Colonization occurs predominantly at the mucosal surfaces of the genital and respiratory tracts and is a prerequisite for infection (Hu et al., 1976; Cassell et al., 1985; Razin et al., 1998; Simmons & Dybvig, 2009). Mycoplasma pulmonis is the causative agent of murine respiratory mycoplasmosis (MRM), which is among the most serious of naturally acquired

diseases of rodent colonies. Exposing the upper respiratory tract of mice to M. pulmonis reveals a classic model of chronic mycoplasmal pneumonic disease, and numerous studies have utilized this model system to better elucidate the host–pathogen interactions in chronic respiratory disease caused by various species of Mycoplasma including the human pathogen M. pneumoniae (Cartner et al., 1998). The capability buy AZD5363 of M. pulmonis to attach to the pulmonary epithelium is one of the critical initial steps in the colonization of the host (Cassell et al., 1985). The size- and phase-variable

Vsa (variable surface antigen) lipoproteins influence virulence and the ability of the mycoplasma to adhere to inert surfaces and hemadsorb (Simmons & Dybvig, 2003; Simmons et al., 2007). In the mycoplasma strains used in this study, there are a suite of seven unique phase-variable Vsa isotypes; VsaA, C, E, F, G, H, and I. Isotype switching occurs when a silent vsa gene is combined with the vsa expression site by means of a site-specific DNA inversion (Shen et al., 2000). Size variation is a result of slipped-strand DNA mispairing during replication of the ABT-888 ic50 tandem Clomifene repeat regions of the vsa gene. Mycoplasmas producing the long form of the Vsa protein, containing about 40–60 tandem repeats, attach to glass and plastic surfaces poorly, while mycoplasmas producing a short Vsa with 0–5 repeats exhibit significantly

greater attachment (Simmons & Dybvig, 2003). It is thought that the innate immune response of the host exerts selection pressure for size variants. For example, exposure to complement can select for mycoplasmas producing a long Vsa protein (Simmons et al., 2004). Both long Vsa- and short Vsa-producing mycoplasmas are readily isolated from infected rats and mice (Gumulak-Smith et al., 2001; Denison et al., 2005). The possible role of the Vsa proteins in modulation of cytoadherence to epithelial cells has not previously been examined. Bacterial polysaccharides are often virulence factors that can contribute to immune modulation, immune evasion, biofilm formation, and cellular adherence (Comstock & Kasper, 2006). In Pseudomonas aeruginosa, polysaccharides have a positive role in both biofilm formation and cellular attachment (Byrd et al., 2009). Streptococcus pneumoniae modulates the adherence to the epithelia of the upper respiratory tract through regulation of the production of its capsular polysaccharide. Reduced production of capsular polysaccharide results in a transparent colony morphology and an enhanced ability to adhere to respiratory epithelium.

The cell suspension was sonicated (8 × 30 s, Sonicator Heat system with 50% duty cycle) on ice in the presence of protease inhibitor PMSF. The cell lysate was centrifuged at 14 000 g for 30 min at 4 °C to remove cell debris. The clear supernatant was loaded on a Q-Sepharose ion exchanger. The cleared supernatant was applied to a 2 cm × 10 cm column packed with an anion-exchanger, Q-Sepharose, previously equilibrated with buffer A. The flow through was collected and passed five to six times from the column. The protein fraction bound to the matrix (including

the target protein) was eluted with 100 mL of a linear 0–1 M NaCl gradient, prepared in the same buffer. The fractions were then run on a 10% sodium dodecyl sulphate (SDS) gel to determine which

fractions contain the full-length squalene synthase. The fractions Daporinad showing SSN activity were pooled and applied on a phenyl superose column for further purification. The protein sample from the preceding step was saturated with 25% ammonium sulphate [(NH4)2SO4] Midostaurin purchase in buffer B (20 mM Tris, 175 mM NaCl, 2 mM EDTA, 5 mM BME, 1% Tween 80) and was applied on pre-equilibrated phenyl superose [with 25% (NH4)2SO4-saturated buffer B]. The column was washed successively with buffer B containing 20%, 15%, 10% and 5% saturated (NH4)2SO4. Finally, the protein was eluted with buffer B. The fractions showing squalene second synthase activity were combined and used for further studies. Protein samples were separated by 10% SDS-PAGE and transferred to a nitrocellulose

membrane. SDS-polyacrylamide gels were stained with Coomassie brilliant blue R-250. Western blots were probed with hexahistidine antibody, following the manufacturer’s recommendation, and immunoreactive proteins were visualized using chemiluminescent substrate Lumigen. Squalene synthase activity of the recombinant protein was determined as reported by Sealey-Cardona et al., 2007. The catalytic activity of SSN was assayed by measuring the conversion of [3H] FPP to [3H] squalene. Final assay concentrations were 50 mM morpholinepropanesulfonic acid–NaOH buffer (pH 7.4), 20 mM MgCl2, 5 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, 1% Tween 80, 10 mM dithiothreitol, 0.025 mg mL1 of bovine serum albumin, 0.25 mM NADPH, 0.10 mg of recombinant protein mL−1 and different concentrations of FPP. The final volume of the reaction was 200 μL. After incubation at 37 °C for 5 min, 40 μL of 10 M NaOH was added, followed by 10 μL of a mixture (50 : 1) of 70% ethanol and squalene. The resulting mixtures were mixed vigorously by vortexing, and then 10-μL aliquots were applied to 2.5 × 10-cm channels of a silica gel thin-layer chromatogram, and the newly formed squalene was separated from unreacted substrates by chromatography in toluene–ethyl acetate (9 : 1). The region of each chromatogram from 2 cm below the squalene band (Rf=0.

, 2003; Dong et al., 2003; Warriner et al., 2003) may present increased nutrient exudation, and hence may function as triggers for mobilization of S. Weltevreden to these sites (Cooley et al., 2003; Dong et al., 2003; Jablasone et al., 2005). Alternatively, S. Weltevreden may associate with the root surface through adhesive properties

(Wachtel et al., 2002), specific selective advantages (Jacobsen, 1997) and well-developed colonization ability (Dong et al., 2003), which may represent a potential strategy for contamination of plants and further dissemination selleck screening library into edible parts along the root and shoot surfaces. As the number of replicate spinach plant roots infected with S. Weltevreden increased in Experiment A during the evaluation period and bacterial numbers in individual root systems tended to increase rather than decrease over time, it is likely that the bacteria observed were viable, rather than resting or dead. In Experiment B, where S. Weltevreden was added in saline solution directly to soil after plant emergence, the pathogen was detected in all replicates at all sampling occasions (Fig. 3). However, bacterial numbers present in the roots decreased significantly from day 0 to day 21 postinoculation, in contrast to the trend of increased cell numbers with time in Experiment

A. When roots started to develop in the pots in Experiment A, S. Weltevreden may have benefited from increased nutrient levels

available in roots accessible via lateral root junctions or breaks, leading to proliferation. The MG-132 more pronounced decline in bacterial numbers in roots in Experiment B, compared with Experiment A, was similar to the trends seen in soil between the two 4��8C experiments. This indicates that S. Weltevreden inoculated into soil after plant emergence faced more competition from the indigenous microbial communities for nutrients and colonization sites compared with S. Weltevreden applied to the soil before planting. Salmonella can contaminate crops in fields through leaves or other aerial surfaces (Doyle & Erickson, 2008). In the current study, no S. Weltevreden cells were recovered above the threshold level on leaves when added to the soil in a manure mixture (Experiment A). However, when bacteria were added in saline solution and added directly to soil 14 days after sowing and fertilization, cells were detected in all replicate pots on days 0 and 7 postinoculation (Experiment B). However, as S. Weltevreden was detected on leaves on the day of bacterial addition, this finding may have been an artifact resulting from negligent inoculation, i.e. unintentional application of bacteria to the leaves. Alternatively, S. Weltevreden may have potentially mobilized to spinach leaves through direct contact between leaves and soil/manure slurry as well as aerosol dispersion (Doyle & Erickson, 2008). Nevertheless, it is interesting to note that S.

pro-saccade trials. While Fig. 5C and E represents the increase in neck EMG above baseline, the absolute level of evoked neck EMG was also greater on anti-saccade vs. pro-saccade trials (data not shown, but note how the divergence in Fig. 5C for the last two stimulation intervals exceeds the divergence in baseline activity). This observation means that ICMS-SEF is not simply driving the muscles to the maximal level of recruitment. Further, note how these EMG increases are much smaller in magnitude than the visual response on neck muscles shown in Fig. 4C, which itself tends to be far less than the DAPT mw neck muscle recruitment that accompanies saccade generation,

even when head-restrained (Corneil et al., 2004, 2008; Chapman & Corneil, 2011). Finally, we analysed the neck EMG responses evoked by ICMS-SEF delivered in the post-cue interval. HDAC cancer These data are further segregated by saccade direction relative to the side of the stimulating electrode, as the evoked neck EMG interacts with the visual response on neck muscles for later stimulation times. Accordingly, we describe the effects of ICMS-SEF at each of the four post-cue intervals in sequence, in reference to the data shown in Fig. 6. Again, Fig. 6 shows data from the representative site (Fig. 6A), and across our sample (Fig. 6B–E). As mentioned above, the response evoked

by SEF stimulation at the earliest post-cue interval (i.e. 10 ms after cue presentation) precedes the visual response Staurosporine on neck muscles. Accordingly, the increase in EMG activity above baseline depended only on task (being greater on anti-saccades), but not on saccade direction (leftmost traces in Fig. 6A; leftmost series of datapoints, Fig. 6C). In contrast, the response evoked by SEF stimulation delivered slightly later (i.e. 43 ms after cue presentation) displayed a marked dependency with both task and saccade direction. At this interval, ICMS-SEF before ipsilaterally directed anti-saccades (dashed lines around empty traces in Fig. 6A; dashed line connecting circles in Fig. 6C) evoked the largest

response, followed by stimulation preceding contralaterally directed pro-saccades (solid traces in Fig. 6A; solid line connecting squares in Fig. 6C). Note that both such trials feature cue presentation on the side of the muscle (i.e. contralateral to the side of the stimulating electrode), and hence the evoked response is interacting with the ongoing visual response on neck muscles. Even here, it is clear that the stimulation-evoked effect is greater on anti- vs. pro-saccades, and the consistency of this effect is demonstrated by the shifts in the frequency histograms in Fig. 6E, which represent the difference in saccade direction for either pro- (upward histrograms) or anti-saccades (downward histograms).

The activity of the soluble protein kinases, 2′3′ cAMP phosphodiesterase (cyclic phosphodiesterase), total phosphodiesterases, AC and the phosphatases was measured in cells recovering from γ radiation effects (Fig. 3). The AC activity increased rapidly following γ irradiation and reached a maximum in 0.5 h PIR (Fig. 3a), during which the activity of phosphodiesterases and phosphatases was low. Whereas the AP did not change significantly during PIR, the acid phosphatase increased nearly 1.5-fold from 1 h PIR (5.146 μmol min−1 mg−1 protein) to 4 h PIR (8.243 μmol min−1 mg−1

protein) (Fig. 3b). The levels of cyclic phosphodiesterase decreased rapidly in 1 h PIR followed by an increase of nearly threefold in 4 h PIR (Fig. 3c). These Birinapant nmr results might support the argument that the net increase in the cAMP levels was due to differential regulation of AC and cyclic phosphodiesterase activities in response to DNA damage. Although, D. radiodurans R1 genome does not annotate the selleck screening library classical bacterial AC and 2′3′ cAMP phosphodiesterase, it encodes for protein with a phosphodiesterase-type functional domain with nearly 30% genome without annotated functions, leaving the strong possibility

that unknown proteins are responsible for these activities. The amino acid sequence of AC from Escherichia coli was subjected to multiple sequence alignment, which showed different levels of amino acid similarities with some of the deinococcal ORFs. Among them, DR_1433 showed close to 75% match with E. coli protein in psiblast analysis. The presence of AC and cyclic phosphodiesterase activities in cell-free extracts of this bacterium suggested the strong possibility of AC and cyclic phosphodiesterase activities containing uncharacterized proteins in bacterial genome and it will be interesting to investigate these activities separately. Aliquots of γ-irradiated cells were collected during PIR and nucleotide-binding proteins were purified by heparin-sepharose affinity chromatography. Fractions

were tested for nucleolytic activity on dsDNA substrate. Results showed the presence of nucleolytic activity in unirradiated and zero PIR-irradiated samples. This Ketotifen activity was completely absent in 1- and 2-h PIR samples (Fig. 4a) but reappeared in 3- and 4-h PIR samples. This indicated that the bacterium has an as yet unidentified mechanism to regulate the nuclease activity during different stages of PIR. It may be speculated that during early PIR, i.e. before 2 h PIR, the bacterium needs to protect its shattered genome and very low nuclease activity might be required for DSB end-joining, whereas at a later stage, i.e. after 2 h PIR, high recombinase functions are needed, which requires the high nuclease activity observed at 3 and 4 h PIR. Except for the unirradiated control, all the samples, including 1 and 2 h PIR, showed inhibition of nucleolytic function with 2 mM ATP (Fig.

These effects on conidiation have been found so far for all pex mutants in which PTS1 protein peroxisomal localization has been lost, but not in the pexG mutant

specifically lacking PTS2 protein import (Hynes et al., 2008). Similar to pexC∷bar, the growth of pexBΔ on the short-chain fatty acid (C4) butyrate and the long-chain fatty acid (C18) Buparlisib in vivo oleate as sole carbon sources was inhibited while growth on acetate was not affected and was only slightly affected on l-proline (Fig. 2b). The pexBΔ strain was crossed to a veA+ strain (MH11283: genotype yA2 pabaA1; veA+). Fifty percent of the progeny had phenotypes corresponding to pexBΔ consistent with a single gene mutation and with fertility in heterozygous crosses. The pexBΔ; veA+ strain shown in Fig. 2a was isolated from this cross. Selfed crosses of both pexBΔ and pexBΔ; veA+ strains were fertile, producing mature cleistothecia. However, as described previously for other pex mutants (Hynes et al., 2008), sexual development was slower than for the wild type and cleistothecia

were smaller (not shown). Strains containing the veA+ allele produce more cleistothecia than veA1 strains (Kim et al., 2002), and this was observed for the pexBΔ; veA+ strain (not shown). The production of mature cleistothecia by pexBΔ; veA+ is shown in Fig. 2c. Selfed crosses RXDX-106 of the pexBΔ strains produced viable progeny, as determined by single colony development from plated ascospores. The release of ascospores from squashed cleistothecia is shown Isotretinoin in Fig. 2d and also for the pexC∷bar strain. Overall, it can be concluded that the loss of PexB results in phenotypes identical to those seen in other pex mutants that cause loss of targeting of proteins to peroxisomes. Neither peroxisomes nor the RING-finger peroxin 2 play essential roles in meiosis in A. nidulans. However, because A. nidulans is homothallic, the generality

of this result for all Eurotiomycetes requires examining the effects of pex mutations on mating in heterothallic species. Previously, it has been suggested that the unusual Cys8 Zn2+-binding sequence in the RING-finger peroxins of filamentous ascomycetes might be involved in a unique meiotic function (Kiel & van der Klei, 2009). In addition, Pex2 and Pex12 have protein ubiquitin ligase activities (Platta et al., 2009) and protein ubiquitination independent of a peroxisomal role has been suggested as possibly being involved in meiosis (Peraza-Reyes et al., 2008). Clearly, neither of these possibilities are generally true for ascomycetes. To our knowledge, the roles of Pex2, Pex10 and Pex12 have not been investigated in Sordariomycetes other than P. anserina. It would be of interest to investigate this in N. crassa and M. grisea. However, differences within Sordariomycetes are already apparent. Loss of the importomer protein Pex14 results in male sterility in N. crassa, but not in P. anserina (Managadze et al., 2007; Peraza-Reyes et al., 2008).

The metabolite was identified as FA by comparing its retention time of HPLC analysis and charge to mass ratio of m/z = 332 of HPLC/MS analysis with the standard sample of FA (Fig. 3b). FA could not be degraded by strain T1 and accumulated gradually in the cultures Lumacaftor clinical trial as shown in Fig. 3a. We speculate that strain T1 degraded FE by the rapid cleavage of ester bonds to give FA. The most rapid degradation of FE and accumulation of FA were observed at 30 °C and pH 8.0. Over 95% and 73% of the FE was degraded within 24 h when the initial concentration of FE was 100 mg L−1 and 200 mg L−1, respectively (Fig. 4a). In addition, at

lower incubation levels (0.2–1%, about 105–106 cells mL−1), 88.38% and 92.72% of 100 mg L−1 PS-341 FE was still degraded (Fig. 4b). To our knowledge, strain T1 exhibits the fastest rate of degradation among the reported FE-degrading bacteria. P. fluorescens strains UA5-40, BD4-13, RA-2 and M-17 can degrade 82–96% of 3.256 mg L−1 FE after a 48 h incubation (Robert & Robert, 1998). Alcaligenes sp. H (Song et al., 2005a) and P. azotoformans

QDZ-1 (Nie et al., 2011) can degrade 45.8% and 90.8% of 100 mg L−1 FE within 5 days, respectively. Strain T1 could also degrade other AOPP herbicides. The degradation rates for haloxyfop-R-methyl, quizalofop-p-ethyl, cyhalofop-butyl and clodinafop-propargyl were 93.2%, 90.1%, 96.8% and 97.9%, respectively, which were superior to the reported strain QZD-1. When 50 μL of CFE was added to 4 mL of reaction buffer containing 25 mg L−1 FE, 90% of the substrate was degraded. Under the same conditions, no substrate was degraded when boiled CFE were added

to the assay mixture. This finding indicates that FE was degraded by soluble enzymes present in the CFE of strain T1. Zymogram analysis of the esterases is shown in Fig. 5a, lane 1, 3. Three purple bands were visualised on the gel after it was stained with α-napthyl acetate and fast blue B. This result shows Rhodococcus sp. T1 had three esterases and esterase band 3 was identified as FE hydrolase for it could form transparent halo on MSM plate containing 200 mg L−1 FE. Many other hydrolases which convert pesticides have been previously described, such as methyl parathion hydrolases (Cui et al., 2001; Fu et al., 2004), Terminal deoxynucleotidyl transferase carbofuran hydrolases (Xu et al., 2006a) and pyrethroid hydrolases (Liang et al., 2005; Guo et al., 2009). For cloning of FE hydrolase gene, a genomic library of strain T1 was constructed as described previously and two positive clones that produced transparent halos were selected on the LB agar plates containing 100 mg L−1 ampicillin and 200 mg L−1 FE. The recombinant plasmids harboured by them carried 4.2 and 4.0 kb inserts, respectively, and sequencing reports indicated that the 4.0 kb fragment was included in the 4.2 kb fragment (data not shown). Nucleotide sequence analysis revealed that 4.2 kb fragment consisted of five ORFs.

All were obtained from the same reputable supplier at different dates, and paired with primer R907 (Teske et al., 1996). Soil community PCR was performed with

a 25-μL reaction mixture containing Buparlisib 1.25 U GoTaq polymerase (Promega), 1 × PCR buffer, 1.5 mM MgCl2, 0.4 mg mL−1 bovine serum albumin, 4 μM each primer, 200 μM dNTPs, and 10 ng of template DNA. Pure-culture PCR was performed using a 30-μL reaction mix using the above concentrations of reagents. Thermocycling conditions were as follows: initial denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 45 s, and 72 °C for 1 min, and a final elongation at 72 °C for 7 min. After PCR, 1 μL of PCR product was resolved in a 1.5% agarose gel to confirm product size and the negative control. DNA concentrations were determined throughout by fluorometry using the HS dsDNA kit and Qubit Fluorometer (Invitrogen). A total of 300 ng of PCR product was loaded into each lane for soil community DGGE, while 50 ng of DNA was loaded for pure-culture DGGE. A denaturing gradient of 35–65% denaturants [100% denaturants is a mixture of 7 M urea and 40% (v/v) formamide] (Muyzer et al., 1993) was used in 6% (w/v) polyacrylamide gels. Electrophoresis was performed in 1 × Tris-acetate EDTA buffer at 60 °C

and at a constant voltage of 70 V for 16 h using a DCode system (BioRad). The DGGE gels were stained in a 1 : 2000-diluted SybrGold (Molecular Probes) in water BAY 57-1293 purchase enough for 30 min. Gel images were captured

using a ChemiDoc XRS (BioRad), and analyzed using quantity one software (BioRad). The background was subtracted using a rolling disk set at 20, and band density at positions was converted to intensity per Rf value between 0 and 1. After normalizing for total intensity across lanes, data were input into the past software package and analyzed using multivariate principal component analysis (Hammer et al., 2001). PCR product from each primer set was ligated into pGEM-T Easy Vector (Promega) and transformed into E. coli JM109 competent cells. A total of 10 clones from each primer set reaction were chosen at random for sequencing. The DNA sequences were determined using the chain termination method by the Nevada Genomics Center, using the T7 primer. Vector sequence and 16S rRNA gene sequences downstream of the respective primer were removed manually. The melting temperature (Tm) was calculated with biomatch (Promega), using the base-stacking algorithm. Empirical observations of differences in DGGE profiles generated using separate GC-clamp primer batches lead us to suspect variation in performance of distinct batches. Therefore, we PCR-amplified two soil DNA extracts using three batches (N1–N3) of the same GC-clamp primer, paired with the same reverse primer R907. To compare the effect of a longer template-directed sequence, primer G1 had the same GC-clamp sequence but an 18- rather than a 15-base 16S rRNA gene sequence (Muyzer et al., 1993).

A meta analysis of DAPT research buy transmission outcomes in several major USA and European studies also demonstrated that an HIV viral load < 1000 HIV RNA copies/mL at delivery was associated with a relatively low risk

of transmission and that antiretroviral prophylaxis offered additional clinically significant protection [162]. Zidovudine has been shown to reduce cervicovaginal shedding of HIV [19] and there are no data to suggest that cART is more effective than zidovudine at reducing cervicovaginal shedding in women with a plasma HIV VL < 50 copies/mL. Therefore zidovudine monotherapy is an option in this setting. There are no data to support the use of intravenous zidovudine infusion during labour in elite controllers. cART may provide more reassurance about prevention of mother-to-child transmission but will also expose both mother and infant to more potential drug toxicities. The choice of cART is as per Recommendation 5.3.3. Data on the mode of delivery in elite controllers are sparse and limited to case reports [163]. The benefits of PLCS at various levels of viraemia are discussed in Section 7.2: Mode of delivery. There are no data to support the use of PLCS for PMTCT when the VL is < 50 HIV RNA copies/mL in women

on antiretroviral therapy. The Writing Group therefore recommends vaginal delivery Selleck Luminespib for all elite controllers on antiretroviral therapy. 5.6.1 The discontinuation of NNRTI-based cART postpartum should be according to the BHIVA guidelines for the treatment of HIV-1 positive adults with antiretroviral therapy 2012 (www.bhiva.org/Guidelines.aspx). Grading: 1C The literature comparing strategies for stopping antiretroviral therapy in pregnant women is limited and therefore no alternative recommendation, compared with non-pregnant women, is made. However, in a randomised controlled study comparing two durations of treatment to prevent NNRTI-related mutations after single-dose nevirapine, 21 days of therapy (0.5% with

mutations on population-based sequencing and 5% detection of minority species by allele-specific PCR) outperformed 7 days’ cover (1.9%; Roflumilast P = 0.37 and 18%; P = 0.02, respectively) regardless of whether cover was provided by a two dual-nucleoside regimen (zidovudine/lamivudine or tenofovir/emtricitabine) or boosted PI monotherapy (lopinavir/ritonavir) [164]. Therefore, 21 days of therapy is preferred following the use of single-dose nevirapine. 5.6.2 Antiretroviral therapy should be continued postpartum in women who commenced cART with a history of an AIDS-defining illness or with a CD4 cell count < 350 cells/μL as per adult treatment guidelines. Grading: 1B Available RCT data to address the question as to whether one should continue or stop cART in women receiving it to prevent MTCT and not for their own health is sparse and has limited applicability to current ART treatment practices.

3c). These differences were not the consequence of different growth rates as both strains showed similar growth curves in minimal medium (data not shown). The M. loti triple mutant also showed a significantly lower competitive ability when co-inoculated with the rhcN mutant strain (Fig. 3a). Different independent experiments (Fig. 3a) indicated a positive role for the protein encoded in mlr6316 in the symbiotic competitiveness on Lo. tenuis cv. Esmeralda: The wt strain showed a slightly higher competitiveness than the mlr6316 mutant, and the same difference was observed when the double mutant mlr6358/mlr6361 was co-inoculated with the triple mutant. The comparison between the results obtained when the wt strain

was co-inoculated with the mlr6316 mutant and those obtained when the wt strain was co-inoculated with the triple mutant indicates Belinostat in vivo that the triple mutation affects competitiveness more drastically than the single mutation in mlr6316 (Fig. 3a and b). This suggests the possibility that the protein encoded in mlr6358 and/or the protein encoded in mlr6361 play a positive role in the symbiotic

competitiveness. Consistent with this, the double mutant mlr6358/mlr6361 was less competitive than the wt strain (Fig. 3a). The triple mutation in mlr6358, mlr6361, and mlr6316 also PF-01367338 in vitro caused a more drastic effect on competitiveness than the combined mlr6316/mlr6361 mutation (Fig. 3a and b). This indirectly indicates that Mlr6358 has a positive effect on competitiveness on Lo. tenuis. No statistically significant differences were observed in competitiveness on Lo. tenuis cv. Esmeralda between the wt and the mlr6361 mutant or between the wt and the double mutant mlr6331/mlr6361 (Fig. 3a). However, the mutant affected in both Mlr6361

and Mlr6331 showed decreased competitiveness compared with the wt strain on Lo. japonicus this website MG-20 (Fig. 3c). To determine which of the two proteins are responsible for the positive effect on this plant, co-inoculation assays of the double mutant with each of the single mutants were performed. Results indicate that the double mutant was less competitive than the single mutant affected in mlr6361 but more competitive than the single mutant affected in mlr6331 (Fig. 3c). This indicates that Mlr6331 has a positive effect and that Mlr6361 has a negative effect on the competitiveness on Lo. japonicus MG-20. We determined the nodulation kinetics for the M. loti wt, the rhcN mutant, and the triple mutant on Lo. tenuis cv. Esmeralda (Fig. 4). Although the rhcN mutant showed greater competitive ability on this plant (Fig. 3a), its nodulation kinetics was negatively affected when compared with the wt strain. On the other hand, in concordance with the competitiveness results, the M. loti triple mutant presented a kinetic phenotype significantly negatively affected compared with the wt strain and a delayed nodulation kinetics compared with the rhcN mutant strain (Fig. 4).