The other possibility is that the BsuM enzyme remained in the cytoplasmic portion of the donor R+ M+ cell in the fusant and that, upon division of the fusants, various quantities of the enzyme were distributed INK-128 to the progeny cells depending on where the cell division took place. It has been demonstrated that L-form colonies of B. subtilis arise among those of the bacillary form

after PEG-induced cell fusion (Hauser & Karamata, 1992), which indicates that cell division can occur while the fusant is still without the cell wall. As the L-form B. subtilis cell divides by an extrusion–resolution mechanism that is independent of FtsZ (Leaver et al., 2009), it is possible that a similar mechanism was involved in the division of the fused cells that were produced by random collision between the donor and the recipient protoplasts. This may result in incorporation of various parts and quantities of the cytoplasm of the R+ M+ cell into that of the

R− M− protoplast. Thus, upon cell fusion and subsequent cell division, different proportions of the donor and the recipient cell cytoplasms will constitute progeny cells. Cell death by restriction of the recipient chromosome will occur if a higher proportion of the cytoplasm from the R+ M+ donor cell occupies the progeny cell after division. There must be a critical level of the BsuM restriction enzyme under which the recipient DNA is saved from the restriction attack, and this may account for the reduced but significant efficiency of plasmid transfer from the R+ M+ donor to the R− M− recipient cell. We note in this Ku-0059436 mw respect that the cotransfer efficiencies of pLS32neo and pHV33 from the R+ M+ to R− M− cells were 1/9 to 1/7 of the levels observed for the

fusion between the homologous pairs (see ‘Results’). As the chromosomal DNA in this fraction of the fused cells escaped the restriction attack, it is tempting Doxacurium chloride to speculate that the cytoplasmic space in the donor R+ M+ cell containing both plasmids but not enough quantities of the BsuM restriction enzyme to destroy the recipient chromosomal DNA corresponds to 1/9 to 1/7 of the space that brings about transfer of both plasmids in the case of homologous pairs. The heterospecific cell fusion between B. subtilis RM125 and either B. stearothermophilus or B. circulans was successful but the efficiencies were relatively low (see ‘Results’). The restriction system(s) in B. stearothermophilus CU21 or B. circulans BM used here has not been studied yet, but it is known that by a rough estimate, most bacteria carry one or more restriction systems (Wilson & Murray, 1991), which may have affected the fusion efficiency in this study. Another possible reason for the low efficiency is that the compositions of the membrane constituents in those bacteria are different from that of B. subtilis RM125, causing inefficient membrane fusion between the heterospecific bacteria.

Sap1 to Sap8 are secreted into the extracellular environment, while Sap9 and Sap10 are retained at the cell surface via a (modified) GPI anchor (Albrecht et al., 2006). Saps are involved in multiple processes, like degradation of host tissues and proteins to facilitate invasion and

nutrient uptake. Furthermore, they can degrade host immune proteins (Gropp et al., 2009). While Sap1 to Sap3 activities are maximal at pH 3–5, Sap4 to Sap6 activities are optimal at pH 5–7, correlating with the fact that Sap4 to Sap6 are essential for systemic infections and were only present in the secretome of hypha-enriched cultures grown in the presence of GlcNAc at pH 7.4 (Felk et al., 2002; Sorgo et al., 2010). Accordingly, Sap2 and Sap3 were exclusively detected at pH 4. Also phospholipases are involved in tissue Selumetinib mw destruction and invasion. All five phospholipase Selleckchem isocitrate dehydrogenase inhibitor B genes in C. albicans contain a signal sequence for secretion, while only

PLB3, PLB4.5, and PLB5 have a putative GPI attachment signal (De Groot et al., 2003). Plb3 has been detected in fluconazole-stressed cultures but only at very low levels (Sorgo et al., 2011), probably because the correct induction conditions were not met. Of the ten lipase genes encoded by C. albicans, all except LIP7 contain an N-terminal signal for secretion. LIP genes were shown to be differentially expressed depending on the growth condition, and expression was independent of lipids (Hube et al., 2000). Nevertheless, until now only Lip4 has been identified at very low levels in exponentially growing cultures with lactate as carbon source (Ene et al., 2012). Apart from hydrolytic enzymes, C. albicans also secretes proteins to sequester metal ions. Zinc is an important trace metal required for microbial growth. Zinc uptake is facilitated by two proteins, the secreted protein Pra1 and the zinc transporter Zrt1 (Citiulo et al., 2012). Pra1 (pH-regulated antigen) is highly expressed at neutral pH and shows negligible expression at acidic pH (Sentandreu et al., 1998). Upon host cell penetration, C. albicans secretes

Pra1 into the host cell cytosol, scavenges available zinc, and re-associates with the fungal cell, where it interacts with the zinc transporter Nitroxoline Zrt1 to enable zinc uptake. Interestingly, Pra1 is recognized by a leukocyte receptor protein, and this probably explains why pra1 mutant cells are more resistant to leukocyte killing and more virulent in a murine model of systemic infection (Soloviev et al., 2011). Freely available iron is also very scarce during infection, and iron is actively scavenged by C. albicans from its host. All five members of the C. albicans Rbt5 family, comprising Csa1, Csa2, Pga7, Pga10, and Rbt5, are CFEM proteins, which are characterized by the possession of one or more 8-cysteine-containing domains.

Efficacy and tolerability are similar to those in treatment-naïve patients. “
“Insulin resistance in viral infections is

common. We have explored the effectiveness of metformin for alleviating insulin resistance in HIV-infected patients and assessed the relevance of the ataxia-telangiectasia mutated (ATM) rs11212617 variant in the clinical response with the rationale that metformin modulates cellular bioenergetics in an ATM-dependent process. HIV-infected patients (n = 385) were compared with controls recruited from the general population (n = 300) with respect to the genotype distribution of the ATM rs11212617 variant and its influence on selected metabolic and inflammatory variables. We also followed up a subset of male patients with HIV and hepatitis C virus (HCV) coinfection (n = 47) who were not receiving antiviral treatment and for whom Selleckchem Ku 0059436 metformin was prescribed for insulin resistance, which tends to have a higher incidence and severity in coinfected patients. Among the HIV-infected patients, human cytomegalovirus (91.9%)

and HCV (62.3%) coinfections were frequent. Selected metabolic and/or inflammatory variables were significantly altered Selleckchem Vincristine in infected patients. Treatment with metformin in HIV and HCV coinfected patients was well tolerated and significantly increased the sensitivity of peripheral tissues to insulin. The minor allele (C)

of the rs11212617 variant was fantofarone associated with treatment success and may affect the course of insulin resistance in response to metformin (odds ratio 1.21; 95% confidence interval 1.07–1.39; P = 0.005). There were no differences between treated and untreated patients in viral loads or variables measuring immune defence, indicating that toxicity is unlikely. We provide novel data suggesting that identification of the ATM rs11212617 variant may be important in assessing the glycaemic response to metformin treatment for insulin resistance in HIV-infected patients. “
“The EuResist expert system is a novel data-driven online system for computing the probability of 8-week success for any given pair of HIV-1 genotype and combination antiretroviral therapy regimen plus optional patient information. The objective of this study was to compare the EuResist system vs. human experts (EVE) for the ability to predict response to treatment. The EuResist system was compared with 10 HIV-1 drug resistance experts for the ability to predict 8-week response to 25 treatment cases derived from the EuResist database validation data set. All current and past patient data were made available to simulate clinical practice. The experts were asked to provide a qualitative and quantitative estimate of the probability of treatment success. There were 15 treatment successes and 10 treatment failures.

These features make NDH-2 a promising target for the development of new drug candidates. High-resolution structural data and deeper understanding of phenothiazine action may facilitate structure-based design of small-molecule NDH-2 inhibitors with improved efficacy and selectivity. Diarylquinolines represent a novel class of antimycobacterial drugs with strong in vitro and in vivo activity against different mycobacterial species (Andries et al., 2005; Ji et al., 2006). Diarylquinolines block ATP synthesis and cause a

decrease of cellular ATP levels (Koul et al., 2007). As the bacterial ATP stores are depleted over a period of time, subsequently pronounced bacterial killing is observed (Koul et al., 2008). Diarylquinolines specifically interact with the oligomeric transmembrane subunit c of mycobacterial ATP synthase (Koul et al., 2007, see also Fig. 2). During enzymatic catalysis, this oligomeric subunit, together with subunits ɛ see more and γ, rotates relative to subunits α3β3δab and in this way couples proton flow to the synthesis of

ATP (Boyer, 1993; Junge et al., 1997). Protons enter from the periplasmic space via an entry channel in subunit a and are then transferred to an essential acidic residue in the membrane-spanning part of subunit c (Fig. 2). After a close to 360° rotation of the cylindrical subunit c oligomer relative to subunit a, the protons are released on the cytosolic side of the membrane via an exit channel in subunit a (Vik & Antonio, 1994; Diez BTK inhibitor screening library et al., 2004). Mutagenesis studies indicate that diarylquinoline lead compound TMC207 binds to the central region of subunit c, close to the essential acidic residue (Koul

et al., 2007). TMC207 may compete with protons for binding to subunit c or may alternatively interfere with the extensive conformational changes of this subunit during catalysis. Whereas typical inhibitors Galeterone of ATP synthase subunit c, such as dicyclohexyl-carbodiimide and oligomycin, are not selective and highly toxic (Matsuno-Yagi & Hatefi, 1993; Wallace & Starkov, 2000; Amacher, 2005), TMC207 displays a surprising selectivity, with only an extremely low effect on human ATP synthesis (Haagsma et al., 2009). Although several residues of subunit c are reported to modulate diarylquinoline sensitivity (Koul et al., 2007), the molecular basis for the observed selectivity needs to be further investigated. No high-resolution structure is available for mycobacterial ATP synthase or its subunits, and structural models for mycobacterial subunit c have only been built based on the known structure of the homologous subunit from E. coli, Ilyobacter tartaricus or Bacillus PS3 (de Jonge et al., 2007; Koul et al., 2007). High-resolution structural data for mycobacterial subunit c and biochemical investigations on drug/target interaction would help to explain drug selectivity and would provide input for docking studies to design new compound derivates.

These features make NDH-2 a promising target for the development of new drug candidates. High-resolution structural data and deeper understanding of phenothiazine action may facilitate structure-based design of small-molecule NDH-2 inhibitors with improved efficacy and selectivity. Diarylquinolines represent a novel class of antimycobacterial drugs with strong in vitro and in vivo activity against different mycobacterial species (Andries et al., 2005; Ji et al., 2006). Diarylquinolines block ATP synthesis and cause a

decrease of cellular ATP levels (Koul et al., 2007). As the bacterial ATP stores are depleted over a period of time, subsequently pronounced bacterial killing is observed (Koul et al., 2008). Diarylquinolines specifically interact with the oligomeric transmembrane subunit c of mycobacterial ATP synthase (Koul et al., 2007, see also Fig. 2). During enzymatic catalysis, this oligomeric subunit, together with subunits ɛ FK866 in vitro and γ, rotates relative to subunits α3β3δab and in this way couples proton flow to the synthesis of

ATP (Boyer, 1993; Junge et al., 1997). Protons enter from the periplasmic space via an entry channel in subunit a and are then transferred to an essential acidic residue in the membrane-spanning part of subunit c (Fig. 2). After a close to 360° rotation of the cylindrical subunit c oligomer relative to subunit a, the protons are released on the cytosolic side of the membrane via an exit channel in subunit a (Vik & Antonio, 1994; Diez PARP inhibitor et al., 2004). Mutagenesis studies indicate that diarylquinoline lead compound TMC207 binds to the central region of subunit c, close to the essential acidic residue (Koul

et al., 2007). TMC207 may compete with protons for binding to subunit c or may alternatively interfere with the extensive conformational changes of this subunit during catalysis. Whereas typical inhibitors Carteolol HCl of ATP synthase subunit c, such as dicyclohexyl-carbodiimide and oligomycin, are not selective and highly toxic (Matsuno-Yagi & Hatefi, 1993; Wallace & Starkov, 2000; Amacher, 2005), TMC207 displays a surprising selectivity, with only an extremely low effect on human ATP synthesis (Haagsma et al., 2009). Although several residues of subunit c are reported to modulate diarylquinoline sensitivity (Koul et al., 2007), the molecular basis for the observed selectivity needs to be further investigated. No high-resolution structure is available for mycobacterial ATP synthase or its subunits, and structural models for mycobacterial subunit c have only been built based on the known structure of the homologous subunit from E. coli, Ilyobacter tartaricus or Bacillus PS3 (de Jonge et al., 2007; Koul et al., 2007). High-resolution structural data for mycobacterial subunit c and biochemical investigations on drug/target interaction would help to explain drug selectivity and would provide input for docking studies to design new compound derivates.

Interestingly, our own predictions of enzyme localization Roscovitine using signalp 3.0 (Bendtsen et al., 2004) and lipop v. 1.0 (Juncker et al., 2003), as well as the locatep database (Zhou et al., 2008) indicate that EF2863 is a secreted protein, whereas the leader peptide of EF0114 seems to have no signal peptidase I cleavage site, meaning that this protein may be N-terminally anchored to the cell membrane. Different localization of the two endoglycosidases may reflect different physiological roles. Proteins with high-mannose N-linked glycans are frequently found in human

glycoproteins (Fujiwara et al., 1988, Furukawa et al., 1989). Even though the release of nutrients from these glycoproteins

seems to be a physiologically important role of enzymes such as EfEndo18A, one may speculate about additional physiological roles such as modulation of the host immune system. Interestingly, it has been shown that EfEndo18A from E. faecalis V583 is up-regulated in blood and urine (Vebo et al., 2009, 2010), where E. faecalis frequently causes infection. The prevalence of endoglycosidases that exploit, alter or inactivate host glycoproteins may give pathogenic bacteria this website an advantage during infection. This work was supported by grant 183637/S10 from the Research Council of Norway. We thank Britt Dahl for technical assistance during the cloning experiments. “
“The Pectobacterium atrosepticum strain SCRI1043 genome contains two complete prophage

sequences. One, ECA41, is Mu-like and is able to integrate into, and excise from, ASK1 various genomic locations. The other, ECA29, is a P2 family prophage, and is also able to excise from the genome. Excision of both prophages is rare and we were unable to induce lysis of cultures. Deletion of the entire prophages, both separately and in combination, did not affect the growth rate or the secretion of plant cell wall-degrading enzymes, but swimming motility was decreased. The virulence of prophage deletion strains in the potato host was decreased. Lysogenization of a bacterial host by temperate bacteriophages can alter bacterial physiology. Most dramatically, this manifests itself as lysogenic conversion, where a previously avirulent strain becomes a serious pathogen. Enterohaemorrhagic Escherichia coli and Vibrio cholerae are prime examples, where Stx phage and CTXΦ provide the Shiga toxin and cholera toxin genes, respectively (O’Brien et al., 1984; Waldor & Mekalanos, 1996). Phage-encoded functions are diverse. Bor and Lom, carried by phage λ, are involved in resistance to the host immune system and cell adhesion, respectively (Barondess & Beckwith, 1990; Pacheco et al., 1997); SopE is an effector protein secreted by the Type III secretion system in Salmonella that activates human Rho GTPases (Hardt et al.

0, 4 °C) at a final concentration of 4 mg protein mL−1. For the membrane CFE, 1% v/v β-dodecyl-d-maltoside was added to the preparation to facilitate the solubilization Idelalisib research buy of the membrane-bound proteins. To ensure optimal protein separation, 4–16% linear gradient gels were cast using the Bio-Rad MiniProtean™ 2 system using 1 mm spacers. Soluble or membrane proteins (60 μg) were loaded into the wells and the gels were electrophoresed under native conditions. Eighty volts were applied for the stacking gel. The voltage was then increased to 300 V

once the running front entered the separating gel. The blue cathode buffer [50 mM Tricine, 15 min Bis-Tris, 0.02% w/v Coomassie G-250 (pH 7) at 4 °C] was changed to a colorless cathode buffer [50 mM Tricine,

15 min Bis-Tris (pH 7) at 4 °C] when the running front was half-way through the gel. Upon completion, the gel slab was equilibrated for 15 min in a reaction buffer (25 mM Tris-HCl, 5 mM MgCl2, at pH 7.4). The in-gel visualization of enzyme activity was ascertained by coupling the formation of NAD(P)H to 0.3 mg mL−1 of phenazine methosulfate (PMS) and 0.5 mg mL−1 of iodonitrotetrazolium (INT). ICDH-NADP activity was visualized using a reaction mixture consisting of reaction buffer, 5 mM isocitrate, 0.1–0.5 mM NADP, INT, and PMS. The same reaction mixture was utilized for ICDH-NAD, except 0.1–0.5 mM NAD was utilized. GDH-NAD activity was visualized using a reaction mixture consisting Selleckchem HSP inhibitor of reaction buffer, 5 mM glutamate, 0.1–0.5 mM NAD, INT, and PMS. GDH-NADP activity

was visualized using a reaction mixture consisting of reaction buffer, 5 mM glutamate, 0.5 mM NADP, INT, and PMS. KGDH activity was visualized using a reaction mixture consisting of reaction buffer, 5 mM KG, 0.5 mM NAD, 0.1 mM CoA, INT, and PMS. Glutamate synthase (GS) activity was determined using a reaction mixture consisting of reaction buffer, 5 mM glutamine, 0.5 mM NADPH, 5 mM KG, 5 U mL−1 GDH, INT, and 0.0167 mg mL−1 Gefitinib supplier of 2,4-dichloroindophenol. Complex I was detected by the addition of 1 mM NADH and INT. Rotenone (40 μM) was added to inhibit the complex. Succinate dehydrogenase was monitored by the addition of 5 mM succinate, INT, and PMS. Complex IV was assayed by the addition of 10 mg mL−1 of diaminobenzidine, 10 mg mL−1 cytochrome C, and 562.5 mg mL−1 of sucrose. KCN (5 mM) was added to the reaction mixture to confirm the identity of Complex IV. Aspartate amino transferase (AST) was monitored by the addition of 5 mM aspartate, 5 mM KG, 0.5 mM NADP, 5 U of GDH, INT, and PMS. The formation of glutamate effected by AST under these conditions was detected by GDH. Reactions were halted using destaining solution (40% methanol, 10% glacial acetic acid) once the activity bands reached their desired intensities. Activity stains performed in the absence of substrate and/or in the presence of inhibitors assured band specificity.

Both AcfB and TcpI are transmembrane Neratinib manufacturer proteins, and the homology with MCPs has been noted previously (Everiss et al., 1994; Harkey et al., 1994). The tcpI and acfB genes were originally identified through TnphoA mutagenesis, and in this study a tcpI:TnphoA V. cholerae strain was found to exhibit wild-type levels of intestinal colonization, while an acfB∷TnphoA V. cholerae strain was approximately 10-fold defective for intestinal colonization (Peterson & Mekalanos, 1988). AcfB and TcpI share 26% amino acid identity over their entire

length, and the segments from aa 463 to 530 in AcfB and aa 453 to 520 in TcpI share 77% identity (Fig. 1 and Supporting Information, Fig. S1). Both proteins are predicted to have signal

peptides, and the N-terminal periplasmic portions contain a Cache motif (Anantharaman & Aravind, 2000), a signaling domain found in chemotaxis receptors. The transmembrane segments are predicted to be located at aa 278–292 in TcpI and aa 286–300 in AcfB (Cserzo et al., VE-821 molecular weight 1997), and the cytoplasmic portions contain a HAMP motif (Aravind & Ponting, 1999) and an MCP signaling domain (PF00015), both typically found in MCPs (Fig. 1). The Cache domain is predicted to be involved in small molecule recognition, while the HAMP domain has been shown to modulate conformation of MCP oligomers in response to ligand binding in the Cache domain and methylation of the MCP domain (Khursigara et al., 2008). To determine the roles of AcfB and TcpI in intestinal colonization, V. cholerae strains containing chromosomal mutations in acfB and tcpI were constructed. The tcpI gene is in a single gene operon, and so a deletion/insertion mutation (ΔtcpI∷Cm) was constructed; however, due to the location of acfB within a multigene operon, an in-frame deletion was constructed (ΔacfB) to prevent deleterious effects on downstream gene expression. We additionally constructed a V. cholerae strain with a

ΔcheY-3 mutation in this genetic background; cheY-3 is essential for V. cholerae chemotaxis (Butler & Camilli, 2004). The acfB, tcpI, and acfB tcpI V. cholerae strains were monitored for swimming behavior Cell press utilizing soft agar plates (Fig. 2). In this assay, the ΔcheY-3 mutant, despite being motile, demonstrates no net movement away from the point of inoculation, and productive movement could be complemented back to wild-type levels by providing cheY-3 in trans, as has been demonstrated previously (Butler & Camilli, 2004). The acfB and tcpI (single) mutants displayed motility patterns that were slightly greater than the wild-type strain, the acfB strain more so than the tcpI strain (Fig. 2); strains containing Tn-phoA fusion insertions in these genes were previously shown to similarly display enhanced motility patterns (Everiss et al., 1994; Harkey et al., 1994). In contrast, the acfB tcpI (double) mutant displayed a slightly smaller motility pattern than the wild-type strain.

A total of 1920 clones resulting from the SSH process were obtained, of which 772 were randomly sequenced, resulting in 296 contigs after removal of redundant sequences. The specificity of the contigs to the bovine EHEC strain (strain 4276) was determined by a blastn search with the human EHEC strain (strain 11368) genome sequenced by Ogura et al. (2009). Of the 296 nonredundant DNA contigs, Selleck ERK inhibitor 115 contained genes different from those of the human EHEC strain (strain 11368). BLASTN and BLASTX against the GenBank were searched for the 115 contigs specific to the bovine strain (Table 1 and Table S3). Several groups of genes were revealed by more than one clone: colicin resistance genes, multiple antibiotic resistance

region from Salmonella enterica,

phages P1 and P7, pathogenicity island (termed PAI ICL3) described in the VTEC O113:H21 E. coli CL3 (containing putative adhesins and hemolysins), genes from the genomic islands GEI 3.21 described in E. coli O111:H−, transposase from Enterobacter cloacae, E. coli and Acinetobacter baumanii, predicted type I restriction-modification enzyme from E. coli 0127:H6 E2348/69, DEAD/DEAH box helicase from Nitromonas europea, SNF2 family helicase from E. coli strain E24377A, plasmid pO111_2 from E. coli O111:H−, and plasmid pSMS35_8 from E. coli SMS-3-5. BLASTN revealed six sequences that are not homologous to any annotated LGK-974 datasheet DNA sequences in GenBank. The other sequences were detected in only one clone and corresponded to genes specific to Klebsiella pneumoniae, Pseudomonas aeruginosa, Citrobacter rotendium, C59 nmr Shigella sonnei, Erwinia sp., Desulfurispirillum indicum, Dickeya zeae, Pantoea ananatis, and several strains of E. coli. Several sequences (in bold in Table 1 and Table S3) were chosen for further characterization based upon the frequency

of the contigs in the subtractive library or upon the putative involvement in adherence to the eukaryotic cells or in host specificity: genes from PAI ICL3, four sequences with no homology, genes from P1 and P7 phages, genes from genomic island GEI 3.21, hypothetical proteins from E23477A strain, DEAD/DEAH box helicase from Nitromonas sp., genes from E. coli O111:H− strain 11128, transposase from A. baumanii., ABC transporter from D. zeae, and avrA genes from E. coli strain CB769. The regions of DNA homologous to that previously identified in the subtractive library were searched for in EHEC and EPEC strains of serogroup O26 isolated from human and from cattle using DNA colony hybridization (Table 2) or using specific PCR for PAI ICL3 locus (Table 3). Statistical analyses were performed to assess differences in the presence of the fragments according to host specificity (human or bovine) and/or pathotype (EHEC or EPEC). Two sequences, both homologous to the genomic island GEI 3.21 from E. coli O111:H−, were statistically associated with EPEC strains in comparison with EHEC strains.

A total of 1920 clones resulting from the SSH process were obtained, of which 772 were randomly sequenced, resulting in 296 contigs after removal of redundant sequences. The specificity of the contigs to the bovine EHEC strain (strain 4276) was determined by a blastn search with the human EHEC strain (strain 11368) genome sequenced by Ogura et al. (2009). Of the 296 nonredundant DNA contigs, find more 115 contained genes different from those of the human EHEC strain (strain 11368). BLASTN and BLASTX against the GenBank were searched for the 115 contigs specific to the bovine strain (Table 1 and Table S3). Several groups of genes were revealed by more than one clone: colicin resistance genes, multiple antibiotic resistance

region from Salmonella enterica,

phages P1 and P7, pathogenicity island (termed PAI ICL3) described in the VTEC O113:H21 E. coli CL3 (containing putative adhesins and hemolysins), genes from the genomic islands GEI 3.21 described in E. coli O111:H−, transposase from Enterobacter cloacae, E. coli and Acinetobacter baumanii, predicted type I restriction-modification enzyme from E. coli 0127:H6 E2348/69, DEAD/DEAH box helicase from Nitromonas europea, SNF2 family helicase from E. coli strain E24377A, plasmid pO111_2 from E. coli O111:H−, and plasmid pSMS35_8 from E. coli SMS-3-5. BLASTN revealed six sequences that are not homologous to any annotated NVP-BEZ235 DNA sequences in GenBank. The other sequences were detected in only one clone and corresponded to genes specific to Klebsiella pneumoniae, Pseudomonas aeruginosa, Citrobacter rotendium, Staurosporine chemical structure Shigella sonnei, Erwinia sp., Desulfurispirillum indicum, Dickeya zeae, Pantoea ananatis, and several strains of E. coli. Several sequences (in bold in Table 1 and Table S3) were chosen for further characterization based upon the frequency

of the contigs in the subtractive library or upon the putative involvement in adherence to the eukaryotic cells or in host specificity: genes from PAI ICL3, four sequences with no homology, genes from P1 and P7 phages, genes from genomic island GEI 3.21, hypothetical proteins from E23477A strain, DEAD/DEAH box helicase from Nitromonas sp., genes from E. coli O111:H− strain 11128, transposase from A. baumanii., ABC transporter from D. zeae, and avrA genes from E. coli strain CB769. The regions of DNA homologous to that previously identified in the subtractive library were searched for in EHEC and EPEC strains of serogroup O26 isolated from human and from cattle using DNA colony hybridization (Table 2) or using specific PCR for PAI ICL3 locus (Table 3). Statistical analyses were performed to assess differences in the presence of the fragments according to host specificity (human or bovine) and/or pathotype (EHEC or EPEC). Two sequences, both homologous to the genomic island GEI 3.21 from E. coli O111:H−, were statistically associated with EPEC strains in comparison with EHEC strains.