coli [49]. For complementation of a Salmonella fliJ mutant (strain MKM40, kind gift from the late Prof. R. M. Macnab), the HP0256 gene was amplified with primer pairs HP0256-QF/HP0256-QR (Table 4). The amplicons were digested with NcoI and BamHI, and ligated to similarly restricted pQE-60. Salmonella was transformed by electroporation using a standard protocol [50]. Electrocompetent Salmonella fliJ mutant cells were then transformed and

transformants were Fedratinib cell line selected on kanamycin (50 μg/ml). For complementation of the HP0256 mutant, a full length copy of the gene was introduced into the HP0203-HP0204 chromosomal intergenic region of a P79 HP0256-KO mutant according to the method described by Langford et al. using the pIR203K04 plasmid [51]. As expression of HP0256 is controlled by a promoter further upstream in a 5-gene operon, the gene was first amplified using the primers HP0256-F2 and HP0256-R and fused to the flaA promoter amplified using the primers FLA-F2 and FLA-R2, by overlap extension PCR. This composite fragment

flaA promoter-HP0256 was then cloned into pIR203K04 as a Cla1/BamH1 fragment. Transmission electron microscopy Cell samples were subjected to negative staining. Whole cells of H. pylori were grown on a plate containing brain heart infusion (BHI) supplemented with 10% foetal calf serum, for 24 h in a micro-aerobic atmosphere. Next, cells were harvested and carefully resuspended in 2% ammonium molybdate (Sigma) with 70 μg/ml this website bacitracin

(Sigma), as a wetting agent. 5 μl cell preparation was applied to a copper grid overlaid with a carbon-coated Formvar film. The excess sample was carefully removed and the copper grid was dried. The copper grids were observed in a JEOL JEM-1200EX transmission electron microscope at an accelerating voltage of 80 kV. Plate motility assay H. pylori strains and mutants were grown for 2 days on CBA plates and then stab inoculated on Brucella soft agar plates containing 0.3% (w/v) agar and 5% (v/v) heat-inactivated foetal bovine serum (Sigma). Motility plates were incubated at 37°C in an atmosphere containing 5% CO2 and periodically observed for halo formation. Protein electrophoresis and blotting A standard protocol was used to perform sodium dodecyl sulfate-polyacrylamide MAPK inhibitor gel electrophoresis [52] and immunoblotting. Proteins from 12.5% acrylamide gels were transferred onto nitrocellulose membrane by electroblotting [53]. Polyclonal antibody directed against H. pylori flagellin and hook protein was used as primary antibody [33]. Anti-rabbit antibody conjugated to horseradish-peroxidase (Sigma) was used as secondary antibody. Hydrogen Barasertib in vivo peroxide and 4-chloro-1-naphtol (Sigma) were employed for colour development. Microarray analysis To compare the transcriptional profiles of the wild-type and HP0256 mutant strains, a H.

Surgery 2010, 147:246–54.PubMedCrossRef 16. Seng KY, Teo WL, Fun CY, Law YL, Lim CL: Interrelations

between plasma caffeine concentrations and neurobehavioural effects in healthy volunteers: model analysis using NONMEM. Biopharm Drug Dispos 2010, 31:316–30.PubMed 17. Harris RC, Nevill M, Harris DB, Fallowfield JL, Wise JA: Absorption of creatine supplied as a drink, in meat or in solid form. J Sports Science 2001, 20:147–151.CrossRef 18. Persky A, Brazeau G: Clinical Pharmacology of the Dietary Supplement Creatine Monohydrate. Pharmacol Rev 2001, 53:161–176.PubMed 19. Hammett ST, Wall MB, Edwards TC, Smith AT: Dietary supplementation of creatine monohydrate reduces the human fMRI BOLD signal. Neurosci Lett 2010, 479:201–5.PubMedCrossRef 20. Selleckchem AZD4547 Blumert PA, Crum AJ, Ernsting M, Volek JS, Hollander DB, Haff EE, Haff GG: The acute effects of twenty-four hours 4SC-202 purchase of sleep loss on the performance of national-caliber male collegiate weightlifters. J Strength Cond Res 2007, 21:1146–54.PubMed 21. Edwards BJ, Waterhouse J: Effects of one night of partial sleep deprivation upon diurnal rhythms of accuracy and consistency in throwing darts. Chronobiol Int 2009, 26:756–68.PubMedCrossRef 22. Oliver SJ, Costa RJ, Laing SJ, Bilzon JL, Walsh NP: One night of sleep deprivation decreases treadmill

endurance performance. Chronobiol Int 2009, 26:756–68.CrossRef 23. Kronholm E, Sallinen M, Suutama T, Sulkava R, Era P, Partonen T: Self-reported sleep duration and cognitive functioning in the general population. J Sleep Res 2009, 18:436–46.PubMedCrossRef 24. Odle-Dusseau HN, Bradley Baf-A1 JL, Pilcher JJ: Subjective perceptions of the effects of sustained performance under sleep-deprivation conditions. Chronobiol Int 2010, 27:318–33.PubMedCrossRef 25. selleck chemicals Maridakis V, Herring MP, O’Connor PJ: Sensitivity to change in cognitive performance

and mood measures of energy and fatigue in response to differing doses of caffeine or breakfast. Int J Neurosci 2009, 119:975–94.PubMedCrossRef 26. Sergi S: An update on the mechanisms of the psychostimulant effects of caffeine. J Neurochem 2008, 105:1067–1079.CrossRef 27. Lara DR: Caffeine, mental health, and psychiatric disorders. J Alzheimers Dis 2010, 20:S239–48.PubMed 28. Rawson ES, Lieberman HR, Walsh TM, Zuber SM, Harhart JM, Matthews TC: Creatine supplementation does not improve cognitive function in young adults. Physiol Behav 2008, 95:130–4.PubMedCrossRef 29. Verbessem P, Lemiere J, Eijnde BO, Swinnen S, Vanhees L, Van Leemputte M, Hespel P, Dom R: Creatine supplementation in Huntington’s disease: a placebo-controlled pilot trial. Neurology 2003, 61:925–30.PubMed 30. Leproult R, Copinschi G, Buxton O, VanCauter E: Sleep loss results in an elevation of cortisol levels the next evening. Sleep 1997, 20:865–70.PubMed 31.

03 μg/ml), using b 0.5%, c 1% or d 2% suspensions of SRBC. The results are the average of P-gp inhibitor three independent experiments, each performed in triplicate ± the standard deviation. Asterisks indicate significant differences according to Student’s t test (*, P < 0.05; **, P < 0.01). Analysis of trapped chromosomal DNA fragments in Selleckchem Liproxstatin 1 strains showing penicillin G-inducible

hly expression The chromosomal fragments carrying penicillin G-inducible promoters were sequenced and compared with the L. monocytogenes EGD-e genome. In the case of seven strains, namely 15, 18, 37, 198, 199, 201 and 203 (Table 2), this analysis identified single genes as the source of the trapped chromosomal DNA fragments. In PF-573228 research buy the case of strain 195, the

trapped fragment was comprised of sequences originating from two genes, lmo2095 and lmo2096, both present in the opposite transcriptional orientation to the reporter gene. It was reasoned that the identified promoter might originate from a divergently transcribed gene positioned immediately upstream of the cloned fragment, but examination of the genome sequence showed that the two preceding genes, lmo2097 and lmo2098, are in the same orientation as lmo2095 and lmo2096. Thus, the identified promoter could not direct the expression of any of these genes and for this reason it

was excluded from further investigations. In the case of strain 41, the trapped chromosomal fragment contained the full sequence of genes lmo0943 (fri) and lmo0944 plus sequences upstream of these genes, as well as a fragment of the sequence preceding gene lmo0945, which is in the same Thiamet G transcriptional orientation. Thus, on the basis of simple sequence analysis it was not possible to identify which promoter was directing hly expression in this strain. In an attempt to clarify this situation, the possible cotranscription of fri, lmo0944 and lmo0945 was examined by RT-PCR. The three anticipated PCR products were amplified from cDNA generated by reverse transcription using primers specific for genes lmo0945 and lmo0944, which demonstrated that fri, lmo0944 and lmo0945 are cotranscribed in both non-stressed cells and in cells grown under penicillin G pressure (Figure 1). Consequently, each of these genes was analyzed further. Table 2 Description of L. .

2007; Le Quere et al. 2009; Manning et al. 2010), there is an urgent need to develop updated planning approaches to provide for biodiversity conservation in the face of altered climates. In this paper, we outline five major approaches for incorporating climate change into conservation plans to improve the chances that these plans and priorities will remain effective as climate

changes. The development of systematic conservation plans helps guide where we should work to efficiently achieve conservation objectives, which of these places are the highest priorities, and increasingly, how we should work in these Nutlin-3 datasheet places (Redford et al. 2003; Wilson et al. 2007). Although early efforts at such planning focused largely on conserving the species, communities, or ecosystems of a specific region, the science of conservation planning is now advancing to better incorporate ecological processes and more recently, ecosystem services (Egoh et al. 2007). Despite these advances, many of the species and ecosystems for which these conservation plans were developed are likely to be facing ever increasing stresses due to the direct and indirect

effects of climate change. The recent Intergovernmental Panel on Climate Change Fourth Assessment Report (IPCC 2007a) suggests that 10–40% of species will be at high risk of extinction as global mean temperature reaches 2–3°C above pre-industrial levels. Under projected future climate changes,

ecosystems will be affected by the Seliciclib resulting changes in sea-level rise, ocean acidification, changes in the pattern and intensity of precipitation, change in wind direction and speed, and reductions in snow/ice cover and permafrost. Clear evidence that climate change is already RG-7388 supplier acting as a stressor include coral reef bleaching, shifts in species ranges, and local extinctions, as well as more subtle changes in growing seasons, drought stress, migration patterns, primary production, and species interactions, just to Immune system name a few (Donner et al. 2005; Parmesan 2006; Foden et al. 2008; Sinervo et al. 2010; Breshears et al. 2009). Conservation planners, scientists, and practitioners are adapting approaches to address both altered ecological systems and human responses to climate-induced changes within these ecosystems (Marshall et al. 2010) to help ensure the continued relevance and effectiveness of conservation efforts. Climate change adaptation refers to the adjustment of natural or anthropogenic systems to a changing climate for the purpose of moderating impacts or capitalizing on novel opportunities (IPCC 2007b). We argue that integrating adaptation into systematic conservation planning is imperative for four reasons. First, systematic planning processes are frequently used to establish conservation priorities of government and non-governmental organizations alike, and adaptation has a central role to play in developing these priorities.

Rhizosphere is the most preferable ecological

niche for microbial dynamics. It is a general assumption that rhizospheric microorganisms are the primary consumers of plant root exudates [18]. Therefore, it is expected that rhizospheric community dynamics will be affected by changes in the physiological activities of the plant as regulated by the genetic modifications induced. Considering above facts, the objective of this study was to assess the community structure (density and diversity) of actinomycetes associated with the rhizospheric soils of Bt transgenic brinjal. In addition, soil chemical properties were also determined as variations therein, are considered as the early indicators of the impact of transgenic crop Androgen Receptor inhibitor on soil fertility [19]. Methods Experimental site and this website crop description Field trials were conducted in the agricultural farm of Indian Institute of Vegetable Research (I. I.V.R.), Varanasi, India (25° 08’ N latitude, 83° 03’ E longitude, 90 m from sea level, average temperature maximum 33°C and minimum 20°C). The site has been used for intensive vegetable production but not for any transgenic crop plantation prior to the present study (during 2010–2011). The soil (WHC 39.9%)

is pale brown silty loam (sand 30%, silt 70%, clay 2%), Inceptisol with pH 6.7, organic C (0.73%) and, total N (0.09%) [20]. Ten- days old seedlings of VRBT-8 Bt transgenic event are selected for the study (data not shown). Genetic transformation was brought up through Agrobacterium tumefaciens LBA4404- mediated gene transfer that harbours pBinAR binary vector for neomycin phosphotransferase (npt-II) gene with neopaline synthase (NOS) promoter and a Cry1Ac gene fused to a constitutive, widely used plant promoter (CAMV35S) and octopine synthase gene (OCS) [21]. Treatments selleck compound consisted of randomised blocks design Succinyl-CoA in six plots of brinjal (Solanum melongena L. var. Kashi Taru), each 12 m2 (3 for transgenic -VRBT-8 and its near-isogenic non-transgenic, respectively) grown in containment condition to conform to bio-safety regulations and simulated agricultural

conditions. Recommended cultivation practices were adopted in which soils prior to transplantation, were added with 25–30 tonnes/ ha farm yard manure (FYM) along with NPK (100–120 kg N, 75–85 kg P and 45–50 kg K) [22]. Irrigation was done at the interval of every 10–15 days to maintain optimum moisture conditions. Soil sampling and analyses Soil sampling (in triplicate for each sampling stage) was done at different crop growth stages (branching, flowering and maturation) including pre-vegetation and post-harvest stage during the consecutive years (2010 and 2011). Rhizospheric soil samples were collected from the branching, flowering and maturation stage of non-Bt and Bt brinjal crop by uprooting the plants.

Development of new nanofabrication methods is always a significant issue of concern. Recently, the friction-induced nanofabrication was proposed to produce

protrusive nanostructures on Si(100) surface by scanning a diamond tip on a target sample without any post-etching [7]. Besides silicon, this method can also enable the fabrication on electrical insulators, such as quartz and glass. As a straightforward and maskless method, the friction-induced nanofabrication points out a new route in fabricating nanostructures on demand. It is well known that monocrystalline silicon has three typical crystal planes, i.e., (100), (110), and (111). As a typically anisotropic material, monocrystalline silicon presents different elastic modulus on various crystal planes, namely 130 GPa on Si(100), 169 GPa on Si(110), and 188 GPa on Si(111), eFT508 datasheet respectively [8]. Experimental results showed that the cutting process GS-1101 mw and friction behavior of silicon were influenced by the crystal anisotropy [9, 10]. Based on pin-on-disk tests, the average friction coefficient measured on Si(100) wafer was about 80% higher than that on Si(110) and Si(111) wafers [10].

Moreover, because of the difference in the density of dangling bonds and PKC inhibitor structure of back bonds, the etching rate of Si(100) or Si(110) was two orders of magnitude faster than that of Si(111) in alkaline solution [11, 12]. These anisotropic properties of monocrystalline silicon may induce the different nanofabrication behavior on silicon surfaces with various crystal planes. Therefore, even though the friction-induced nanofabrication enables producing protrusive nanostructures on Si(100) surface, it remains unknown whether the same nanofabrication method can be realized on other silicon crystal planes. In the present study, the effect of crystal plane orientation on the friction-induced Sodium butyrate nanofabrication on monocrystalline silicon was investigated. To verify whether the friction-induced fabrication can be realized on various silicon crystal planes, scratch tests at a linearly increasing load were performed on Si(100), Si(110), and Si(111)

surfaces, respectively. The effect of crystal plane orientation on the formation of friction-induced hillocks was further detected by scanning three silicon crystal planes under a constant normal load. Finally, the formation mechanism of the hillock on various silicon crystal planes was discussed based on their mechanical performance and bond structure. Methods Materials Si(100), Si(110), and Si(111) wafers were purchased from MCL Electronic Materials Ltd., Luoyang, China. The surface root-mean-square roughness of the wafers was measured as less than 0.2 nm over a square of 2 × 2 μm2 by an atomic force microscope (AFM; SPI3800N, Seiko Instruments Inc., Tokyo, Japan). The mechanical properties of the wafers were detected by a triboindenter (TI750, Hysitron Inc.

However, the function of miR-203 in breast cancer remains unclear, especially in TNBC. In this paper, we showed that miR-203 was down-regulated in TNBC cell lines and that the ectopic over-expression of miR-203 blocked tumor cell proliferation and migration in vitro. Furthermore, BIRC5 and LASP1 were identified as two direct functional click here targets of miR-203 in TNBC cells. These data suggest that the reduced expression of miR-203 facilitates the development and metastasis of TNBC. Materials and methods Cell culture and treatment Human triple-negative breast cancer cell lines

(MDA-MB-468 and MDA-MB-231) and normal breast cell line MCF-10A, were purchased from the American Type Culture Collection. MDA-MB-468 and MDA-MB-231 cells were maintained in DMEM (Gibco) supplemented with

10% FBS and 100 U/ml penicillin and 100 μg/ml streptomycin. MCF-10A cells were maintained in DMEM/F-12 supplemented with 10% FBS, insulin (10 μg /ml), hydrocortisone (500 ng/ml) and EGF (20 ng/ml). The cells were collected using 0.05% trypsin EDTA following the specified incubation period. Precursor miRNA/siRNA/plasmid transfection Cells were seeded in 6-well plates PU-H71 mouse at a concentration of 1 × 105 and cultured in medium selleck inhibitor without antibiotics for approximately 24 h before transfection. Cells were transiently transfected with miR-203 precursor (Applied Biosystems) or negative control miRNA, BIRC5 siRNA (Sigma), LASP1 siRNA (Sigma) or control siRNA at a final concentration of 200nM. PcDNA-BIRC5 or pcDNA-LASP1 plasmid was also transfected into MDA-MB-231 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

Real-time PCR assay Total RNA was extracted from cultured cells using the TRIzol reagent (Invitrogen). cDNA was obtained by reverse transcription of total RNA using a TaqMan Reverse Transcription Kit (Applied Biosystems) and iScript cDNA Synthesis kit (BIO-RAD), respectively. The expression level of mature miR-203 was measured using a TaqMan miRNA assay (Applied Biosystems) according to the provided check protocol and using U6 small nuclear RNA as an internal control. Expression of BIRC5 and LASP1mRNA was detected using Power SYBR Green kit (Applied Biosystems). All experiments were performed in triplicate. Colony formation assay Cells were seeded into a 12-well cell culture plate and incubated for 2 weeks at 37 °C after treatment. Then, cells were washed twice with PBS, fixed with cold methanol, stained with 0.1% crystal violet, washed and air dried. Migration assay Cells were harvested and re-suspended in serum-free DMEM medium. For the migration assay, 5 × 104 cells were added into the upper chamber of the insert (BD Bioscience, 8 μm pore size). Cells were plated in medium without serum, and medium containing 10% fetal bovine serum in the lower chamber served as the chemoattractant. After 6 h of incubation, cells were fixed with 3.

When biofilms grown in iron supplemented media were treated with cobalt as well as phage in combination, a negligible amount of biofilm formation consisting of mostly of red and yellow regions was seen on day 3 [Figure 5(d)] as well as on day 7 [Figure 5(d´)] when compared

with 3rd and 7th day biofilms treated with cobalt as shown in [Figure 5(b) and Figure 5(b´)] respectively or phage alone [Figure 5(c) and Figure 5(c´)]. Figure 5 K. pneumoniae B5055 biofilm developed on coverslip (a/ a´) 3/ 7 day biofilm grown in 10  μM FeCl 3 supplemented media (b/ b´) 3/ 7 day biofilm grown in 10  μM FeCl 3  + 500  μM cobalt salt supplemented media PF-01367338 solubility dmso (c /c´) 3/ 7 day biofilm grown in 10  μM FeCl 3 supplemented media followed by treatment with phage KPO1K2 (d/ d´) 3/ 7 day biofilm grown in 10  μM FeCl 3   +  500  μM cobalt salt supplemented media followed by treatment with phage KPO1K2. Discussion Biofilms are recalcitrant to antibiotics as their higher concentrations are needed to eradicate bacterial cells in this mode of growth. Attempts have been made in the past to evolve alternate strategies to destroy biofilms. Since bacteria, both in planktonic and biofilm mode require iron for their growth [14] hence, iron chelating IWR-1 mw agents

have been reported to inhibit biofilm growth. Hancock et al. [15] have reported that since Zn (II) and Co (II) have a higher than iron affinity for the master controller protein of iron uptake i.e. ‘Fur’ thus they reduce biofilm formation by infectious Selleck Screening Library E. coli. In this study, a significant reduction (p < 0.05) was observed in the growth of younger biofilms (1–3 day old) when 500 μM CoSO4 and 10 μM FeCl3 supplemented media was used. This might be because of the elevated levels of metals which could Afatinib nmr interfere with normal iron regulation by shutdown of Fur-controlled iron uptake systems like enterobactin, ferric dicitrate, aerobactin as well as additional downstream effects on putative adhesion factors

involved in biofilm establishment thereby resulting in deleterious effect on biofilm formation [2, 22] as well as pathogenicity of the organism. No previous reports are available involving the use of phage and iron antagonizing molecules in combination on biofilm kinetics. Thus, we studied the efficacy of depolymerase producing phage (KPO1K2) in eradicating the biofilms of K. pneumoniae B5055 grown in minimal media supplemented with 500 μM CoSO4 and iron. A complete eradication of the younger biofilms (upto 2 day old) given combination treatment was observed. This was possibly due to the degradation of exopolysaccharide matrix encompassing the biofilm structure by the phage encoded depolymerase [7, 17] which facilitated the process of bacterial growth inhibition by phage as well as CoSO4.

PubMedCrossRef 35. Rigano LA, Siciliano F, Enrique R, Sendin L, Filippone P, Torres PS, Questa J, Dow JM, Castagnaro AP, Vojnov AA, et al.: Biofilm formation, epiphytic fitness, and canker development in Xanthomonas axonopodis pv. citri. Mol Plant Microbe Interact 2007,20(10):1222–1230.PubMedCrossRef 36. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ:

Basic local alignment search tool. J Mol Biol 1990,215(3):403–410.PubMed Authors’ contributions LAR designed the experiments, performed the experimental work and drafted the manuscript; MRM and APC contributed to coordinate the study and to draft the manuscript; AMDA isolated the DNA sample from Candidatus Liberibacter asiaticus used for specificity tests and critically revised the manuscript; AAV participated in the analysis and interpretation of the data and prepared the final version of the manuscript. All authors read and approved the final version of the manuscript.”
“Background Celiac disease (CD) selleck chemicals llc is an immune-mediated

enteropathy triggered by the ingestion of gluten-containing grains (including wheat, rye, and barley) in genetically susceptible individuals [1]. Its estimated prevalence in Western Countries is near 1% [2]. It is generally agreed that CD is a T-cell mediated disorder in which gliadin derived peptides activate lamina propria T lymphocytes which release proinflammatory cytokines [3]. To date, several peptides including alpha- and gamma-gliadins, have been reported to activate CD4+ lymphocytes via their interaction with HLA-DQ2 and -DQ8 heterodimer on antigen presenting cells (APC) [4]. Recently, scientific evidence showed microecological changes in the intestinal AZD1480 mouse tract of celiac infants, suggesting a potential role

of gut microbiota in CD. Alterations in the composition of faecal short-chain fatty acids in CD patients compared with those of healthy controls have been demonstrated [5]. Imbalance in the composition of duodenal microbiota or in faecal bacterial communities of children with CD has also been reported [6–9]. Rod-shaped bacteria have been observed in both gluten-free diet (GFD)-treated and untreated pediatric patients’mucosa, along with a distinctive lectin pattern [10]. The present study was carried out to oxyclozanide add further information on the characterization of intestinal microbiota of CD patients, a variable that may represent a new piece of the intriguing puzzle of CD illness. For this purpose we analyzed by TTGE the composition of duodenal mucosa-associated microbiota in the same cohort of GFD untreated and treated CD children and in controls. This prospective study was performed to compare the influence of the disease status on gut microbial composition and to study whether the microbial imbalance could be a peculiar characteristic of the disease. Results Agglomerative hierarchical classification (AHC) The TTGE profiles of PCR amplicons obtained with universal primers were firstly analyzed by XLStat Citarinostat nmr software.

This strain was used as receptor to select transconjugants carrying the Tn5mob-labeled pSym of

GR64 (CFN2001-1), the Tn5mob-labeled pSym of GR64 and Tn5-GDYN-labeled pRet42a of R. etli CFN42 (CFN2001-2), and both plasmids of GR64 (CFN2001-3). Other derivatives carried either pSfr64a::Tn5-GDYN or pSfr64b::Tn5mob in Agrobacterium strain GMI9023 genomic background. Plasmid profiles Plasmid profiles were visualized by the Eckhardt technique [38], as modified by Hynes and McGregor [39]. Filter blot hybridization and plasmid visualization For Southern-type hybridizations [40], Eckhardt type gels, or 1% agarose gels where restricted DNA was electrophoresed, were blotted onto nylon membranes, and hybridized under stringent conditions, as previously reported [41], by using Rapid-hyb buffer. Probes were linearized by digesting them with #XAV-939 mouse randurls[1|1|,|CHEM1|]# appropriate restriction enzymes and were labeled with [α32P]dCTP by using Repotrectinib chemical structure a Rediprime DNA labeling system. All restriction endonucleases, [α -32P]dCTP, hybridization buffer, and labeling systems were purchased from Amersham Pharmacia Biotech. Nodulation assays Overnight cultures were used to inoculate surface-sterilized Phaseolus vulgaris cv. Negro Jamapa seeds. Plants were grown in 250-ml Erlenmeyer flasks with Fahraeus agar medium [42], without added nitrogen, at 28°C. Nodulation was scored at day 15 after inoculation. Surface-sterilized nodules were crushed on PY plates,

and the plasmid pattern of single colonies was checked on Eckhardt type gels. Amplification and sequencing of recA, rpoB, and nifH gene fragments Partial nifH, recA and rpoB fragments were amplified with the primer pairs nifH40F/nifH817R, recA41F/recA640R and rpoB454F/rpoB 1364R as previously tuclazepam described [43, 44]. All amplifications were performed with Taq polymerase (USB-Amersham). Amplification products were purified

using Roche’s PCR product purification system. Both strands were commercially sequenced by Macrogen, Korea. Phylogenetic inference Reference nifH, recA, rpoB and repB sequences were retrieved via BLASTP searches from a locally maintained BLAST database containing all fully sequenced Rhizobiales genomes, and via remote BLASTP searches against NCBI’s non-redundant database. The query sequences for nifH, recA and rpoB used in the BLASTP searches were those obtained from the sequenced PCR amplicons from strain GR64, while that of repB was obtained from the sequence of pSfr64a. Nucleotide sequences were translated and aligned using muscle 3.7 [45]. The resulting protein multiple sequence alignments were used as masks to generate the underlying codon alignments using custom Perl scripts. Models of nucleotide substitution were selected by the Akaike information criterion (AIC), using MODELTEST3.7 [46]. Among-site rate variation was modelled by a gamma distribution, approximated with 4 rate categories, each category being represented by its mean.