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Background: Escherichia coli (E. coli) comprise part of the normal vaginal microflora. Transfer from mother to neonate can occur during delivery resulting, sometimes, in neonatal bacterial disease. Here, we aim to report the first outbreak of CTX-M ESBL-producing E. coli with evidence of mother-to-neonate transmission in an Irish neonatal intensive care unit (NICU) followed by patient-to-patient transmission., Methods: Investigation including molecular typing was conducted. Infection was defined by clinical and laboratory criteria and requirement for antimicrobial therapy with or without positive blood cultures. Colonisation was determined by isolation without relevant symptoms or indicators of infection., Results: Index case was an 8-day-old baby born at 34 weeks gestation who developed ESBL-producing E. coli infections at multiple body sites. Screening confirmed their mother as colonised with ESBL-producing E. coli. Five other neonates, in the NICU simultaneously with the index case, also tested positive. Of these, four were colonised while one neonate developed sepsis, requiring antimicrobial therapy. The second infected neonate's mother was also colonised by ESBL-producing E. coli. Isolates from all eight positive patients (6 neonates, 2 mothers) were compared using pulsed-field gel electrophoresis (PFGE). Two distinct ESBL-producing strains were implicated, with evidence of transmission between mothers and neonates for both strains. All isolates were confirmed as CTX-M ESBL-producers. There were no deaths associated with the outbreak., Conclusions: Resources were directed towards control interventions focused on hand hygiene and antimicrobial stewardship, which ultimately proved successful. Since this incident, all neonates admitted to the NICU have been screened for ESBL-producers and expectant mothers are screened at their first antenatal appointment. To date, there have been no further outbreaks.

BipA is a highly conserved protein with global regulatory properties in Escherichia coli. We show here that it functions as a translation factor that is required specifically for the expression of the transcriptional modulator Fis. BipA binds to ribosomes at a site that coincides with that of elongation factor G and has a GTPase activity that is sensitive to high GDP:GTP ratios and stimulated by 70S ribosomes programmed with mRNA and aminoacylated tRNAs. The growth rate-dependent induction of BipA allows the efficient expression of Fis, thereby modulating a range of downstream processes, including DNA metabolism and type III secretion. We propose a model in which BipA destabilizes unusually strong interactions between the 5' untranslated region of fis mRNA and the ribosome. Since BipA spans phylogenetic domains, transcript-selective translational control for the 'fast-track' expression of specific mRNAs may have wider significance.

The cheA gene of Escherichia coli encodes two proteins from in-frame tandem translation start sites. The long form of CheA (CheA(L)) is the histidine kinase responsible for phosphorylating the response regulator, CheY. The short form of CheA (CheA(S)) is identical in domain structure to CheA(L) except that it is missing the first 97 amino acids. Reduced CheA(S) bound to and enhanced the activity of the phosphatase of phospho-CheY, CheZ. Oxidized CheA(S) was unable to interact with CheZ. Oxidized CheA(S) formed covalent dimers, whereas CheA(L) did not. This property was believed to be the result of an intermolecular disulfide bond. The CheA proteins contain three cysteine residues, one of which likely lies within the CheZ binding region of CheA(S) and is exposed to solvent. We identified the CheZ binding domain of CheA(S) by testing the various fragments of CheA(S) that contain cysteine residues for CheZ binding activity in an ELISA-based CheA(S)-CheZ binding assay. Fragments of CheA(S) lacking the truncated P1 domain of CheA(S) ('P1) were unable to bind CheZ. We also found that a fusion of the first 42 amino acids of CheA(S) ('P1 domain) to GST bound CheZ and enhanced its activity. The interaction between the GST-CheA[98-139] fusion protein and CheZ was dependent on the accessibility of a cysteine residue (Cys-120) located in the 'P1 domain.

BipA is a novel member of the ribosome binding GTPase superfamily and is widely distributed in bacteria and plants. We report here that it regulates -multiple cell surface- and virulence-associated -components in the enteropathogenic Escherichia coli (EPEC) strain E2348/69. The regulated components include bacterial flagella, the espC pathogenicity island and a type III secretion system specified by the locus of enterocyte effacement (LEE). BipA positively regulated the espC and LEE gene clusters through transcriptional control of the LEE-encoded regulator, Ler. Additionally, it affected the pattern of proteolysis of intimin, a key LEE-encoded adhesin specified by the LEE. BipA control of the LEE operated independently of the previously characterized regulators Per, integration host factor and H-NS. In contrast, it negatively regulated the flagella-mediated motility of EPEC and in a Ler-independent manner. Our results indicate that the BipA GTPase functions high up in diverse regulatory cascades to co-ordinate the expression of key pathogenicity islands and other virulence-associated factors in E. coli.

One of the commonly used resins for immobilized metal affinity purification of polyhistidine-tagged recombinant proteins is TALON resin, a cobalt (II)--carboxymethylaspartate-based matrix linked to Sepharose CL-6B. Here, we show that TALON resin efficiently purifies the native form of Lac repressor, which represents the major contaminant when (His)(6)-tagged proteins are isolated from Escherichia coli host cells carrying the lacI(q) gene. Inspection of the crystal structure of the repressor suggests that three His residues (residues 163, 173, and 202) in each subunit of the tetramer are optimally spaced on an exposed face of the protein to allow interaction with Co(II). In addition to establishing a more efficient procedure for purification of the Lac repressor, these studies indicate that non-lacI(q)-based expression systems yield significantly purer preparations of recombinant polyhistidine-tagged proteins., (Copyright 2001 Academic Press.)

For a sustained infection, enteric bacterial pathogens must evade, resist or tolerate a variety of antimicrobial host defence peptides and proteins. We report here that specific organic acids protect stationary-phase Escherichia coli and Salmonella cells from killing by a potent antimicrobial peptide derived from the human bactericidal/permeability-increasing protein (BPI). BPI-derived peptide P2 rapidly halted oxygen consumption by stationary-phase cells preincubated with glucose, pyruvate or malate and caused a 109-fold drop in cell viability within 90 min of addition. In marked contrast, O2 consumption and viability were not significantly affected in stationary-phase cells preincubated with formate or succinate. Experiments with fdhH, fdoG, fdnG, selC and sdhO mutants indicate that protection by formate and succinate requires their oxidation by the Fdh-N formate dehydrogenase and succinate dehydrogenase respectively. Protection was also dependent on the BipA GTPase but did not require the RpoS sigma factor. We conclude that the primary lesion caused by this cationic peptide is not gross permeabilization of the bacterial cytoplasmic membrane but may involve specific disruption of the respiratory chain. Because P2 shares sequence similarity with a range of other antimicrobial peptides, its cytotoxic mechanism has broader significance. Additionally, protective quantities of formate are secreted by E. coli and Salmonella during growth suggesting that such compounds are important determinants of bacterial survival in the host.

We report the functional characterization of BipA, a GTPase that undergoes tyrosine phosphorylation in an enteropathogenic Escherichia coli (EPEC) strain. BipA mutants adhere to cultured epithelial cells but fail to trigger the characteristic cytoskeletal rearrangements found in cells infected with wild-type EPEC. In contrast, increased expression of BipA enhances actin remodelling and results in the hyperformation of pseudopods. BipA appears to be the first example of a new class of virulence regulator, as it also controls flagella-mediated cell motility and resistance to the antibacterial effects of a human host defence protein. Its striking sequence similarity to ribosome-binding elongation factors suggests that it uses a novel mechanism to modulate gene expression.

The interaction of propionyl-CoA and acetyl-CoA with E. coli citrate synthase has been studied in order to gain insight into the structural requirements for substrate binding by this enzyme. In contrast to the enzyme from pig heart, the E. coli enzyme was unable to catalyse significant exchange of the methylene protons of propionyl-CoA while overall activity was very low with this enzyme. Carboxymethyl-CoA is a presumptive transition state analogue of acetyl-CoA using pig heart citrate synthase. The effect of carboxymethyl-CoA on both the native enzyme from E. coli and a catalytically active aspartate mutant (D362E) was investigated. Whereas the native enzyme was inhibited by carboxymethyl-CoA, the mutant enzyme (D362E) shows either no inhibition or minimal inhibition depending on the assay conditions. The binding of acetyl-CoA is not inhibited as a result of the mutation. The results with propionyl-CoA and carboxymethyl-CoA suggest that the active site of the E. coli enzyme is more restricted as compared with the enzyme from pig heart and, in the case of propionyl-CoA, this restriction prevents the formation of a catalytically productive enzyme-substrate complex.

The TraT protein is a highly cell-surface-exposed lipoprotein specified by F-like plasmids that confers serum resistance and blocks the conjugative transfer of plasmids to cells bearing identical or closely related plasmids, a process known as surface exclusion. The TraT protein specified by the antibiotic-resistance plasmid R6-5 was purified to apparent homogeneity. When added to mating mixtures, TraT blocked the transfer of plasmids belonging to Surface Exclusion Group IV (Sfx IV) but had no significant effect on the transfer of plasmids belonging to other groups. Additionally, the purified protein has a protective effect on bacterial cells incubated in serum, indicating that it does not have to be located on the cell surface to mediate serum resistance. To localize regions of the protein that were responsible for surface exclusion specificity, the amino acid sequence of the TraT protein specified by CoIB2-K98 (Sfx II) was determined by cloning and sequencing of the corresponding gene. Comparison of the derived sequence with those of the F and R100-1 proteins indicated that surface exclusion specificity of TraT is determined by single alterations in a five-amino-acid region (residues 116-120). This was confirmed by segment swapping experiments in which the specificity of the R6-5 TraT protein (Sfx IV) was switched to that of the CoIB2-K98 protein (Sfx II). Our results suggest that the region defined by residues 116-120 is located on the external face of the outer membrane and interacts specifically with the donor cell in surface exclusion.

A 1.2 kb BamHI fragment from pDK30 [Robinson, Kenan, Sweeney & Donachie (1986) J. Bacteriol. 167, 809-817] was cloned in pDOC55 [O'Connor & Timmis (1987) J. Bacteriol. 169, 4457-4482] to give two constructs, pDOC89 and pDOC87, in which the Escherichia coli D-alanine:D-alanine ligase (EC 6.3.2.4) gene (ddl) was placed under the control of the lac and lambda PL promoters respectively. Both constructs, when used to transform E. coli M72, gave similar levels of expression of the ddl gene. The expressed enzyme was purified to homogeneity and the amino acid sequence of its N-terminal region was found to be consistent with that predicted from the gene sequence, except that the N-terminal methionine was not present in the mature protein. [1(S)-Aminoethyl][(2RS)2-carboxy-1-octyl]phosphinic acid (I), previously shown to bind tightly to Enterococcus faecalis and Salmonella typhimurium D-alanine:D-alanine ligases following phosphorylation Parsons, Patchett, Bull, Schoen, Taub, Davidson, Combs, Springer, Gadebusch, Weissberger, Valiant, Mellin & Busch (1988) J. Med. Chem. 31, 1772-1778; Duncan & Walsh (1988) Biochemistry 27, 3709-3714], was found to be a classical slow-binding inhibitor of the E. coli ligase.

We have previously described defined mutants of the TraT protein, an outer membrane lipoprotein specified by F-like plasmids, which sensitize Escherichia coli and Salmonella typhimurium to antibiotics that are normally excluded from the cell. In this paper, the isolation, characterization and molecular cloning of suppressors of one such mutant (pDOC40) is reported. The suppressors, which were isolated by selection for vancomycin-resistant revertants, also restored resistance to several hydrophobic antibiotics although there were no detectable changes in lipopolysaccharides (LPS), phospholipids or outer membrane proteins. Three suppressor loci, provisionally designated sip, for suppression of increased permeability, were cloned in cosmids and mapped by a novel approach involving random sequencing of cloned DNA to identify flanking genes with known map positions. Our results indicate that the sipB locus is located in the 11 min region (485-510 kb) whereas sipC and sipD both map to 82 min (3850-3885 kb). Additionally, the previously sequenced nlpA gene was also mapped to the 82 min region. The cloned suppressor loci were specific for the permeability phenotype caused by the mutant R6-5 TraT protein and had no effect on the permeability phenotype caused by a related TraT mutant of S. typhimurium.

The TraT protein is a surface-exposed lipoprotein, specified by plasmids of the IncF group, that mediates serum resistance and surface exclusion. The structure and function of the TraT protein determined by plasmid R6-5 was probed by genetic insertion of a foreign antigenic determinant, the C3 epitope of polio virus, at residues 61, 125, 180, 200 or 216 of the protein. The chimaeric proteins were transported to the outer membrane and, in three cases, immunoassays with an anti-C3 monoclonal antibody indicated that the C3 epitope was exposed on the cell surface. Three of the hybrids, with insertions at residues 125, 180 and 200, assembled into the trypsin-resistant oligomeric form characteristic of the wild-type protein, which suggested that these regions are not involved in TraT subunit:subunit interactions. Additionally, the hybrid protein carrying the C3 epitope at position 180 functioned in a genetic suppression assay and retained partial surface-exclusion activity. Thus, its localization, folding and organization does not appear to be grossly altered from that of the wild-type protein. Applications of the protein for the transport of foreign antigenic determinants to the cell surface are discussed.

The genes encoding the Eco RI restriction-modification (R/M) system have been separately cloned onto compatible plasmids. We have shown that the Eco RI restriction gene is expressed in the total absence of methylase enzyme and confirmed that a temperature-sensitive mutant is defective in Eco RI modification activity at higher temperatures. Insertion of transcriptional terminators into the restriction gene had no detectable effect on Eco RI modification activity. This strongly suggests that a separate promoter exists for the methylase gene. Analysis of the published sequence shows that the methylase gene promoter may overlap with the COOH-terminal region of the endonuclease structural gene. The temperature-sensitive Eco RI system has been exploited in the construction of two plasmid cloning vectors, pLV57 and pLV59, which can be used to select positively for transformants bearing recombinant plasmids; cloning of a DNA fragment into pLV57 or pLV59 at the unique HindIII, Bg/II, or PstI sites inactivates the Eco RI restriction gene and permits the hybrid plasmid to survive at 37 degrees C. The temperature-sensitive modification activity of these vectors should also facilitate the introduction of Eco RI linkers into DNA cloned in this way.

A highly repressible expression vector system that allows the cloning of potentially deleterious genes has been constructed. Undesired expression of a cloned gene was prevented (i) at the level of initiation of transcription, by the presence of the strong but highly repressible leftward promoter of bacteriophage lambda, lambda pL, and (ii) at the level of transcript elongation or translation, through synthesis of antisense RNA complementary to the mRNA of the cloned gene. The system was tested by measuring the inhibition of expression of traT, the gene for the TraT major outer membrane lipoprotein. Direct detection and functional assays indicated that an essentially complete inhibition of traT expression was obtained. As a further test of the system, the gene encoding the EcoRI restriction endonuclease was cloned in the absence of the gene of the corresponding protective EcoRI modification methylase. Transformants harboring this construct were only viable when both repression controls were operational.

The prevalence of Escherichia coli O157:H7 infection in birds is low but several deliberate inoculation studies show that poultry are readily and persistently infected by this organism indicating a possible threat to public health. The mechanisms of colonisation of poultry are not understood and the aim is to establish models to study the interaction of E. coli O157:H7, at the cellular and whole animal levels. A non-toxigenic E. coli O157:H7 (NCTC 12900) was used in adherence assays with an avian epithelial cell line (Div-1) and used to inoculate 1-day-old SPF chicks. In vitro, NCTC 12900 induced micro-colonies associated with cytoskeletal arrangements and pedestal formation with intimate bacterial attachment. In the 1-day-old SPF chick, a dose of 1×105 cfu resulted in rapid and extensive colonisation of the gastrointestinal tract and transient colonisation of the liver and spleen. The number of E. coli O157:H7 organisms attained approximately 108 cfu/ml caecal homogenate 24 h after inoculation and approximately 107 cfu/ml caecal homogenate was still present at day 92. Faecal shedding persisted for 169 days, ceasing 9 days after the birds came into lay and 6% of eggs were contaminated on the eggshell. Histological analysis of tissue samples from birds dosed with 1×107 cfu gave evidence for E. coli O157:H7 NCTC 12900 induced micro-colonies on the caecal mucosa, although evidence for attaching effacing lesions was equivocal. These models may be suitable to study those factors of E. coli O157:H7 that mediate persistent colonisation in avian species. [Copyright &y& Elsevier]