Antonio Juárez

Distribution and characterization of faecal verotoxin-producing Escherichia coli (VTEC) isolated from healthy cattle.

Faecal swabs obtained from a random sample of 268 cows and 90 calves on 19 Lugo farms were examined for verotoxin-producing Escherichia coli (VTEC). We found VTEC on 95% of the farms. The prevalence rates of VTEC infection in asymptomatic cows and calves were estimated to be 35 and 37%, respectively. The proportion of animals infected on each farm ranged from 0 to 100%. VTEC strains isolated from healthy cattle belonged to 27 O serogroups; however, 57% (85 of 149) were of one of 8 serogroups (O2, O8, O22, O77, O82, O105, O113 and O171). Nearly 60% of the bovine VTEC strains belonged to serogroups that cause haemorrhagic colitis and haemolytic uraemic syndrome in humans. The VTEC were all non-O157:H7; 91% were eae-negative and 86% produced VT2 or VT1 and VT2. These characteristics are different from those of VTEC isolated from calves with diarrhoea.

Expression of the hemolysin operon in Escherichia coli is modulated by a nucleoid-protein complex that includes the proteins Hha and H-NS

Abstract The Escherichia coli protein Hha is a temperature- and osmolarity-dependent modulator of the expression of the hemolysin operon. The Hha protein was purified and its DNA-binding properties analyzed. Hha binds in a non-specific manner throughout the upstream regulatory region of the hemolysin operon in the recombinant hemolytic plasmid pANN202-312. A search for interacting proteins revealed that Hha interacts with H-NS. DNA-binding studies showed that, in vitro, Hha and H-NS together form a complex with DNA that differs from those formed with either protein alone. These data, together with the effects of hha and hns mutations on the expression of the hemolysin genes, suggest that in vivo H-NS and Hha form a nucleoid-protein complex that accounts for the thermo-osmotic regulation of the hemolysin operon in E. coli.

Temperature- and H-NS-dependent regulation of a plasmid-encoded virulence operon expressing Escherichia coli hemolysin.

Proteins H-NS and Hha form a nucleoprotein complex that modulates expression of the thermoregulated hly operon of Escherichia coli. We have been able to identify two H-NS binding sites in the hly regulatory region. One of them partially overlaps the promoter region (site II), and the other is located about 2 kbp upstream (site I). In contrast, Hha protein did not show any preference for specific sequences. In vitro, temperature influences the affinity of H-NS for a DNA fragment containing both binding sites and H-NS-mediated repression of hly operon transcription. Deletion analysis of the hly regulatory region confirms the relevance of site I for thermoregulation of this operon. We present a model to explain the temperature-modulated repression of the hly operon, based on the experiments reported here and other, preexisting data.

Characterization of a sequence (hlyR) which enhances synthesis and secretion of hemolysin in Escherichia coli

SummaryA sequence (hlyR) of about 600 bp which enhances the expression of hemolysin (HlyA) more than 50-fold was identified in the plasmid pHly152-specific hemolysin (hly) determinant. Deletion of this entire hlyR sequence led to the same low level of hemolysin synthesis and excretion as that expressed by the recombinant plasmid pANN202-312. HlyR was active in cis but its activity was orientation-dependent. The enhancing sequence, hlyR, is separated from the promoter phlyI transcribing hlyC, hlyA and possibly hlyB by more than 1.5 kb including an IS2 element. Stepwise removal of the hlyR sequence from its 5′ end by exonuclease III (ExoIII) digestion yielded several types of deletion mutants which expressed decreasing amounts of hemolysin. A similar observation was made when hlyR was shortened by ExoIII from its 3′ end, which suggests that more than one functional region may be present in the hlyR sequence. A deletion of 717 bp within the adjacent IS2 element reduced the activity of hlyR only slightly, indicating that IS2 is not directly involved in the enhancement mechanism but that it may support an optimal positioning in hlyR relative to the hly promoter. The nucleotide sequence of hlyR is rich in A+T and does not contain an extended open reading frame, but exhibits several sequence motives that may represent sites for protein binding and DNA bending.

The hha gene modulates haemolysin expression in Escherichia coli

A mutation in the hha allele results in a large increase in the production of intracellular as well as extracellular haemolysin in Escherichia coli cells harbouring the haemolytic recombinant plasmid pANN202‐312. This single gene mutation was located between 490 and 491.6kb on the physical map of the E. coli chromosome. From the DNA sequence of hha a small polypeptide of 8629 Da was predicted and was expressed in minicells. The deduced polypeptide sequence did not show significant similarities to other characterized proteins related to the regulation of gene expression in E. coli, although it was shown that the hha mutation increases cyloplasmic synthesis of haemolysin.

Detection of pap, sfa and afa adhesin-encoding operons in uropathogenic Escherichia coli strains: Relationship with expression of adhesins and production of toxins**

A total of 243 Escherichia coli strains isolated from patients with urinary tract infections (UTI) were investigated for the presence of pap, sfa and afa adhesin-encoding operons by using the polymerase chain reaction. It was found that 54%, 53% and 2% of the strains exhibited the pap, sfa and afa genotypes, respectively. Pap+ and/or sfa+ strains were more frequent in cases of acute pyelonephritis (94%) than in cases of cystitis (67%) (P < 0.001) and asymptomatic bacteriuria (57%) (P < 0.001). The pap and/or sfa operons were found in 90% of strains expressing mannose-resistant haemagglutination (MRHA) versus 37% of MRHA-negative strains (P < 0.001). The presence of pap and sfa operons was especially significant in strains belonging to MRHA types III (100%) (without P adhesins) and IVa (97%) (expressing the specific Gal-Gal binding typical of P adhesins). Both pap and sfa operons were closely associated with toxigenic E. coli producing alpha-haemolysin (Hly+) and/or the cytotoxic necrotizing factor type 1. There was an apparent correlation between the pap and sfa operons and the O serogroups of the strains. Thus, 93% of strains belonging to O1, O2, O4, O6, O7, O14, O15, O18, O22, O75 and O83 possessed pap and/or sfa operons, versus only 32% of strains belonging to other serogroups (P < 0.001). The results obtained in this study confirm the usefulness of our MRHA typing system for presumptive identification of pathogenic E. coli exhibiting different virulence factors. Thus, 85% of strains that possessed both pap and sfa adhesin-encoding operons showed MRHA types III or IVa previously associated with virulence of E. coli strains that cause UTI and bacteraemia.

Differential Regulation of Horizontally Acquired and Core Genome Genes by the Bacterial Modulator H-NS

Horizontal acquisition of DNA by bacteria dramatically increases genetic diversity and hence successful bacterial colonization of several niches, including the human host. A relevant issue is how this newly acquired DNA interacts and integrates in the regulatory networks of the bacterial cell. The global modulator H-NS targets both core genome and HGT genes and silences gene expression in response to external stimuli such as osmolarity and temperature. Here we provide evidence that H-NS discriminates and differentially modulates core and HGT DNA. As an example of this, plasmid R27-encoded H-NS protein has evolved to selectively silence HGT genes and does not interfere with core genome regulation. In turn, differential regulation of both gene lineages by resident chromosomal H-NS requires a helper protein: the Hha protein. Tight silencing of HGT DNA is accomplished by H-NS-Hha complexes. In contrast, core genes are modulated by H-NS homoligomers. Remarkably, the presence of Hha-like proteins is restricted to the Enterobacteriaceae. In addition, conjugative plasmids encoding H-NS variants have hitherto been isolated only from members of the family. Thus, the H-NS system in enteric bacteria presents unique evolutionary features. The capacity to selectively discriminate between core and HGT DNA may help to maintain horizontally transmitted DNA in silent form and may give these bacteria a competitive advantage in adapting to new environments, including host colonization.

The novel Hha/YmoA family of nucleoid‐associated proteins: use of structural mimicry to modulate the activity of the H‐NS family of proteins

The Hha/YmoA family of proteins is a group of conserved, low‐molecular‐weight proteins involved in the regulation of gene expression. Studies performed in Escherichia coli, Salmonella sp. and Yersinia sp. highlight the contribution of these proteins in regulating bacterial virulence, horizontal gene transfer and cell physiology. Genes encoding such proteins are located on chromosomes and plasmids in different genera of Gram‐negative bacteria. Their mode of action is currently being analysed by studying direct binding of Hha to DNA and as a component of protein complexes with regulatory functions. Recent data on the interaction of Hha with the H‐NS family of proteins and structural information suggest a physiological role for such protein complexes in many aspects of gene regulation.

Polymerase chain reaction for detection of Escherichia coli strains producing cytotoxic necrotizing factor type 1 and type 2 (CNF1 and CNF2)

Abstract Cytotoxic necrotizing factors type 1 (CNF1) and type 2 (CNF2) are dermonecrotic protein toxins produced by human and animal clinical isolates of Escherichia coli . In this study, two pairs of oligonucleotide primers were designed to amplify fragments of the genes for CNF1 and CNF2 by the polymerase chain reaction (PCR). We also described a further primer pair which amplify fragments of both CNF1 and CNF2 genes. Amplification products of the expected size (543-bp for CNF1, 543-bp for CNF2 and 533-bp for CNFs) were detected in all 240 necrotoxigenic Escherichia coli (NTEC) strains tested. In contrast, no amplification products were seen when the PCR reaction was performed with DNA obtained from 69 non-NTEC bacterial strains (including 13 enterotoxigenic, 11 verotoxigenic and 45 non-toxigenic strains). Furthermore, the toxin genotypes determined by PCR corresponded to the phenotypic results of HeLa cell assay. Therefore, the specificity and sensitivity for the three pairs of oligonucleotide primers designed in this study was 100%. The PCR protocol describe here permits rapid and accurate detection of CNF1 and CNF2 genes in Escherichia coli strains isolated from clinical material and contaminated foods.

YdgT, the Hha paralogue in Escherichia coli , forms heteromeric complexes with H-NS and StpA

In enteric bacteria, proteins of the Hha/YmoA family play a role in the regulation of gene expression in response to environmental factors. Interaction of both Hha and YmoA with H‐NS has been reported, and an Hha/H‐NS complex has been shown to modulate expression in Escherichia coli of the haemolysin operon of plasmid pHly152. In addition to the hns gene, the chromosome of E. coli and other enteric bacteria also includes the stpA gene that encodes the StpA protein, an H‐NS paralogue. We report here the identification of the Hha paralogue in E. coli, the YdgT protein. As Hha paralogue, YdgT appears to fulfil some of the functions reported for StpA as H‐NS paralogue: YdgT is overexpressed in hha mutants and can compensate, at least partially, some of the hha‐induced phenotypes. We also demonstrate that YdgT interacts both with H‐NS and with StpA. Protein cross‐linking studies showed that YdgT/H‐NS heteromeric complexes are generated within the bacterial cell. The StpA protein, which is subjected to Lon‐mediated turnover, was less stable in the absence of Hha or YdgT. Our findings suggest that Hha, YdgT and StpA may form complexes in vivo.