Standard curves generated with concentrations of ATP from 0.1 to 100 nM were used to calculate the ATP concentrations in each sample. The results are expressed as the fold increase against the ATP level in culture supernatants of untreated cells. Prior to infection, differentiated THP-1 macrophages were treated with 10 μM diphenyleneiodonium chloride (DPI) (Sigma Aldrich), a potent inhibitor of reactive oxygen species (ROS) production (Hancock & Jones, 1987), for 1 h, and the cells were then infected with viable S. sanguinis AZD1208 datasheet SK36 (MOI 50, 100, or 200) for 2 h in the presence of DPI. The cells were washed with PBS, and cultured in fresh medium containing DPI and antibiotics for 18 h.

Viability was determined as described above.

Macrophages were lysed with PBS containing 1% Triton X100 and a protease inhibitor cocktail (Nakalai Tesque, Kyoto, Japan). Clarified lysates were resolved using gel electrophresis with a sodium dodecyl sulfate polyacrylamide 4–15% gradient gel (SDS-PAGE) (Bio-Rad Laboratories, Hercules, CA), and then transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Uppsala, Sweden). After incubation with 5% non-fat skimmed milk in PBS containing 0.1% Tween-20 for 1 h, the membranes were reacted Seliciclib in vivo with a goat anti-p10 subunit of human caspase-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Antibodies next bound to the immobilized proteins were detected using horseradish-conjugated antigoat IgG (Santa Cruz) and an ECL-plus Western blot detection kit (GE Healthcare). Statistical analyses were performed using QuickCalcs

software (GraphPad Software, La Jolla, CA). Experimental data are expressed as the mean ± SD of triplicate samples. Statistical differences were examined using an independent Student’s t-test, with P < 0.05 considered to indicate statistical significance. To determine whether S. sanguinis induces foam cell formation, differentiated THP-1 macrophages were exposed to viable or heat-inactivated S. sanguinis SK36. The cells were further cultured in the presence of LDL for 2 days, and stained with oil-red O to detect foam cells containing cytoplasmic lipid droplets (Fig. 1a). Foam cell formation by infection with viable S. sanguinis occurred in a dose-dependent manner with maximum induction at an MOI of 50 (Fig. 1b). At an MOI of more than 100, viable S. sanguinis-induced cell death of macrophages (data not shown, and see below). Exposure to heat-inactivated S. sanguinis or E. coli LPS also promoted foam cell formation (Fig. 1b). Our study of foam cell formation suggested that infection with viable S. sanguinis also induces cell death of macrophages at an MOI of more than 100. At first, bacterial internalization of S. sanguinis was confirmed by adhesion and internalization assay (Fig. 2a).

An alternative route is via Access to Science courses designed for students who do not have traditional university entry requirements. The purpose of this research was to examine the MPharm admissions criteria and student progression to identify variables that maybe indicative of degree success. Four datasets corresponding to four concurrent years of admissions to the MPharm programme were examined (N = 381); Cohort 1 (N = 70), Cohort 2 (N = 107), Cohort 3 (N = 83) and Cohort 4 (N = 121). These cohorts were followed through their degree programme and data were captured both prior to admission (via UCAS forms) and during their studies (via academic records) including: age, gender,

nationality, Epacadostat chemical structure prior qualifications, GCSE and A-level subjects and grades, years on the MPharm course, module and end of year results. All data were coded and entered IBM SPSS Statistics (version 17). Data cleaning was undertaken to ensure consistency between variables and coding regularity. Each cohort was analysed separately and in combination to enable both inter- and intra-cohort exploration. Data were examined using independent sample t-tests, one-way ANOVA, chi-square and Kruskal-Wallis tests. Statistical significance was set at p < 0.05 for all tests. Students who achieved an ‘A’ grade at A-level chemistry were significantly more likely to attain a better degree classification (p = 0.005). Students with A-level

mathematics or biology showed no difference in degree attainment. No learn more advantage in final degree attainment was seen with students having A-levels in chemistry, biology and maths, compared to those with alternative A-level combinations. Students with prior degrees had no advantage over those with A-levels or those entering through the Access Course. Students going straight to university with A-levels were significantly different (p = 0.001) from those on the Access course, with a final degree percentage of 61.8 ± 5 compared to 57.7 ± 5 respectively. An

examination of factors predisposing students towards success or failure in their degree can help improve the Farnesyltransferase MPharm admissions process. There was a trend linking A-level grades, but not A-level choices, with degree classification, suggesting A-level grades are a good predictor of academic success, particularly in the first year of the programme. However this trend lessened after year one, suggesting the structure of the first year allowed students to improve their weaker areas. There was a significant difference between the degree classification of Access to Science students and A-level students. Further work is required to ascertain the additional needs of Access students to help them through the degree programme, and how they access the many support resources already in place within the college. The main limitation of the study was missing data in the admission records, which led to reduced sample sizes and possibly skewed results.

An alternative route is via Access to Science courses designed for students who do not have traditional university entry requirements. The purpose of this research was to examine the MPharm admissions criteria and student progression to identify variables that maybe indicative of degree success. Four datasets corresponding to four concurrent years of admissions to the MPharm programme were examined (N = 381); Cohort 1 (N = 70), Cohort 2 (N = 107), Cohort 3 (N = 83) and Cohort 4 (N = 121). These cohorts were followed through their degree programme and data were captured both prior to admission (via UCAS forms) and during their studies (via academic records) including: age, gender,

nationality, AC220 clinical trial prior qualifications, GCSE and A-level subjects and grades, years on the MPharm course, module and end of year results. All data were coded and entered IBM SPSS Statistics (version 17). Data cleaning was undertaken to ensure consistency between variables and coding regularity. Each cohort was analysed separately and in combination to enable both inter- and intra-cohort exploration. Data were examined using independent sample t-tests, one-way ANOVA, chi-square and Kruskal-Wallis tests. Statistical significance was set at p < 0.05 for all tests. Students who achieved an ‘A’ grade at A-level chemistry were significantly more likely to attain a better degree classification (p = 0.005). Students with A-level

mathematics or biology showed no difference in degree attainment. No BIBF 1120 cost advantage in final degree attainment was seen with students having A-levels in chemistry, biology and maths, compared to those with alternative A-level combinations. Students with prior degrees had no advantage over those with A-levels or those entering through the Access Course. Students going straight to university with A-levels were significantly different (p = 0.001) from those on the Access course, with a final degree percentage of 61.8 ± 5 compared to 57.7 ± 5 respectively. An

examination of factors predisposing students towards success or failure in their degree can help improve the Linifanib (ABT-869) MPharm admissions process. There was a trend linking A-level grades, but not A-level choices, with degree classification, suggesting A-level grades are a good predictor of academic success, particularly in the first year of the programme. However this trend lessened after year one, suggesting the structure of the first year allowed students to improve their weaker areas. There was a significant difference between the degree classification of Access to Science students and A-level students. Further work is required to ascertain the additional needs of Access students to help them through the degree programme, and how they access the many support resources already in place within the college. The main limitation of the study was missing data in the admission records, which led to reduced sample sizes and possibly skewed results.

In M-Nha, most Asp residues (14/19) were predicted to be in the hydrophobic region, while the alignment of M-Nha with Na+/H+ antiporters of another six microorganisms indicated that three aspartates, including Asp-138, Asp-167 and Asp-224, were conserved in M-Nha (Fig. 3). The protein encoded by m-nha gene showed a high similarity of 92%, 86% and 62% to NhaH from H. dabanensis D-8T, H. aidingensis AD-6T and B.

subtilis, respectively. Interestingly, M-Nha has a long carboxyl terminal hydrophilic tail (140 amino acid residues), similar to Nhap and NhaG type Na+/H+ antiporters, whereas NhaH does not. It was reported that both the ion specificity and activity of an Na+/H+ antiporter were partially determined by the structural properties of the C-terminal hydrophilic tail (Hamada et al., 2001; Waditee et al., 2001). NhaG from B. subtilis possesses a hydrophilic segment with >100 amino acid selleck residues at the carboxyl terminal region (Gouda et al., 2001), and such a long hydrophilic domain is not present in any other microbial Na+/H+ antiporter except SynNhaP (NhaS1) in Synechocystis sp. (Hamada et al., 2001) and ApnhaP in A. halophytica (Waditee et al., 2001). The activities of NhaG decreased

when 26 residues in the C-terminal of the protein were lost (Gouda et al., 2001), and 56 residues in the C-terminal region of SynNhaP were necessary for antiporter activity (Hamada et al., 2001). Hydropathy analysis BYL719 usually showed that the Na+/H+ antiporter had 10–12 hydrophobic and also probably membrane-spanning regions (Majernik et al., 2001; Yang et al., 2006). Our results also revealed that m-nha gene product fits well into this model. The NhaH PIK3C2G and NhaG had 12 TMS, but M-Nha had only 10 TMS, although they all had high similarity of amino acid sequence. Consequently, the mechanism of ion transport by M-Nha from the Dagong Ancient Brine Well should be different from that of NhaH, NhaG and SynNhaP. With the differences of amino acid sequence and the putative secondary structure of the protein encoded

by m-nha from those Na+/H+ antiporter genes reported previously, it can be proposed that m-nha is a novel Na+/H+ antiporter gene. This study was significant in not only helping us understand the necessity of the existence of Na+/H+ antiporter in the Dagong Ancient Brine Well to maintain the intracellular environment homeostasis for halophiles, but also enriches our knowledge about the different mechanisms of Na+/H+ antiporter in halophiles in such an extreme environment. We thank Dr Terry A. Krulwich (Department of Biochemistry, Mount Sinai School of Medicine of the City University, New York) and Prof. Susheng Yang (Department of Microbiology, College of Biological Sciences, China Agricultural University, Beijing, China) for donating the strain E. coli KNabc.

Finally, it is interesting to notice that of the five identified stress-related

heat shock proteins, GroES, GroEL1, GroEL2, grpE and DnaK2, only GroES is differentially more abundant (Fig. 2d). As GroES interacts with GroEL to form a complex, which assists in correcting misfolded proteins, this result is surprising, particularly when compared with MED4 subjected to high light Stress, whereby both GroES and GroEL12 proteins were more abundant (Pandhal et al., 2007). Another protein identified as being stress response related, a histone-like DNA-binding protein (PMM1321), was more abundant in the P-stressed phenotype (Fig. 2e). These proteins are known to wrap DNA and stabilize it from denaturation under extreme environmental conditions (Pettijohn, 1988). Indeed, a homologue of this protein (HU) was more abundant in Synechocystis sp. PCC6803 under P-deplete see more conditions (Gan, 2006), but surprisingly, was not observed in MED4 under light stress (Pandhal et al., 2007). This observation suggests specificity in stress response for this protein, possibly nutrient starvation; however, a more detailed examination of the overall stress responses within this organism is required. It is clear that MED4 acclimates to long-term P starvation through activating and also suppressing a wide range of cellular processes. Important selleck kinase inhibitor metabolic mechanisms such as glycolysis are depressed, while other systems, most notably P-acquisition

mechanisms, are considerably elevated. Photosynthesis and carbon fixation are reduced, while the structures of the photosystems are reinforced. This, in particular, is an indication of the stressed cell reducing its metabolic activities while simultaneously maintaining cellular integrity. Specific Niclosamide amino acid biosynthesis mechanisms are either reinforced or reduced. This may be an indication of individual amino acid requirements, which could well be linked to intracellular recycling efficiency and/or specificity. Indeed, translation, indicated through ribosome levels, appears

to be increased, indicating an active, ongoing response. Specific chaperonins and protein-folding proteins, particularly membrane-associated ones, are more abundant, while DNA integrity is reinforced. Interestingly, there does appear to be a specificity of the stress response to P starvation, whereby under conditions of nitrogen deprivation, ribosomal genes as well as the carboxysome shell protein genes csoS12 and photosystem genes were all repressed, whereas Rubisco is repressed under both N starvation (Tolonen et al., 2006) and P starvation (this study). However, the response to N deprivation was measured over a 48-h period and may not reflect longer term acclimation. The environmental conditions that MED4 are exposed to in situ are considered to be consistent and unchanging; however, these results appear to suggest that MED4 exhibits a capability to withstand long periods of P starvation and recover.

Leishmania donovani promastigotes strain AG83 (MHOM/IN/83/AG83) was grown in M199 medium (Sigma-Aldrich) supplemented with 10% FBS (Invitrogen) and penicillin–streptomycin mixture at 22 °C with slow shaking. To prepare GST-LdCyc1-CRK3 kinase complex, bacterially expressed GST-LdCyc1 was first bound to glutathione–sepharose beads (GE Healthcare Lifesciences), and the bead-bound GST-LdCyc1 was then incubated with an extract of Sf9 cells expressing LdCRK3 in the binding buffer (50 mM Tris-HCl, pH 8.0 containing

50 mM NaCl, 5 mM NaF, 1 mM Na3VO4, 0.1 mM EDTA, 0.1% Triton X-100, 10% TSA HDAC ic50 glycerol, 2 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl flouride (PMSF) and protease inhibitors) on a rotating wheel at 4 °C for overnight. The beads were washed three times with the same binding buffer, and the kinase complex was eluted with 50 mM Tris-HCl, pH 8.0, containing 10% glycerol, 10 mM reduced glutathione and PMSF. The complete ORF of LdHAT1 was cloned into pET21b vector, and the C-terminal 6His-tagged chimera was expressed in pG-JKE8 (TAKARA)-transformed Escherichia coli strain BL21 cells (expressing GroEL and GroES chaperons for greater solubility of the over-expressed protein) by induction

with 1 mM isopropyl β-D-1-thiogalactopyranoside at 37 °C ABT-199 mouse for 3 h. Two mutants of LdHAT1, viz., LdHAT1ΔCy and LdHAT1-T394A were also cloned into pET21b vector and expressed as mentioned above. All 6His-tagged proteins were purified over Ni-NTA agarose beads. To perform interaction assay between LdCyc1 and LdHAT1, 5 μg of bacterially purified LdHAT1 protein was incubated on a rotating wheel at 4 °C for 1 h with glutathione beads bound to 0.2 μg of either GST or GST-LdCyc1 proteins in 50 mM Na-phosphate (pH 8.0) containing 250 mM NaCl, 0.5% Triton X-100, 10% glycerol, 1 mM EDTA, 2 mM DTT and protease inhibitors. Subsequently, the beads were washed six times with the same buffer, and the bound proteins were analysed by immunoblot analysis with appropriate antibodies. Similar experiments were carried out with the mutant LdHAT1 proteins. To synchronize L. donovani

promastigotes, exponentially growing cells were blocked with 10 mM hydroxyurea (HU) for 36 h followed by releasing PAK5 the arrest by re-suspending the cells in equal volume of growth medium, and cells were collected at different intervals. The synchronicity of cell cycle progression was confirmed by analysis in a flow cytometer (Supporting Information, Fig. S3). The population of cells from each time point was also examined by analysing the fluorescence and differential interference contrast (DIC) images of 4′,6-diamidino-2-phenylindole (DAPI)-stained cells captured by a Zeiss Axio-observer Z1 inverted microscope (Fig. S3). Cells from different time intervals were lysed in 50 mM Tris-HCl (pH 8.0) containing 150 mM NaCl, 50 mM NaF, 1 mM Na3VO4, 0.

, 1995; Kominkova et al., 2000). Most works reveal fungi, especially aquatic Selleck Natural Product Library hyphomycetes, as the dominant players, in terms of activity

and biomass increase, during early decomposition of leaf litter in aquatic ecosystems (Baldy et al., 1995; Romaní et al., 2006). However, phenol-degrading bacteria may also be involved in decomposition of recalcitrant plant material in aquatic environments, although their potential role is much less investigated. Phenol-degrading bacteria are highly adaptive, as observed through the analysis of key functional genes in communities growing in biological wastewater treatment plants (Futamata et al., 2003; Basile & Erijman, 2010). Phenol hydroxylases, which convert phenol into catechol derivatives via hydroxylation, are specific phenol oxidases generally involved in the degradation of organic compounds. These enzymes have been extensively studied at the molecular level, and they can now be detected in natural samples by high-throughput analytical methods. Multicomponent phenol hydroxylases (mPHs) are considered to be predominant in nature (Nordlund et al., 1993; Watanabe et al., 2002). The largest subunit of multicomponent phenol hydroxylases (LmPHs) has been used as a molecular marker to assess the functional

and genetic diversities of biotechnologically relevant phenol-degrading bacteria (Futamata et al., 2005; Viggor et al., 2008). Moreover, phenol-degrading Adriamycin bacteria have been isolated and characterized from the phyllosphere of trees showing that leaves may contain a significant bacterial diversity with respect to LmPH sequence similarities (Sandhu et al., 2009). However, to the best Protirelin of our knowledge, no experimental report exists describing the change in the bacterial phenol-degrading community during leaf litter by the use of selected molecular markers targeting to functional genes. In this study, we have used the LmpH gene as a molecular proxy to analyze the changes in the phenol-degrading bacterial community during the decomposition of submersed

Platanus acerifolia [Aiton] Willd. leaves in a forested stream. We hypothesize that phenol-degrading bacteria might contribute to leaf litter breakdown and that their community structure might change throughout the decomposition process as higher amounts of free phenolic compounds are available. To test this hypothesis, three discrete sampling dates were chosen according to mass weight and enzymatic activity data from a previous experiment of leaf litter decomposition. Selected samples covered the main observed changes in microbial activity and biomass. The observed changes of the bacterial community indicate that a specialization of potential phenol-degrading bacteria exists during the decomposition of leaves.

Symptomatic patients with hyperlactataemia were defined as having SHL. LA was defined as SHL with

either (1) an arterial pH less than normal (<7.38) or (2) a plasma anion gap >16 mEq/L and/or serum bicarbonate <24 mmol/L. Routine lactate measurements were not scheduled in INITIO and only performed at the investigators' discretion. In the clinical substudy, data from all randomized patients (except those randomized in error) were used to examine which baseline clinical and biochemical parameters were associated with subsequent development of LA or SHL. In the molecular substudy, mtDNA and mtRNA from PBMCs were examined in a nested case–control study of cases of SHL and LA. Two controls (subjects without SHL or LA) were randomly selected for each case matched for time of event, duration on ddI+ d4T and BMI. A BMI >25 kg/m2 was considered overweight as per World Health Dabrafenib datasheet Organization definitions [21]. BMI was included in matching after the initial analysis of the clinical parameters. Frozen PBMC pellets were re-suspended in phosphate-buffered saline (PBS) and www.selleckchem.com/products/AG-014699.html split into two aliquots. Genomic DNA (gDNA)

was extracted from one aliquot using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) and quantified using Sybr Green I (Molecular Probes, Eugene, OR, USA) against DNA standards of known concentrations. Samples were then adjusted to a standard concentration of 2.5 ng/μL.

RNA was extracted from the other aliquot using the RNeasy Mini Kit (Qiagen) with on-column gDNA digestion using RNase-free DNase (Qiagen) and quantified using Sybr Green II (Molecular Probes) against RNA standards of known concentration. Olopatadine Aliquots (200 ng) of RNA were reverse-transcribed into cDNA using the Superscript III system (Invitrogen, Carlsbad, CA, USA). To adjust for variability among reverse transcriptase (RT) reactions, four RT reactions per sample were performed. All samples were checked for cDNA quality and then pooled [22,23]. Any sample with insufficient cDNA quality was excluded. gDNA aliquots of 2 μL (5 ng) or pooled cDNA aliquots of 2 μL were analysed using real-time quantitative polymerase chain reaction (PCR) on the Lightcycler 2.0 platform (Roche Diagnostics, Mannheim, Germany). Samples were run in duplicate, with internal positive and negative controls. mtDNA copy number per cell was calculated by comparing the gDNA copy numbers of a region distal to the site of initiation of replication of the mitochondrial genome (region 2) and a region close to the site of initiation of replication (region 1) with the copy number of a nuclear gene [peroxisome proliferator-activated receptor gamma (PPARG)] (2 copies/cell). Two separate regions of the mitochondrial genome were chosen to improve sensitivity [24].

, 2010). Random DNA fragments

were generated with RsaI, ligated into SmaI digested cloning vector pBluescript SK and transformed in Escherichia coli XL1-blue (Stratagene) with electroporation according to the manufacturer’s instructions (Bio-Rad micropulser). The positive clones were sequenced by capillary sequencer; these DNA fragments served as the initial templates for the genome walking. Overlapping sequencing reactions (141 reactions using Life Tech’s Genetic Analyzer 3500) were used to cover the whole phage DNA, the mean usable read length was 861 bases, so that each nucleotide of the 40 058 bp phage DNA was covered on the average 3.03 times (each nucleotide was covered buy Doramapimod selleck compound at least by two capillary sequencing reads). NGS data (2 827 891 reads with a mean read length of 45.15 bases, altogether 127 685 564 bases) were used to correct the possible sequencing errors of the sequence backbone revealed by capillary sequencing method. The average coverage of each nucleotide by SOLiD reads was 3187. CLC Genomics Workbench software (CLC Bio, Aarhus, Denmark) was used to generate contigs and

to assemble NGS and capillary sequencing data. GenBank accession number: JN991020. ORFs were assigned using the ORF finder programs RAST (Aziz et al., 2008; http://rast.nmpdr.org/ ), Glimmer (http://www.ncbi.nlm.nih.gov/genomes/MICROBES/glimmer_3.cgi), NCBI ORF finder (http://www.ncbi.nlm.nih.gov/projects/gorf/), and GeneMark (Lukashin & Borodovsky, 1998, http://exon.biology.gatech.edu/). Sclareol Translated sequences identified by ORF finders were further analyzed by homology searching using NCBI blastp (Altschul et al., 1997). Molecular masses, isoelectric points, and codon statistics

were calculated using CLC Genomics Workbench. For the comparative genomic studies, we screened for sequence homologies with NCBI blastx (Altschul et al., 1997) against the Entrez Query ‘Viruses (ORGN)’ databases to looking for potential relationships as recommended by Lavigne et al., 2003. To confirm the resulted relations, we used CoreGenes 3.1 program to create pairwise comparisons, using default stringency setting (‘75’) at http://binf.gmu.edu:8080/coreGenes3.1/. Sixteen broad host range bacteriophages against P. tolaasii LMG 2342T and P. putida DSM 9278 were obtained after the repeated isolation cycles. The results of the spot lysis assay against different pseudomonads are summarized in Table 2. The most effective phages were Bf3 and Bf7, which infected 16 strains of the treated pseudomonads, but Bf10 and Bf16 had also remarkable abilities (infecting 15 and 13 strains, respectively). The nucleic acid of the phages proved to be DNA as they were susceptible to deoxyribonuclease but not to ribonuclease digestions. The genome sizes were approximately 38 kb based on restriction enzyme digestion experiments.

Briefly, bacteria were grown in 150 mL of THB in the presence of 0.05% Tween 80 and 20 mM dl-threonine until the culture reached the early-exponential phase with an OD600 nm of 0.2. The culture was chilled on ice for 30 min, and the bacteria were harvested ABT-737 in vivo by centrifugation and washed extensively with ice-cold sterile distilled water and 10% glycerol in distilled H2O. Cells from the 150 mL culture were suspended in 0.6 mL of 10% glycerol. One hundred

microliters of suspended cells were used for each electroporation, which was conducted in a chilled 2-mm Gap cuvette using a Pulser model of ECM630 (BTX, San Diego, CA) with the following settings: 2.5 kV, 25 μF capacitor and 400 Ω resistor. One milliliter of THB with 0.05% Tween 80 was added to the pulsed cells. After 2-h incubation at 37 °C, the samples were plated on TH agar plates with appropriate selective substance(s). Nine plasmid

p6Srt derivatives were created with a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA): H184A, H204A, F213A, Y236A, L263A, T265A, C266A, R275A and R282A using the primer sets listed in Supporting Information, Table S1. The presence of the desired mutation in each plasmid was confirmed by sequencing the mutagenized plasmids. Actinomyces oris mutants were constructed by transforming check details SrtC1-deficient strain A. orisΔSrtC1 with corresponding p6Srt derivative plasmids based on the allelic-exchange mechanism. Surface proteins were solubilized from A. oris T14V and its mutants using a procedure modified from a mutanolysin digestion method as described previously (Demuth et al., 1996). Briefly, cells from a 10-mL overnight culture were harvested by centrifugation and washed twice with sterile water. The washed cells were suspended in the extraction buffer at a ratio of 4 μL of buffer per milligram of wet cells. The extraction buffer consisted of 26% C1GALT1 melezitose, 10 mM MgCl2, 10 mM phosphate buffer (pH 7.0) and 1000 U mL−1

mutanolysin. After a 5-h incubation at 37 °C, the suspension was centrifuged (10 000 g, 10 min, 4 °C). The supernatant was dialyzed against distilled water using a 10-kDa molecular weight cut off mini Dialysis Units (Pierce, Rockford, IL) and stored at −20 °C for analyses. All chemicals used in the extraction were obtained from Sigma-Aldrich Corp. (St. Louis, MO). Extracted surface proteins were separated on 3–8% Tris-Acetate NuPAGE gels (Invitrogen) and transferred onto nitrocellulose membranes. These membranes were incubated with 1 μg mL−1 monoclonal antibody C8A4 directed against the structural subunit (FimP) of T14V type 1 fimbriae (Cisar et al., 1991). Membranes were washed, incubated with a secondary antibody and developed according to the instructions of WesternBreeze Chromogenic Immunodetection System kit (Invitrogen). Previously, we identified three essential genes (fimQ, fimP and srtC1) for the biosynthesis of type 1 fimbriae in A. oris T14V (Chen et al.