Fractionation of soluble proteins in Escherichia coli using DEAE-, SP-, and phenyl sepharose chromatographies.

In an effort to simplify a complex mixture of soluble proteins from Escherichia coli, methods to fractionate the samples prior to two-dimensional (2D) gel electrophoresis were developed. These methods involve the use of DEAE-Sepharose, SP-Sepharose, and phenyl Sepharose chromatographic columns and the fractionation of the protein mixtures based on differential anionic, cationic, and hydrophobic properties of the proteins, respectively. Fractionation of the soluble proteins from an E. coli extract with DEAE-Sepharose resulted in a threefold increase in the number of detectable 2D gel spots. These gel spots were amenable to protein identification by using in-gel trypsin digestions, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and peptide mass fingerprinting. Significantly, the DEAE-Sepharose column fractionation effectively partitioned the soluble proteins from the cell extracts. Similarly, an SP-Sepharose column was used to fractionate the soluble proteins from E. coli and resulted in over a twofold increase in the number of detectable gel spots. Lastly, fractionation of the cell extract with the phenyl Sepharose column resulted in a threefold increase in the number of detectable 2D gel spots. This work describes an easy, inexpensive way to fractionate the soluble proteins in E. coli and a way to better profile the E. coli proteome.     

Efficient disinfection of Escherichia coli in water by silver loaded alumina

The catalytic inactivation of Escherichia coli (E. coli) in water by silver loaded alumina as catalyst was investigated. Ag/Al₂O₃ and AgCl/Al₂O₃ catalysts exhibited high bactericidal activity at room temperature in water with no need for any light or electrical power input. Dissolved oxygen which can be catalyzed to reactive oxygen species (ROS) was found to be essential for the strong bactericidal activities of the catalysts. Decomposition of the cell wall leading to leakage of the intracellular component and the complete lysis of the whole cell were directly observed by transmission electron microscopy (TEM). The resultant change in cell permeability was confirmed by potassium ion leakage. The different morphological changes between E. coli cells treated with the catalysts and Ag⁺ were also observed. The formation of ROS involved in the bactericidal process by AgCl/Al₂O₃ was confirmed by addition of catalase and OH scavenger. Higher temperature and pH value were found to have positive effect on the bactericidal activity of AgCl/Al₂O₃. All these results indicated that the bactericidal effect of the catalyst was a synergic action of ROS and Ag⁺, not an additive one. A possible mechanism is proposed.     

Survival of Escherichia coli under lethal heat stress by L-form conversion

Transition of bacteria to cell wall deficient L-forms in response to stress factors has been assumed as a potential mechanism for survival of microbes under unfavorable conditions. In this article, we provide evidence of paradoxal survival through L-form conversion of E. coli high cell density population after lethal treatments (boiling or autoclaving). Light and transmission electron microscopy demonstrated conversion from classical rod to polymorphic L-form shape morphology and atypical growths of E. coli. Microcrystal formations observed at this stage were interpreted as being closely linked to the processes of L-form conversion and probably involved in the general phenomenon of protection against lethal environment. Identity of the morphologically modified L-forms as E. coli was verified by species specific DNA-based test. Our study might contribute to a better understanding of the L-form phenomenon and its importance for bacterial survival, as well as provoke reexamination of the traditional view of killing strategies against bacteria.     

Substrate specificity and properties of the Escherichia coli 16S rRNA methyltransferase, RsmE

The small ribosome subunit of Escherichia coli contains 10 base-methylated sites distributed in important functional regions. At present, seven enzymes responsible for methylation of eight bases are known, but most of them have not been well characterized. One of these enzymes, RsmE, was recently identified and shown to specifically methylate U1498. Here we describe the enzymatic properties and substrate specificity of RsmE. The enzyme forms dimers in solution and is most active in the presence of 10-15 mM Mg(2+) and 100 mM NH(4)Cl at pH 7-9; however, in the presence of spermidine, Mg(2+) is not required for activity. While small ribosome subunits obtained from an RsmE deletion strain can be methylated by purified RsmE, neither 70S ribosomes nor 50S subunits are active. Likewise, 16S rRNA obtained from the mutant strain, synthetic 16S rRNA, and 3' minor domain RNA are all very poor or inactive as substrates. 30S particles partially depleted of proteins by treatment with high concentrations of LiCl or in vitro reconstituted intermediate particles also show little or no methyl acceptor activity. Based on these data, we conclude that RsmE requires a highly structured ribonucleoprotein particle as a substrate for methylation, and that methylation events in the 3' minor domain of 16S rRNA probably occur late during 30S ribosome assembly.     

Feedback control of ribosome function in Escherichia coli

We have previously proposed that the rate of ribosome function during balanced growth in E. coli, expressed as the rate of peptide chain elongation, is adjusted by a feedback mechanism: whenever that rate is submaximal (i.e. below 22 amino acid residues polymerized per active ribosome at 37 degrees C), the feedback signal ppGpp is generated by an activation of the ppGpp synthetase expressed from the spoT gene. The accumulation of ppGpp reduces the synthesis of additional ribosomes and thereby reduces the consumption of amino acids which, in turn, allows the remaining ribosomes to function at a higher rate. Here we have described with supporting evidence the proposed feedback loop in greater detail and provided a mathematical analysis which predicts that the SpoT ppGpp synthetase activity should be highest when the ribosomes function at their half-maximal rate. This prediction is consistent with reported observations and is independent of the particular (unknown) mechanism by which the rate of translation controls the ppGpp synthetase activity of SpoT.     

Simultaneous detection of virulence factors from a colony in diarrheagenic Escherichia coli by a multiplex PCR assay with Alexa Fluor-labeled primers

We have developed simultaneous detection of eight genes associated with the five categories of diarrheagenic Escherichia coli by the multiplex PCR assay with Alexa Fluor-labeled primers. This assay can easily distinguish eight genes based on the size and color of amplified products without gel staining.     

The relationship between lay and technical views of Escherichia coli O157 risk

Here, we bring together and contrast lay (accessible primarily through social science methodologies) and technical (via risk assessment and epidemiological techniques) views of the risk associated with the Escherichia coli O157 pathogen using two case study areas in the Grampian region of Scotland, and North Wales. Epidemiological risk factors of contact with farm animals, visiting farms or farm fields and having a private water supply were associated with postcode districts of higher than average disease incidence in the human population. However, this was not the case for the epidemiological risk factor of consumption of beef burgers, which was independent of disease incidence in the postcode district of residence. The proportion of the population expressing a high knowledge of E. coli O157 was greatest in high-incidence disease districts compared with low-incidence areas (17% cf. 7%). This supports the hypothesis that in high-disease-incidence areas, residents are regularly exposed to information about the disease through local cases, the media, local social networks, etc. or perhaps that individuals are more likely to be motivated to find out about it. However, no statistically significant difference was found between high- and low-incidence postcode districts in terms of the proportion of the population expressing a high likelihood of personal risk of infection (10% cf. 14%), giving a counterintuitive difference between the technical (epidemiological and quantitative microbiological risk assessment (QMRA)) and the lay assessment of E. coli O157 risk. This suggests that lay evaluations of E. coli O157 risk reflect intuitive and experience-based estimates of the risk rather than probabilistic estimates. A generally strong correspondence was found in terms of the rank order given to potential infection pathways, with environment and foodborne infection routes dominating when comparing public understanding with technical modelling results. Two general conclusions follow from the work. First, that integrative research incorporating both lay and technical views of risk is required in order that informed decisions can be made to handle or treat the risk by the groups concerned (e.g. the public, policy makers/risk managers, etc.). Second, when communicating risk, for example, through education programmes, it is important that this process is two-way with risk managers (e.g. including Food Standards Agency officials and communications team, public health infection control and environmental health officers) both sharing information with the public and stakeholder groups, as well as incorporating public knowledge, values and context (e.g. geographical location) into risk-management decisions.     

抑制E.coli的SOS应答对恩诺沙星抗药性的影响

通过敲除SOS应答启动蛋白基因rec A,探讨SOS应答对E.coli恩诺沙星抗药性的影响,并体外评价Rec A抑制剂和恩诺沙星联用对细菌协同抑制作用的影响.利用Red重组系统,构建E.coli ATCC 25922的rec A缺失菌株E.coli ATCC 25922/?rec A;在恩诺沙星压力下,利用荧光定量PCR测定SOS应答系统相关基因rec A和umu C表达量的变化.用微量肉汤稀释法测定恩诺沙星等常用抗生素对两个菌株的MIC变化;利用梯度平板法测定恩诺沙星对两个菌株抗药性变异的影响;合成Rec A抑制剂,并评估其与恩诺沙星联合抑制E.coli生长及其抗药性的作用.结果表明,E.coli ATCC 25922/?rec A菌株对恩诺沙星的最低抑菌浓度值降低至原始菌株的1/8;经药物处理后,在梯度平板上,rec A缺失菌株较野生型不易产生抗药性;荧光定量PCR表明,rec A缺失菌株或在Rec A抑制剂作用下,SOS应答系统受到一定的抑制.敲除rec A,使菌株对恩诺沙星的抗药性和抗药率均明显降低;Rec A抑制剂在一定程度上能抑制SOS应答,起到协同抑菌作用.     

Survival of Escherichia coli in the environment: fundamental and public health aspects

In this review, our current understanding of the species Escherichia coli and its persistence in the open environment is examined. E. coli consists of six different subgroups, which are separable by genomic analyses. Strains within each subgroup occupy various ecological niches, and can be broadly characterized by either commensalistic or different pathogenic behaviour. In relevant cases, genomic islands can be pinpointed that underpin the behaviour. Thus, genomic islands of, on the one hand, broad environmental significance, and, on the other hand, virulence, are highlighted in the context of E. coli survival in its niches. A focus is further placed on experimental studies on the survival of the different types of E. coli in soil, manure and water. Overall, the data suggest that E. coli can persist, for varying periods of time, in such terrestrial and aquatic habitats. In particular, the considerable persistence of the pathogenic E. coli O157:H7 is of importance, as its acid tolerance may be expected to confer a fitness asset in the more acidic environments. In this context, the extent to which E. coli interacts with its human/animal host and the organism''s survivability in natural environments are compared. In addition, the effect of the diversity and community structure of the indigenous microbiota on the fate of invading E. coli populations in the open environment is discussed. Such a relationship is of importance to our knowledge of both public and environmental health.     

The solution structure of the oxidative stress-related protein YggX from Escherichia coli

YggX is a highly conserved protein found only in eubacteria and is proposed to be involved in the bacterial response to oxidative stress. Here we report the solution structure of YggX from Escherichia coli determined by nuclear magnetic resonance spectroscopy. The structure of YggX displays a fold consisting of two N-terminal antiparallel beta-sheets and three alpha-helices, which shares significant structural similarity to the crystal structure of a hypothetical protein PA5148 from Pseudomonas aeruginosa. Previous studies propose YggX as an iron binding protein that is involved in cellular iron trafficking. Our data indicate that the protein alone does not bind iron in vitro, suggesting other cofactors or different conditions may be necessary for metal binding.     

SOE-LRed: A simple and time-efficient method to localize genes with point mutations onto the Escherichia coli chromosome

We use a powerful method to replace wild-type genes on the chromosome of Escherichia coli. Using a unique form of PCR, we generate easily constructible gene fusions bearing single point mutations. Used in conjunction with homologous recombination, this method eliminates cloning procedures previously used for this purpose.     

Multiple equilibria of the Escherichia coli chaperonin GroES revealed by mass spectrometry

Nanospray time-of-flight mass spectrometry has been used to study the assembly of the heptamer of the Escherichia coli cochaperonin protein GroES, a system previously described as a monomer-heptamer equilibrium. In addition to the monomers and heptamers, we have found measurable amounts of dimers and hexamers, the presence of which suggests the following mechanism for heptamer assembly: 2 Monomers <--> Dimer; 3 Dimers <--> Hexamer; Hexamer + Monomer <--> Heptamer. Equilibrium constants for each of these steps, and an overall constant for the Monomer <--> Heptamer equilibrium, have been estimated from the data. These constants imply a standard free-energy change, DeltaG(0), of about 9 kcal/mol for each contact surface formed between GroES subunits, except for the addition of the last subunit, where DeltaG(0) = 6 kcal/mol. This lower value probably reflects the loss of entropy when the heptamer ring is formed. These experiments illustrate the advantages of electrospray mass spectrometry as a method of measuring all components of a multiple equilibrium system.     

Enteropathogenic Escherichia coli activates the RhoA signaling pathway via the stimulation of GEF-H1

Enteropathogenic Escherichia coli delivers a subset of effectors into host cells via a type III secretion system, and this step is required for the progression of disease. Here, we show that the type III effectors, EspG and its homolog Orf3, trigger actin stress fiber formation and the destruction of the microtubule networks beneath adherent bacteria. Both effectors were shown to possess the ability to interact with tubulins, and to stimulate microtubule destabilization in vitro. A recent study showed that microtubule-bound GEF-H1, a RhoA-specific guanine nucleotide exchange factor, was converted to its active form by microtubule destabilization, and this sequence of events resulted in RhoA stimulation. Indeed, EspG- and Orf3-induced stress fiber formation was inhibited by the expression of dominant-negative forms of GEF-H1 and RhoA, but not of Rac1 and Cdc42, and by treatment with a ROCK inhibitor. These results indicate that the impact of EspG/Orf3 on microtubule networks triggers the activation of the RhoA-ROCK signaling pathway via GEF-H1 activity. This report reveals for the first time that a pathogen can exploit the host factor GEF-H1.     

Whole genome sequence comparison of vtx2-converting phages from Enteroaggregative Haemorrhagic Escherichia coli strains

Background

Enteroaggregative Haemorrhagic E. coli (EAHEC) is a new pathogenic group of E. coli characterized by the presence of a vtx2-phage integrated in the genomic backbone of Enteroaggregative E. coli (EAggEC). So far, four distinct EAHEC serotypes have been described that caused, beside the large outbreak of infection occurred in Germany in 2011, a small outbreak and six sporadic cases of HUS in the time span 1992–2012. In the present work we determined the whole genome sequence of the vtx2-phage, termed Phi-191, present in the first described EAHEC O111:H2 isolated in France in 1992 and compared it with those of the vtx-phages whose sequences were available.

Results

The whole genome sequence of the Phi-191 phage was identical to that of the vtx2-phage {"type":"entrez-protein","attrs":{"text":"P13374","term_id":"138227","term_text":"P13374"}}P13374 present in the EAHEC O104:H4 strain isolated during the German outbreak 20 years later. Moreover, it was also almost identical to those of the other vtx2-phages of EAHEC O104:H4 strains described so far. Conversely, the Phi-191 phage appeared to be different from the vtx2-phage carried by the EAHEC O111:H21 isolated in the Northern Ireland in 2012.The comparison of the vtx2-phages sequences from EAHEC strains with those from the vtx-phages of typical Verocytotoxin-producing E. coli strains showed the presence of a 900 bp sequence uniquely associated with EAHEC phages and encoding a tail fiber.

Conclusions

At least two different vtx2-phages, both characterized by the presence of a peculiar tail fiber-coding gene, intervened in the emergence of EAHEC. The finding of an identical vtx2-phage in two EAggEC strains isolated after 20 years in spite of the high variability described for vtx-phages is unexpected and suggests that such vtx2-phages are kept under a strong selective pressure.The observation that different EAHEC infections have been traced back to countries where EAggEC infections are endemic and the treatment of human sewage is often ineffective suggests that such countries may represent the cradle for the emergence of the EAHEC pathotype. In these regions, EAggEC of human origin can extensively contaminate the environment where they can meet free vtx-phages likely spread by ruminants excreta.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-574) contains supplementary material, which is available to authorized users.     

Structural and kinetic studies of induced fit in xylulose kinase from Escherichia coli

The primary metabolic route for D-xylose, the second most abundant sugar in nature, is via the pentose phosphate pathway after a two-step or three-step conversion to xylulose-5-phosphate. Xylulose kinase (XK; EC 2.7.1.17) phosphorylates D-xylulose, the last step in this conversion. The apo and D-xylulose-bound crystal structures of Escherichia coli XK have been determined and show a dimer composed of two domains separated by an open cleft. XK dimerization was observed directly by a cryo-EM reconstruction at 36 A resolution. Kinetic studies reveal that XK has a weak substrate-independent MgATP-hydrolyzing activity, and phosphorylates several sugars and polyols with low catalytic efficiency. Binding of pentulose and MgATP to form the reactive ternary complex is strongly synergistic. Although the steady-state kinetic mechanism of XK is formally random, a path is preferred in which D-xylulose binds before MgATP. Modelling of MgATP binding to XK and the accompanying conformational change suggests that sugar binding is accompanied by a dramatic hinge-bending movement that enhances interactions with MgATP, explaining the observed synergism. A catalytic mechanism is proposed and supported by relevant site-directed mutants.     

Comparative genome analysis identifies few traits unique to the Escherichia coli ST131 H30Rx clade and extensive mosaicism at the capsule locus

Background

E.coli ST131 is a globally disseminated clone of multi-drug resistant E. coli responsible for that vast majority of global extra-intestinal E. coli infections. Recent global genomic epidemiological studies have highlighted the highly clonal nature of this group of bacteria, however there appears to be inconsistency in some phenotypes associated with the clone, in particular capsule types as determined by K-antigen testing both biochemically and by PCR.

Results

We performed improved quality assemblies on ten ST131 genomes previously sequenced by our group and compared them to a new reference genome sequence JJ1886 to identify the capsule loci across the drug-resistant clone H30Rx. Our data shows considerable genetic diversity within the capsule locus of H30Rx clone strains which is mirrored by classical K antigen testing. The varying capsule locus types appear to be randomly distributed across the H30Rx phylogeny suggesting multiple recombination events at this locus, but that this capsule heterogeneity has little to no effect on virulence associated phenotypes in vitro.

Conclusions

Our data provides a framework for determining the capsular genetics of E. coli ST131 and further beyond to ExPEC strains, and highlights how capsular mosaicism may be an important strategy in becoming a successful globally disseminated human pathogen.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-830) contains supplementary material, which is available to authorized users.     

Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD

Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ∼2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280–320-kDa oligomeric structure consisting of ∼6–7 subunits.     

A complete view of the genetic diversity of the Escherichia coli O-antigen biosynthesis gene cluster

The O antigen constitutes the outermost part of the lipopolysaccharide layer in Gram-negative bacteria. The chemical composition and structure of the O antigen show high levels of variation even within a single species revealing itself as serological diversity. Here, we present a complete sequence set for the O-antigen biosynthesis gene clusters (O-AGCs) from all 184 recognized Escherichia coli O serogroups. By comparing these sequences, we identified 161 well-defined O-AGCs. Based on the wzx/wzy or wzm/wzt gene sequences, in addition to 145 singletons, 37 serogroups were placed into 16 groups. Furthermore, phylogenetic analysis of all the E. coli O-serogroup reference strains revealed that the nearly one-quarter of the 184 serogroups were found in the ST10 lineage, which may have a unique genetic background allowing a more successful exchange of O-AGCs. Our data provide a complete view of the genetic diversity of O-AGCs in E. coli showing a stronger association between host phylogenetic lineage and O-serogroup diversification than previously recognized. These data will be a valuable basis for developing a systematic molecular O-typing scheme that will allow traditional typing approaches to be linked to genomic exploration of E. coli diversity.     

Optimizing expression and purification of an ATP-binding gene gsiA from Escherichia coli k-12 by using GFP fusion

The cloning, expression and purification of the glutathione (sulfur) import system ATP-binding protein (gsiA) was carried out. The coding sequence of Escherichia coli gsiA, which encodes the ATP-binding protein of a glutathione importer, was amplified by PCR, and then inserted into a prokaryotic expression vector pWaldo-GFPe harboring green fluorescent protein (GFP) reporter gene. The resulting recombinant plasmid pWaldo-GFP-GsiA was transformed into various E. coli strains, and expression conditions were optimized. The effect of five E. coli expression strains on the production of the recombinant gsiA protein was evaluated. E. coli BL21 (DE3) was found to be the most productive strain for GsiA-GFP fusion-protein expression, most of which was insoluble fraction. However, results from in-gel and Western blot analysis suggested that expression of recombinant GsiA in Rosetta (DE3) provides an efficient source in soluble form. By using GFP as reporter, the most suitable host strain was conveniently obtained, whereby optimizing conditions for overexpression and purification of the proteins for further functional and structural studies, became, not only less laborious, but also time-saving.     

Drosophila Cu,Zn superoxide dismutase gene confers resistance to paraquat in Escherichia coli

Superoxide dismutase (SOD) is known to protect organisms from reactive oxygen metabolites. We tested the hypothesis that the Drosophila Cu,Zn SOD is capable of protecting Escherichia coli from oxidative damage caused by the herbicide paraquat. The Cu,Zn Sod gene of Drosophila sechellia was subcloned into pET-20b(+) expression vector. Transformation of E. coli with the constructed vector resulted in an overexpression of this eukaryotic superoxide dismutase, as evidenced by dramatically increased levels of the Cu,Zn SOD polypeptide in bacterial cytosolic extracts. As well, the E. coli transformants showed resistance to paraquat-mediated inhibition of growth and survival. Paraquat is known to promote formation of the superoxide radical anion inside cells and thus the data have been interpreted as indicating that the cloned superoxide dismutase provides protection in E. coli against damage attributable to free radicals.