Easterly winds prevailed in the northern region (78±13°) and north-easterly winds in the south (46.1±12°). During 25–28 January, a major dust deposition event occurred, while the ship was in the southwest of the region, making the sky brown and covering the ship in a layer of red-brown dust. The dust cloud was clearly visible in satellite images and back trajectories for these dates show that

the air mass came from the Sahara region. Seawater samples were collected using a trace metal clean technique from 20 m depth, to minimize iron contamination from the ship’s hull, using a rosette of 20-L Niskin bottles mounted on a titanium frame with a CTD profiler (Sea-Bird Electronics) in polyoxymethylene plastic and titanium casing. Samples were decanted AZD6244 chemical structure into 1-L HCl-cleaned polycarbonate bottles. The experiments commenced within an hour of sampling. Dust samples were collected daily, at sea, onto polypropylene filters (47 mm, 0.45 μm, Sterlitech). Rotary vein vacuum pumps filtered aerosol at 25–30 L min−1 for periods of typically 24 h, although this was reduced to 4–6 h during the major dust event on 25–28 January. The instantaneous dissolution of metals and nutrients was simulated

AG-014699 price by quickly passing 100 mL of deionized water (milli-Q) through the dust-loaded filter (Buck et al., 2006) and the leachate was subsampled into sterile 2-mL polypropylene vials. The bacterioplankton response to dust and leachate additions was determined by time-course sampling during incubations lasting 24 h. Four incubations were performed, two in the southwest of the region and two in the north (Fig.

1). Seawater samples (34 mL) were incubated in HCl-cleaned 35-mL PTFE bottles with dust, leachate or no (control) additions. Dust was added with the polypropylene filter onto which it was collected; additions were calculated postcruise to be 0.3 mg L−1 (incubation 1), 1.5 mg L−1 (incubation 2) or 4.7 mg L−1 (incubations 3 and 4). A further control of a blank polypropylene filter was used to ensure that the bacterioplankton response was due to the dust and not the filter. Leachate additions of 700 μL supplied 100 nM inorganic N and 10 nM P to all incubations. Bottles were placed in 17-DMAG (Alvespimycin) HCl on-deck incubators screened to allow 20% surface irradiance and cooled to in situ temperature. The uptake rate of 35S-methionine (35S-Met) was measured at t=0, 2, 4, 6 and 24 h to determine the bacterioplankton community metabolic response to treatments (+Leachate or +Dust) as compared with controls. Two incubations were also sampled at t=8 h. At t=0 and 6 h, samples were taken to measure cellular uptake by sorted bacterioplankton groups. A further eight t=0 h samples were collected throughout the cruise to measure the cellular uptake in response to natural dust deposition in the ocean.

www.nice.org.uk/CG87 [accessed 16 July 2010]. 4. Wespes E, et al. EAU guidelines

on erectile dysfunction: an update. BYL719 molecular weight Eur Urol 2006; 49: 806–15. 5. Corona G, et al. Association of hypogonadism and type II diabetes in men attending an outpatient erectile dysfunction clinic. Int J Impot Res 2006; 18: 190–7. 6. Kalinchenko S, et al. Oral testosterone undecanoate reverses erectile dysfunction associated with diabetes mellitus in patients failing on sildenafil citrate alone. Aging Male 2003; 6: 94–9. 7. Traish AM, et al. Mechanisms of obesity and related pathologies: androgen deficiency and endothelial dysfunction may be link between obesity and erectile dysfunction. FEBS J 2009; 276: 5755–67. 8. Ding EL, et al. Sex differences of endogenous sex hormones and risk of type 2 diabetes. JAMA 2006; 295: 1288–99. 9. Kapoor D, et al. Clinical and biochemical assessment of hypogonadism in men with type 2 diabetes: Correlations with bioavailable testosterone and visceral adiposity. Diabetes Care 2007; 30: 911–17. 10. Jones TH. Hypogonadism in men with type 2 diabetes.

Pract Diabetes Int 2007; 24: 269–77. 11. Wang C, et al. Investigation, treatment, and monitoring of late-onset hypogonadism in males: ISA, ISSAM, EAU, EAA, and ASA recommendations. J Androl 2009; 30(1): 1–9. 12. Wu FC, et al., the European Male Aging Study Group. Hypothalamic-pituitary-testicular axis disruptions in older men are differentially linked either to age and modifiable risk factors: the European Male Aging Study. J Clin Endocrinol Metab 2008; 93: 2737–45. 13. Jones TH, Saad F. The effects of testosterone on risk factors Venetoclax for, and the mediators of, the atherosclerotic

process. Atherosclerosis 2009; 207: 318–27. 14. Jones TH. Testosterone deficiency: a risk factor for cardiovascular disease? Trends Endocrinol Metab 2010; 21(8): 496–503. 15. Malkin CJ, et al. Low serum testosterone and increased mortality in men with coronary heart disease. Heart 2010; 96: 1821–5. 16. Muralheedharan V, et al. Low testosterone level is associated with significant increase in all cause and cardiovascular mortality in men with type 2 diabetes. The 92nd Annual meeting of the Endocrine Society, San Diego, USA abstract book, 2010; OR17-6. 17. Keating NL, et al. Diabetes and cardiovascular disease during androgen deprivation therapy for prostate cancer. J Clin Oncol 2006; 24: 4448–56. 18. Greenstein A, et al. Does sildenafil combined with testosterone gel improve erectile dysfunction in hypogonadal men in whom testosterone supplement therapy alone failed? J Urol 2005; 173: 530–2. 19. Jones TH, et al. Testosterone improves glycaemic control, insulin resistance, body fat and sexual function in men with metabolic syndrome and/or type 2 diabetes: A multi-centre European clinical trial: the TIMES2 study. Endocrine Abstracts 2010; 21: OC1.6. 20. Kapoor D, et al.

Our results showed the potential of wheat bran to produce laccase by white-rot fungi, because high laccase activities were obtained. However, the use of different strains

for the laccase production clearly modifies the culture conditions, especially for the close-to-the-substrate hyphae. In this region, the hyphae density and the number of layers clearly depend on the strain. On the one hand, P. ostreatus www.selleckchem.com/products/chir-99021-ct99021-hcl.html presented the highest hypha density and the largest number of layers in the interface structure, showing a tendency for a hair-like growing morphology. This morphology could be linked to the low oxygen diffusion presented in the inner layers. On the other hand, C. unicolor presented the lowest hypha density and the smallest number of layers in the interface structure; thus, this fungus was expected to have

the best oxygen diffusion into its inner layers. However, there is no clear relationship between fungal morphology, hypha density and laccase production. Pleurotus ostreatus produced nearly 50% more laccase per gram of total dry matter than the one produced by C. unicolor. Trametes versicolor, which presented medium hypha density, produced the highest activity of laccase per gram of total dry matter, but T. pubescens, with similar growth morphology likely related to their common genus (Trametes), produced the lowest quantity of laccase per gram of total dry matter among all the strains studied. This indicates that, although all strains presented differences in the hypha size, clump size, morphology and the number of layers in the interface structure, see more the laccase production in terms of activity per gram of total dry matter cannot be related to these characteristics of the culture, but directly to the strain. This is in agreement with the review by Grimm et al. (2005), which stated that no simple relationship was found between morphology and productivity in filamentous fungi. This research was financed by the Spanish Ministry of Education

and Science (Project CTQ2007-66541). We thank Isotretinoin Dr A. Hatakka from the Department of Food and Environmental Sciences at the University of Helsinki (Helsinki, Finland) for the Trametes versicolor K120a2 (FBCC564), Cerrena unicolor T71 (FBCC744) and Pleurotus ostreatus DSM 11191 (FBCC375) fungal strains and Erika Winquist from the Aalto University School of Science and Technology (Aalto, Finland) for her help and support reading the manuscript. “
“Here, we describe mutants of Mycoplasma pulmonis that were obtained using a minitransposon, Tn4001TF1, which actively transposes but is then unable to undergo subsequent excision events. Using Tn4001TF1, we disrupted 39 genes previously thought to be essential for growth. Thus, the number of genes required for growth has been overestimated. This study also revealed evidence of gene duplications in M.

PYY3-36-HSA is a large molecule that does not penetrate the blood–brain barrier, and thus provides a useful tool to discriminate between the central (brain) and peripheral

actions of this peptide. PYY3-36-HSA induced significant reductions in food and body weight gain up to 24 h after administration. The anorectic effect of PYY3-36-HSA was delayed for 2 h in rats in which both AP and SFO were ablated, while lesion of either of these circumventricular organs in isolation did not influence the feeding responses to PYY3-36-HSA. The PYY3-36-HSA-induced anorectic effect was also reduced during the 3- to 6-h period following subdiaphragmatic vagotomy. Lesions of AP, SFO and AP/SFO as well as subdiaphragmatic vagotomy blunted PYY3-36-HSA-induced expression of c-fos Selleck GSK-3 inhibitor mRNA in specific brain structures including the bed nucleus of stria terminalis, central amygdala, lateral–external parabrachial nucleus and medial nucleus of the solitary tract. In addition, subdiaphragmatic vagotomy inhibited the neuronal activation induced by PYY3-36-HSA in AP and SFO. These findings suggest that the anorectic effect and brain neuronal activation induced by PYY3-36-HSA are dependent on integrity of AP, SFO and subdiaphragmatic vagus nerve. “
“Striatal medium-sized ALK cancer spiny neurons (MSSNs) receive glutamatergic inputs modulated presynaptically and postsynaptically by

dopamine. Mice expressing the gene for enhanced green fluorescent protein next as a reporter gene to identify MSSNs containing D1 or D2 receptor subtypes were used to examine dopamine modulation of spontaneous excitatory postsynaptic currents (sEPSCs) in slices and postsynaptic N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) currents in acutely isolated cells. The results demonstrated dopamine

receptor-specific modulation of sEPSCs. Dopamine and D1 agonists increased sEPSC frequency in D1 receptor-expressing MSSNs (D1 cells), whereas dopamine and D2 agonists decreased sEPSC frequency in D2 receptor-expressing MSSNs (D2 cells). These effects were fully (D1 cells) or partially (D2 cells) mediated through retrograde signaling via endocannabinoids. A cannabinoid 1 receptor (CB1R) agonist and a blocker of anandamide transporter prevented the D1 receptor-mediated increase in sEPSC frequency in D1 cells, whereas a CB1R antagonist partially blocked the decrease in sEPSC frequency in D2 cells. At the postsynaptic level, low concentrations of a D1 receptor agonist consistently increased NMDA and AMPA currents in acutely isolated D1 cells, whereas a D2 receptor agonist decreased these currents in acutely isolated D2 cells. These results show that both glutamate release and postsynaptic excitatory currents are regulated in opposite directions by activation of D1 or D2 receptors. The direction of this regulation is also specific to D1 and D2 cells.

, 1992; Mayer et al., 1993; Igietseme et al., 1998). Removal of the substrate l-arginine (which would be degraded to Screening Library ic50 agmatine and pumped back into the cytosol in counter exchange for arginine by AaxC) could therefore promote Chlamydia survival and/or fitness, particularly in strains that are known to infect and replicate within these specialized host cells, such as C. pneumoniae and C. psittaci (Wyrick & Brownridge, 1978; Redecke

et al., 1998). The timing of cleavage, and presumably corresponding activity, of AaxB in these strains may correlate with optimal iNOS activation in infected macrophages and ultimately allow Chlamydia to avoid the detrimental consequences of NO production prior to bacterial exit from the host cell. Alternatively, the presence of processed AaxB in EBs may indicate that EBs are ‘preloaded’ with functional AaxB that is used to protect against NO production during the immediate early stage of infection. This study was supported by grants DAPT supplier AI44033 from the National Institute of Allergy and Infectious

Diseases (Maurelli), 1F32AI078655-01 from the National Institute of Allergy and Infectious Diseases (Fisher), and the USUHS Graduate Education Office (Bliven). The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the Department of Defense or the Uniformed Services University. K.A.B. and D.J.F. contributed equally to this work. “
“Expression of adhesin to collagen of Enterococcus faecalis (ace), a known virulence factor, is increased by environmental signals such as the presence of serum, Cyclic nucleotide phosphodiesterase high temperature, and bile salts. Currently, the enterococcal regulator of survival (Ers) of E. faecalis strain JH2-2 is the only reported repressor of ace. Here, we show that for strain OG1RF, Ers is not involved in the regulation of ace. Our data showed similar levels of ace expression by OG1RF and its Δers derivative in the presence of bile salts, serum, and high temperature. Using ace promoter-lacZ fusions and site-directed mutagenesis,

we confirmed these results and further showed that, while the previously designated Ers box is important for increased expression from the ace promoter of OG1RF, the region responsible for the increase is bigger than the Ers box. In summary, these results indicate that, in strain OG1RF, Ers is not a repressor of ace expression. Although JH2-2 and OG1RF differ by six nucleotides in the region upstream of ace as well as in production of Fsr and gelatinase, the reason(s) for the difference in ace expression between JH2-2 and OG1RF and for increased ace expression in bile, serum and at 46 °C remain(s) to be determined. “
“Mycobacterium smegmatis contains three chaperonin (cpn60) genes homologous to the Escherichia coli groEL gene. One of these (cpn60.

, 1992; Mayer et al., 1993; Igietseme et al., 1998). Removal of the substrate l-arginine (which would be degraded to Selleck MK 1775 agmatine and pumped back into the cytosol in counter exchange for arginine by AaxC) could therefore promote Chlamydia survival and/or fitness, particularly in strains that are known to infect and replicate within these specialized host cells, such as C. pneumoniae and C. psittaci (Wyrick & Brownridge, 1978; Redecke

et al., 1998). The timing of cleavage, and presumably corresponding activity, of AaxB in these strains may correlate with optimal iNOS activation in infected macrophages and ultimately allow Chlamydia to avoid the detrimental consequences of NO production prior to bacterial exit from the host cell. Alternatively, the presence of processed AaxB in EBs may indicate that EBs are ‘preloaded’ with functional AaxB that is used to protect against NO production during the immediate early stage of infection. This study was supported by grants Buparlisib cell line AI44033 from the National Institute of Allergy and Infectious

Diseases (Maurelli), 1F32AI078655-01 from the National Institute of Allergy and Infectious Diseases (Fisher), and the USUHS Graduate Education Office (Bliven). The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the Department of Defense or the Uniformed Services University. K.A.B. and D.J.F. contributed equally to this work. “
“Expression of adhesin to collagen of Enterococcus faecalis (ace), a known virulence factor, is increased by environmental signals such as the presence of serum, crotamiton high temperature, and bile salts. Currently, the enterococcal regulator of survival (Ers) of E. faecalis strain JH2-2 is the only reported repressor of ace. Here, we show that for strain OG1RF, Ers is not involved in the regulation of ace. Our data showed similar levels of ace expression by OG1RF and its Δers derivative in the presence of bile salts, serum, and high temperature. Using ace promoter-lacZ fusions and site-directed mutagenesis,

we confirmed these results and further showed that, while the previously designated Ers box is important for increased expression from the ace promoter of OG1RF, the region responsible for the increase is bigger than the Ers box. In summary, these results indicate that, in strain OG1RF, Ers is not a repressor of ace expression. Although JH2-2 and OG1RF differ by six nucleotides in the region upstream of ace as well as in production of Fsr and gelatinase, the reason(s) for the difference in ace expression between JH2-2 and OG1RF and for increased ace expression in bile, serum and at 46 °C remain(s) to be determined. “
“Mycobacterium smegmatis contains three chaperonin (cpn60) genes homologous to the Escherichia coli groEL gene. One of these (cpn60.

The relative amount of these localizations varied between experiments. As a control of free

cytoplasmic GFP, a culture that has been previously reported to produce GFP in all cells of N. punctiforme was used (Fig. 3b) (Huang et al., 2010). This GFP control culture produced homogeneously distributed GFP in the cytoplasm of the heterocysts. In the cytoplasm of vegetative cells, the fluorescence was clearly obstructed by the presence of thylakoid membranes. The presence of thylakoids is seen in the red autofluorescence (magenta) stemming from the thylakoid-attached phycobilisome/photosystem II complexes (Cardona et al., 2009) and in the limited overlap between autofluorescence and GFP fluorescence (Fig. 3b). The full-length HupS–GFP protein required strong denaturing Selleckchem Etoposide conditions (2% SDS) for efficient

extraction (Fig. 1b), whereas most of the HupS–GFP degradation products could be extracted without detergent (Fig. S1). To examine the potential formation of a complete uptake hydrogenase by HupS–GFP and HupL, efforts were made to prepare native NVP-LDE225 solubility dmso extracts of these proteins from N2-fixing cultures of SHG. However, none of these attempts were successful. To examine the solubility of HupS–GFP, anti-GFP Western blots were performed with proteins extracted using buffers containing no detergents, mild nonionic detergents, or strongly denaturing additives. The results show that HupS–GFP could only be efficiently extracted using the strongly denaturing additives (Fig. 1b and Fig. S1). To identify any cell structure differences caused by potential HupS–GFP protein inclusions, isolated heterocysts

from N2-fixing Resminostat cultures of SHG and WT were compared using TEM. The resulting images did not reveal any structural differences between SHG and WT heterocysts (Fig. S2). This study shows that the small subunit of the uptake hydrogenase, HupS, in N. punctiforme is solely localized to the heterocysts. The localization of the uptake hydrogenase in N. punctiforme has been under debate for a long time since previous immunolocalization studies have identified the large subunit, HupL, in both vegetative cells and heterocysts (Lindblad & Sellstedt, 1990; Tamagnini et al., 2002, 2007; Seabra et al., 2009). Interestingly, these studies used several different HupL antibodies and the results are not fully correlating. Both Seabra et al. (2009) and Lindblad & Sellstedt (1990) show vegetative cell localization of HupL, but the subcellular localization to the cytoplasmic membranes between vegetative cells clearly seen in Lindblad & Sellstedt (1990) is missing in Seabra et al. (2009), which argued for a subcellular localization of an inactive form to the vegetative cell thylakoid membranes as well as to what is described as the vesicular region of the heterocysts (Seabra et al., 2009).

Half a decade has

gone by since the publication of the genome sequence of Xac (da Silva et al., 2002), and apparently, Selleckchem Trametinib its large-scale proteome analyses are confined to a few reports using techniques such as two-dimensional protein gels, yeast two-hybrid, and nuclear magnetic resonance scans for folded proteins (Mehta & Rosato, 2001, 2003; Galvao-Botton et al., 2003; Alegria et al., 2004, 2005; Khater et al., 2007). Moreover, the two last methods used heterologous expression of proteins in organisms different from the one under investigation. Most of the limitation to explore the biology of Xac lies in a complete lack of protein expression systems adapted to this bacterium. Here, we showed

the construction and test of new protein expression vectors dedicated to Xac, and the subsequent utilization of our system to characterize the hypothetical protein XAC3408. XAC3408 is 30% identical (at the amino acid level) to the B. subtilis cell division protein ZapA, and our subcellular localization experiments using GFP-XAC3408 support the hypothesis that XAC3408 is the Xac orthologue of ZapABsu. RG7204 cost ZapA-like proteins are conserved among bacteria, in which they function by promoting the FtsZ bundling and stabilization of FtsZ polymers (Gueiros-Filho & Losick, 2002; Low et al., 2004; Scheffers, 2008). ZapAXac exhibited a localization pattern similar to that observed for ZapABsu (Gueiros-Filho & Losick, 2002), and the availability of Xac mutants labeled at the septum

provides a new perspective for antimicrobial drug development trials with Xac aimed to disrupt cell division. The vectors described here are Ixazomib cell line integrative and allow the ectopic expression of proteins from the amy locus of Xac. Such a strategy has been used extensively in the Gram-positive rod B. subtilis (Lewis & Marston, 1999; Gueiros-Filho & Losick, 2002), and it is believed to avoid disturbances to genes/chromosomal regions that might produce undesirable effects in cell growth and altered phenotypes. Besides, integration into amy has the advantage of allowing the characterization of essential genes, which may not accommodate changes in their coding sequences. Finally, the disruption of amy produces a bacterial phenotype easily detectable on a plate and allows the distinction of insertions that had occurred in amy from those in the gene being under investigation. In the present work, we showed that the α-amylase gene was not essential for Xac to grow on a plate, neither was it found to play any key role during infection, an outcome somewhat expected since it has been demonstrated that in bacteria α-amylases may be essential for growth on starch, but dispensable for growth on other carbon sources (Worthington et al., 2003).

Putative transconjugants were confirmed by BOX-PCR typing. Profiles were generated by PCR amplification in 25 μL reaction mixtures containing 3.75 mm MgCl2, 0.2 mm dNTPs, 1× Stoffel buffer, 0.2 μm of primer BOX-AIR (5′-CTACGGCAAGGCGACGCTGACG-3′; Versalovic et al., 1991), 2.5 U Stoffel Taq polymerase (Applied Biosystems) and 1 μL of cell suspension prepared in 100 μL of distilled GSK126 supplier water (~ 1.0 McFarland turbidity standard). Amplification was carried out as follows: initial denaturation for

7 min at 94 °C, then 35 cycles of denaturation at 94 °C for 7 min, followed by annealing at 53 °C for 1 min and extension at 65 °C for 8 min, and a final extension at 65 °C for 16 min. Generated profiles were separated in 1.5% agarose gels in 0.5× TBE buffer (50 mm Tris, 50 mm boric acid, 0.5 mm EDTA), at 50 V for 95 min, and stained with ethidium bromide. Plasmid DNA from donors and transconjugants was purified using Qiagen Plasmid Mini-kit (Qiagen GmbH, Germany). Diversity of plasmids was evaluated by plasmid restriction analysis using 5 U of PstI (CTGCAG) and 5 U of Bst1770I (GTATAC), according to the manufacturer’s instructions (Fermentas, Lithuania). Restriction patterns were visualized in 0.8% agarose gels. Electrophoresis was run at 40 V for 3 h in 0.5× TBE buffer and stained using ethidium bromide. Restriction

this website patterns were compared using GelCompar II software (Applied Maths, SintMartens-Latem, Belgium). Detection Rucaparib purchase of IncP-1, IncQ, IncN and IncW replicons and integrase genes was performed as previously described (Götz et al., 1996; Moura et al., 2010). Briefly, gels were transferred onto nylon membranes (Hybond-N, Amersham,

Germany) and hybridized in middle stringency conditions with PCR-derived specific digoxigenin-labelled probes for intI1, intI2, IncP-1 (trfA), IncQ (oriV), IncN (rep) and IncW (oriV) (Moura et al., 2010). Detection of IncA/C, IncB/O, IncF (FIA, FIB, FIC, FIIA, FrepB subgroups), IncHI1, IncHI2, IncI1-Iγ, IncK, IncL/M, IncU, IncT and IncY replicons was performed by PCR, using primers and conditions previously described (Carattoli et al., 2005). Results were confirmed by sequencing, except for IncFrep replicons, which were confirmed by Southern hybridization with digoxigenin-labelled probes generated by PCR from positive controls (Carattoli et al., 2005). The aim of this study was to evaluate the occurrence, diversity and conjugative potential of plasmids in integron-carrying bacteria from wastewater environments. The presence of plasmid DNA was confirmed in 77% (51 out of 66) of the strains. In the remaining 15 strains (~ 23%), no plasmids were detected by the plasmid extraction method used. Thus, most of the strains analysed harboured at least one plasmid, these strains being retrieved from all stages of the treatment process, including from final effluents (Table 1). Nevertheless, the presence of additional plasmids cannot be excluded.

Putative transconjugants were confirmed by BOX-PCR typing. Profiles were generated by PCR amplification in 25 μL reaction mixtures containing 3.75 mm MgCl2, 0.2 mm dNTPs, 1× Stoffel buffer, 0.2 μm of primer BOX-AIR (5′-CTACGGCAAGGCGACGCTGACG-3′; Versalovic et al., 1991), 2.5 U Stoffel Taq polymerase (Applied Biosystems) and 1 μL of cell suspension prepared in 100 μL of distilled Dasatinib manufacturer water (~ 1.0 McFarland turbidity standard). Amplification was carried out as follows: initial denaturation for

7 min at 94 °C, then 35 cycles of denaturation at 94 °C for 7 min, followed by annealing at 53 °C for 1 min and extension at 65 °C for 8 min, and a final extension at 65 °C for 16 min. Generated profiles were separated in 1.5% agarose gels in 0.5× TBE buffer (50 mm Tris, 50 mm boric acid, 0.5 mm EDTA), at 50 V for 95 min, and stained with ethidium bromide. Plasmid DNA from donors and transconjugants was purified using Qiagen Plasmid Mini-kit (Qiagen GmbH, Germany). Diversity of plasmids was evaluated by plasmid restriction analysis using 5 U of PstI (CTGCAG) and 5 U of Bst1770I (GTATAC), according to the manufacturer’s instructions (Fermentas, Lithuania). Restriction patterns were visualized in 0.8% agarose gels. Electrophoresis was run at 40 V for 3 h in 0.5× TBE buffer and stained using ethidium bromide. Restriction

PI3K assay patterns were compared using GelCompar II software (Applied Maths, SintMartens-Latem, Belgium). Detection 3-oxoacyl-(acyl-carrier-protein) reductase of IncP-1, IncQ, IncN and IncW replicons and integrase genes was performed as previously described (Götz et al., 1996; Moura et al., 2010). Briefly, gels were transferred onto nylon membranes (Hybond-N, Amersham,

Germany) and hybridized in middle stringency conditions with PCR-derived specific digoxigenin-labelled probes for intI1, intI2, IncP-1 (trfA), IncQ (oriV), IncN (rep) and IncW (oriV) (Moura et al., 2010). Detection of IncA/C, IncB/O, IncF (FIA, FIB, FIC, FIIA, FrepB subgroups), IncHI1, IncHI2, IncI1-Iγ, IncK, IncL/M, IncU, IncT and IncY replicons was performed by PCR, using primers and conditions previously described (Carattoli et al., 2005). Results were confirmed by sequencing, except for IncFrep replicons, which were confirmed by Southern hybridization with digoxigenin-labelled probes generated by PCR from positive controls (Carattoli et al., 2005). The aim of this study was to evaluate the occurrence, diversity and conjugative potential of plasmids in integron-carrying bacteria from wastewater environments. The presence of plasmid DNA was confirmed in 77% (51 out of 66) of the strains. In the remaining 15 strains (~ 23%), no plasmids were detected by the plasmid extraction method used. Thus, most of the strains analysed harboured at least one plasmid, these strains being retrieved from all stages of the treatment process, including from final effluents (Table 1). Nevertheless, the presence of additional plasmids cannot be excluded.