberculosis (MTB) through construction of a target based bio-screen to facilitate rapid screening of anti-TB plant compounds. To achieve this, construction of a genetically modified model system was attempted in fast growing, non-pathogenic, Escherichia coli in which experimental testing is relatively easier and rapid as compared to M. tuberculosis, which is pathogenic and slow growing in nature. The exquisitely high sensitivity of M. tuberculosis to isoniazid (INH) has been attributed to lesions in oxyR, a gene that positively regulates the expression of a set of hydrogen peroxide-inducible genes in E. coli and S. typhimurium. Moreover in the mechanism of emergence of INH resistance in M. tuberculosis, oxidative stress response has been implicated. In this study, mutants of E. coli defective in oxidative stress response function were derived and used to screen plant compounds, which might interfere with the oxidative stress response in MTB. Since MTB is inherently known to be oxyR defective and thus being highly sensitive to INH, mutants defective in oxidative stress response were isolated to construct a model system in E. coli, which is otherwise INH resistant, having functional oxyR. These mutants showed simultaneous sensitivity to oxidative stress-causing agents like hydrogen peroxide and cumene hydroperoxide. To further define the mutational lesions, complementation studies were carried out through mobilization of cloned wild type genes involved in the oxidative stress response and in this way a biological screen was constructed to identify plant compounds/essential oils/extracts/oil components which induce oxidative stress. The positives were finally tested for activity against M. tuberculosis strain H37Rv using the radiometric BACTEC 460 TB system. Interestingly, the bioactives were found to be active against the pathogen with marked potency, as the reduction in δGI values for the identified bioactives against M. tuberculosis were significant. The study demonstrates application of a specific target-based genetic model system in E. coli as a rapid high throughput screen in identifying anti-mycobacterials from plants.     

Characterization of the heat shock response inEnterococcus faecalis

We have characterized the general properties of the heat shock response of the Gram-positive hardy bacteriumEnterococcus faecalis. The heat resistance (60°C or 62.5°C, 30 min) of log phase cells ofE. faecalis grown at 37°C was enhanced by exposing cells to a prior heat shock at 45°C or 50°C for 30 min. These conditioning temperatures also induced ethanol (22%, v/v) tolerance. The onset of thermotolerance was accompanied by the synthesis of a number of heat shock proteins. The most prominent bands had molecular weights in the range of 48 to 94kDa. By Western blot analysis two of them were found to be immunologically related to the well known DnaK (72 kDa) and GroEL (63 kDa) heat shock proteins ofEscherichia coli. Four other proteins showing little or no variations after exposure to heat are related to DnaJ, GrpE and Lon (La)E. coli proteins and to theBacillus subtilis ⁴³ factor. Ethanol (2% or 4%, v/v) treatments elicited a similar response although there was a weaker induction of heat shock proteins than with heat shock.     

携带Paenibacillus polymyxa WLY78固氮基因簇(nif)的重组大肠杆菌Escherichia coli78-7转录组分析

[目的]来自Paenibacillus polymyxa WLY78的固氮基因簇（nifBHDKEfNXhesAnifV）可以转化入Escherichia coli中表达并使重组大肠杆菌合成有固氮活性的固氮酶。本文拟通过对重组大肠杆菌E.coli 78-7的转录组分析以提高其固氮能力。[方法]对固氮条件（无氧无NH₄⁺）和非固氮条件（空气和100 mmol/L NH₄⁺）培养的重组大肠杆菌E.coli 78-7进行转录组分析。[结果]nif基因在两种培养条件下显著表达,说明在重组大肠杆菌中可规避原菌中氧气和NH₄⁺对nif基因的负调控。对于固氮过程必需的非nif基因,如参与钼、硫、铁元素转运的mod、cys和feoAB,这些基因在两种培养条件下表达水平有差异。而参与铁硫簇合成的suf和isc基因簇在两条件下表达水平差异巨大。此外,参与氮代谢的基因在固氮条件下显著上调。[结论]重组大肠杆菌中与固氮相关的非nif基因在该菌的固氮过程中具有较大影响,本文对在异源宿主中调高固氮酶活性研究具有重要意义。     

Eukaryotic integral membrane protein expression utilizing the <Emphasis Type="Italic">Escherichia coli</Emphasis> glycerol-conducting channel protein (GlpF)

A fusion protein expression system is described that allows for production of eukaryotic integral membrane proteins in Escherichia coli (E. coli). The eukaryotic membrane protein targets are fused to the C terminus of the highly expressed E. coli inner membrane protein, GlpF (the glycerol-conducting channel protein). The generic utility of this system for heterologous membrane-protein expression is demonstrated by the expression and insertion into the E. coli cell membrane of the human membrane proteins: occludin, claudin 4, duodenal ferric reductase and a J-type inwardly rectifying potassium channel. The proteins are produced with C-terminal hexahistidine tags (to permit purification of the expressed fusion proteins using immobilized metal affinity chromatography) and a peptidase cleavage site (to allow recovery of the unfused eukaryotic protein).     

Molecular cloning of a maltose transport gene from Bacillus stearothermophilus and its expression in Escherichia coli K-12

Genes responsible for maltose utilization from Bacillus stearothermophilus ATCC7953 were cloned in the plasmid vector pBR325 and functionally expressed in Escherichia coli. The 4.2 kb Bacillus DNA insert in clone pAM1750 suppressed the growth defects on maltose caused by mutations in E. coli maltose transport genes (malE, malK or complete malB deletion) but not mutations in genes affecting intracellular maltose metabolism (malA region). Transport studies in E. coli and B. stearothermophilus suggested that pAM1750 codes for a high affinity transport system, probably one of two maltose uptake systems found in B. stearothermophilus ATCC7953. Nucleotide sequence analysis of a 3.6 kb fragment of pAM 1750 revealed three open reading frames (ORFs). One of the ORFs, malA, encoded a putative hydrophobic protein with 12 potential transmembrane segments. MalA showed amino acid sequence similarity to proteins in the superfamily containing LacY lactose permease and also some similarity to MaIG protein, a member of a binding protein-dependent transport system in E. coli. The products of two other ORFs were not hydrophobic, did not show similarity to other known sequences and were found not to be essential for maltose utilization in transport-defective E. coli mutants. Hence MalA protein was the only protein necessary for maltose transport, but despite giving a detectable but low level of transport function in E. coli, the protein was very poorly expressed and could not be identified.     

Roles of Bcl-2 family members,PI3K and NF-κB pathways in <Emphasis Type="Italic">Escherichia coli</Emphasis>-induced apoptosis in human monocytic U937 cells

We previously showed that infection of human monocytic U937 cells with nonpathogenic Escherichia coli (E. coli) induced rapid apoptosis in a dose- and time-dependent manner. We also found that E. coli increase p38 mitogen-activated protein Kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK), and decrease extracellular-Regulated Kinase1/2 (ERK1/2) phosphorylation and increase caspase-3 and -9 activity in U937 cells. The current study determines if Bcl-2, Bax, the phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor kappa B (NF-κB) regulates E. coli–induced U937 cell apoptosis. Studying the underlying mechanisms we found that the E. coli-induced apoptosis in U937 cells was associated with a more prominent reduction in expression of Bcl-2, levels of P-Akt and NF-κB. Because levels of inhibition of apoptosis protein (cIAP), and X-chromosomelinked inhibitor of apoptosis protein (XIAP) are regulated by NF-κB, E. coli decreased the levels of these proteins in U937 cells through inhibition of NF-κB. Moreover, E. coli markedly elevated Bax expression and cytochrome c redistribution. LY294002, PDTC and Embelin, specific inhibitors of PI3K, NF-κB and XIAP, induced U937 cell apoptosis and the apoptosis is dependent on activity of caspase-3 and -9 in E. coli-treated U937 cells. Through using LY294002 and western blotting, we identified NF-κB was the downstream Akt target regulated by E. coli. Taken together, these results clearly indicate reduced activation of NF-κB via impaired PI3K/Akt activation could result in increased apoptosis of U937 cells infected by E. coli. Moreover, E. coli can induce apoptosis with an increased expression of Bax and a reduced expression of Bcl-2, which resulted in increased levels of cytochrome c release and increase caspase-3 and -9 in U937 cells.     

Enhanced tolerance against freezing stress in<Emphasis Type="Italic">Escherichia coli</Emphasis> cells expressing an algal cyclophilin gene

Members of the cyclophilin (Cyp) family are known to function as co-chaperones, interacting with chaperones such as heat shock protein 90, and perform important roles in protein folding under high temperature stress. In addition, they have been isolated from a wide range of organisms. However, there have been no reports on the functions of algal Cyps under other stress conditions. To study the functions of the cDNAGjCyp-1 isolated from the red alga (Griffithsia japonica), a recombinant GjCyp-1 containing a hexahistidine tag at the amino-terminus was constructed and expressed inEscherichia coli. Most of the gene product expressed inE. coli was organized as aggregate insoluble particles known as inclusion bodies. Thus, the optimal time, temperature, and concentration ofl(+)-arabinose for expressing the soluble and nonaggregated form of GjCyp-1 inE. coli were examined. The results indicate that the induction of Cyp, at 0.2%l(+)-arabinose for 2 h at 25°C, had a marked effect on the yield of the soluble and active form of the co-chaperone as PPlase. An expressed fusion protein, H₆GjCyp-1, maintained the stability ofE. coli proteins up to-75°C. In a functional bioassay of the recombinant H₆GjCyp-1, the viability ofE. coli cells overexpressing H₆GjCyp-1 was compared to that of cells not expressing H₆GjCyp-1 at −75°C. For all the cycles of a freeze/thaw treatment, a significant increase in viability was observed in theE. coli cells overexpressing H₆GjCyp-1. The results of the GjCyp-1 bioassays, as well asin vitro studies, strongly suggest that the algal Cyp confers freeze tolerance toE. coli.     

The adaptive acid tolerance response in root nodule bacteria and Escherichia coli

Root nodule bacteria and Escherichia coli show an adaptive acid tolerance response when grown under mildly acidic conditions. This is defined in terms of the rate of cell death upon exposure to acid shock at pH 3.0 and expressed in terms of a decimal reduction time, D. The D values varied with the strain and the pH of the culture medium. Early exponential phase cells of three strains of Rhizobium leguminosarum (WU95, 3001 and WSM710) had D values of 1, 6 and 5 min respectively when grown at pH 7.0; and D values of 5, 20 and 12 min respectively when grown at pH 5.0. Exponential phase cells of Rhizobium tropici UMR1899, Bradyrhizobium japonicum USDA110 and peanut Bradyrhizobium sp. NC92 were more tolerant with D values of 31, 35 and 42 min when grown at pH 7.0; and 56, 86 and 68 min when grown at pH 5.0. Cells of E. coli UB1301 in early exponential phase at pH 7.0 had a D value of 16 min, whereas at pH 5.0 it was 76 min. Stationary phase cells of R. leguminosarum and E. coli were more tolerant (D values usually 2 to 5-fold higher) than those in exponential phase. Cells of R. leguminosarum bv. trifolii 3001 or E. coli UB1301 transferred from cultures at pH. 7.0 to medium at pH 5.0 grew immediately and induced the acid tolerance response within one generation. This was prevented by the addition of chloramphenicol. Acidadapted cells of Rhizobium leguminosarum bv. trifolii WU95 and 3001; or E. coli UB1301, M3503 and M3504 were as sensitive to UV light as those grown at neutral pH.     

Green fluorescent protein is superior to blue fluorescent protein as a quantitative reporter of promoter activity in <Emphasis Type="Italic">E. coli</Emphasis>

Fluorescent proteins related to and derived from green fluorescent protein (GFP) are widely used as tools for investigating a wide range of biological processes. In particular, GFP and its relatives have been used extensively as qualitative reporters of gene expression in many different organisms, but relatively few studies have investigated fluorescent proteins as quantitative reporters of gene expression. GFP has some limitations as a reporter gene, including possible toxicity when expressed at high levels. Therefore, it would be useful if other fluorescent proteins could be identified for use as quantitative reporters. Toward this end, we investigated BFP as a quantitative reporter of promoter activity in E. coli and directly compared it with GFPuv using a set of well-characterized synthetic constitutive promoters. The fluorescence produced in E. coli strains expressing GFPuv or BFP grown on solid medium was quantified using a CCD camera and fluorimetry. GFPuv consistently gave more reliable and statistically significant results than did BFP in all assays. Correspondingly, we found that the signal-to-noise ratio for GFPuv fluorescence is substantially higher than for BFP. We conclude that, under the conditions assessed in this study, GFPuv is superior to BFP as a quantitative reporter of promoter activity in E. coli. J. Bayes, M. Calvey, L. Reineke, A. Colagiavanni, and M. Tscheiner made equivalent contributions to this work.