In API 20NE, cells are positive for the oxidase test, hydrolysis of esculin and gelatin, assimilation of d-glucose, and negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, β-galactosidase, assimilation of l-arabinose, d-mannose, d-mannitol, N-acetyl-glucosamine, d-maltose, potassium gluconate, capric acid, adipic acid, malic

acid, trisodium citrate, and phenyl acetic acid. In API 20E, cells show negative reactions for lysine decarboxylase, ornithine RGFP966 nmr decarboxylase, tryptophan deaminase, H2S production, and citrate utilization, and positive for acetoin production. In API ZYM, alkaline and acid phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, naphthol-AS-BI-phosphohydrolase, and β-glucuronidase are positive

for this strain, whereas the strain is negative for lipase (C14), α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, Selleckchem PI3K Inhibitor Library N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase. In API 50 CH, acid is produced from d-xylose, d-galactose, d-glucose, l-rhamnose, amygdalin, esculin, d-cellobiose, sucrose, and gentiobiose; no acid is produced from remaining substrates. Polar lipids and the major fatty acids are listed in the genus description. Major respiratory quinone is MK-6, and DNA G+C content is 33.7 mol%. The type strain is CC-SAMT-1T (BCRC 80315T = JCM 17682T), isolated from coastal seawater collected at China Sea (Changhua County, Siansi Township, XianGong South 4th Road), Taiwan. The authors thank the reviewers for critical comments. We gratefully acknowledge Prof. Hans G. Trüper

for bacterial nomenclature. Hsin-I. HuangI, Wai-Shuo Huang, and You-Cheng Liu are thanked for their help during sample collection, electron microscopy, and G+C assay. We thank Dr Ren-Jye Lee for LC-MS/MS. This work was supported in part by the Ministry of Education, Tryptophan synthase Taiwan under the ATU plan. M.S. is grateful to Ministry of Economic Affairs, Taiwan, for awarding scholarship. The authors declare no conflict of interest. A.H. and M.S. contributed equally to this work. “
“Herbicides have the potential to impair the metabolism of soil microorganisms. The current study addressed the toxic effect of bentazon and 4-chloro-2-methylphenoxyacetic acid on aerobic and anaerobic Bacteria that are involved in cellulose and cellobiose degradation in an agricultural soil. Aerobic saccharide degradation was reduced at concentrations of herbicides above environmental values. Microbial processes (e.g.

Cells grown to the stationary phase in M9 succinate minimal liquid medium were harvested and washed three times with M9 medium without carbon sources. A 1 : 1 mixture of the mutant (LacZ−) and control cells

(ATCC17616cox::lacZ; LacZ+) was inoculated into 2.7 g of soil sample in a 50-mL test tube, and the water content was adjusted to 60% of the maximum water-holding capacity. Approximately 50 tubes were prepared for each mixture, and three tubes were used for each time point. At different time points after the incubation at 30 °C, M9 minimal medium was added to the tube, vigorously vortexed, and treated mildly by sonication. The sample was left standing still for 30 min, and the supernatant was recovered and plated onto an M9 succinate minimal agar plate containing X-gal. check details The colony-forming units (CFUs) g−1 of soil were measured, and the ratio of white (mutant) to white plus blue (control) colonies was calculated. The LacZ activities of cells in the soil and in the laboratory medium were measured as described previously (Nishiyama et al., 2010). For the measurement of LacZ activity

in the laboratory medium, one-percent volume of an overnight culture (M9 succinate minimal medium) was transferred to fresh M9 medium, and the cells were incubated for 24 h and harvested. For the measurement of LacZ activity Selleck RG7422 in the soil, the cells in the soil were harvested as described (Nishiyama et al., 2010). In brief, the tube, into which M9 medium was added, was vortexed vigorously for 30 s, shaken for 30 min, and mildly sonicated for 15 s. After leaving for 30 min for sedimentation of

the soil particles. the cells were collected from the supernatant by Carnitine palmitoyltransferase II centrifugation at 7500 r.p.m. (5500 g) for 6 min. The harvested cells were disrupted by sonication, and cell debris was removed by centrifugation. The crude extract thus obtained was used to measure the LacZ activity. The activity was normalized by the amount of protein in the reaction mixture that was measured using a Protein Assay kit (Bio-Rad Laboratories). Genomic sequence of ATCC 17616 predicted a pathway for the catabolism of tryptophan and anthranilate (Fig. 1b). In this pathway, the three enzymes KynA (tryptophan 2,3-dioxygenase), KynB (kynurenine formamidase), and KynU (kynureninase) convert tryptophan to anthranilate, which is next converted to catechol by the four-component anthranilate dioxygenase (AndAc AndAd AndAb AndAa). Catechol is then converted to TCA cycle compounds by the activities of CatA, CatB, and CatC. The genomic locus for the catabolism of anthranilate and catechol in ATCC 17616 is shown in Fig. 1a. An andAc mutant (17616ΔandAc) of ATCC 17616 was tested for its ability to utilize tryptophan and anthranilate as a sole carbon source. The wild-type strain, but not 17616ΔandAc, grew on both compounds.

Briefly, these data include comprehensive demographic and exposure category information

on all adults diagnosed with HIV infection [10] and prospective clinical information obtained at least annually from all HIV specialized clinics to form a national HIV cohort [11]. In addition, results of all sequential CD4 counts are reported directly by laboratories [12]. Death reports are obtained from clinicians and record linkage with the death register of the Office for National Statistics (ONS). Limited patient identifiers (surname soundex, sex and date of birth) are used to link individual records across data sets across years, to create a cohort and to estimate establishment and retention in care [13]. Data on persons aged ≥ 15 years diagnosed in 2010 and accessing HIV care in 2011 as buy Daporinad well as those diagnosed in 2011 were included in the analyses. A ‘late HIV diagnosis’ was defined as a diagnosis with a CD4 count < 350 cells/μL reported within 3 months of diagnosis. This is also the threshold under which national guidelines recommend treatment should begin [14]. Data are presented as proportions or rates

for those diagnosed during 2011. Patients with no CD4 count reported within 3 months of diagnosis were excluded. Guidelines recommend that patients should have a CD4 count within 14 days of diagnosis [6]. The first CD4 test was therefore used as a proxy for integration into HIV Nivolumab purchase care. The proportions of adults diagnosed in 2011 with a CD4 test reported within 1 and 3 months of HIV diagnosis were calculated. Patients with no CD4 count reported within 12 months of HIV diagnosis were excluded. The retention rate was calculated by determining the proportion of patients diagnosed in 2010 seen again for HIV care in 2011. Patients who died were excluded from the analyses as were those diagnosed in Scotland (due to limited linkage information). Treatment coverage rates in 2011 were calculated for adults diagnosed

in 2010 stratified by CD4 count at diagnosis. One-year mortality was defined as death within 1 year of HIV diagnosis. Rates are presented per 1000 of population among adults diagnosed in 2010, stratified by CD4 count at diagnosis. Proportions are presented among persons for whom the relevant Baricitinib information was available. The emphasis of this paper is descriptive, but key findings have been supported by χ2 tests and t-tests for trend where appropriate. In 2011, 6219 adults were newly diagnosed with HIV infection compared with 6299 in 2010. The completeness of demographic and epidemiological data for persons diagnosed in 2011 was as follows: sex, 100%; ethnicity, 95%; age, 100%; exposure category, 92%; region of residence, 99%; and region of birth, 80%. Similar levels of completeness were observed among those diagnosed in 2010.

, 2010). Optimal PCR conditions utilized 30 cycles of 94 °C (30 s), 52 °C (30 s), and 72 °C (30 s), with an initial denaturation at 95 °C (5 min) and a final extension at 72 °C (5 min), and the same concentrations of reagents as used for 16S rRNA gene PCR. Clone libraries based on the 16S rRNA gene and the GHF48 gene were constructed by pooling amplicon DNA, purifying from PCR and cloning into a pMD18-T

vector (TaKaRa Biotechnology Co. Ltd., Dalian, China). Two vector-specific primers were used for the amplification CH5424802 in vivo of the DNA inserts: M13-47 (5′-CGCCAGGGTTTTCCCAGTCACGAC-3′) and RV-M (5′-GAGCGGATAACAATTTCACACAGG-3′). PCR amplification was using 30 cycles of 94 °C (30 s), 54 °C (45 s), and 72 °C (2 min), with an initial denaturation at 95 °C (5 min) and a final extension at 72 °C (10 min). Clones were screened by agarose gel electrophoresis to check the inserts were the correct size. The PCR products were purified using the TaKaRa agarose gel DNA purification kit (TaKaRa Biotech Co.) and were sequenced by Shanghai Biosune (Shanghai, China) with an Applied

Biosystems automatic sequencer (ABI3730). A total of 50 clones from each clone ATPase inhibitor library were screened. dnastar lasergene software was used for manual editing of the amplified 16S rRNA and GHF48 gene sequences. Operational taxonomic units (OTUs) definition at 97% sequence similarity was determined using the dotur software package (Schloss & Handelsman, 2005). The rarefaction curve was generated by past software package with a confidence threshold of 95% (Hammer et al., 2001). The

identification of phylogenetic neighbors and the calculation of pairwise 16S rRNA and GHF48 gene sequence similarities were achieved by blasting in EzTaxon-E database and NCBI (Kim et al., 2012). Sequences were classified into different bacterial taxa by RDP naive Bayesian rRNA classifier Version 2.4 Staurosporine in vivo with a confidence threshold of 80% (Cole et al., 2009). Phylogenetic analysis was performed with the software package mega version 4.0 (Tamura et al., 2007) after multiple alignment of data by clustalx (Chenna et al., 2003). The phylogenetic trees were constructed using neighbor-joining (NJ) methods. Bootstrap values were calculated based on 1000 replicates. The nucleotide sequences of both the 16S rRNA genes and GHF48 genes from the clone libraries have been deposited in the GenBank database under accession numbers JQ741978–JQ741999. The isolated microbial community could degrade FP and Avicel under anaerobic conditions at 60 °C within 3 days, as shown in Fig. 1a. Initially, the FP became soft, then sticky, and eventually it dissolved completely. The phenomenon of the FP decomposition differed from that of C. thermocellum LQR1, in which the FP initially became thin and then dissolved. The fermentation products of the cellulolytic culture were detected by HPLC for 6 days.

Typhimurium; Prigent-Combaret et al., 2001) and for the P-pili of UPEC (Jones et al.,

1997). Bundle-forming pili are pivotal for EPEC to form microcolonies and to attach to host cells (Tobe & Sasakawa, 2001). The Cpx-TCS is induced by overexpression of the BFP subunit BfpA and by mature BFP (Nevesinjac & Raivio, 2005). This finding strongly indicates that intermediates other than unprocessed BfpA are also sensed by the Cpx-TCS (Nevesinjac & Raivio, 2005). The Cpx system controls curli fimbriae expression, which are involved in forming surface amyloidal fibres important for biofilm formation and host cell adhesion (Dorel et al., 1999; Jubelin et al., 2005; Barnhart & Chapman, 2006), and curli overexpression PTC124 molecular weight BYL719 manufacturer induces the Cpx response (Prigent-Combaret et al., 2001). P-pili are crucial for kidney colonization by UPEC strains and belong to the group of chaperone-usher pili (CU pili; reviewed in Waksman & Hultgren, 2009). Essential for the formation of CU pili is a periplasmic chaperone that guides the single subunits after release from the SecYEG

translocase across the periplasmic space to the usher in the outer membrane. Deletion of the chaperone PapD results in misfolded P-pilus subunits that become toxic for the cell and induces the Cpx response (Jones et al., 1997). Overexpression of CpxP suppresses the lethal phenotype by causing the misfolded pilus subunits to be degraded by DegP (Isaac et al., 2005). Because the induction

of the Cpx-TCS by PapG does not depend on the DegP protease (Hung et al., 2001) but rather on click here CpxP, it was suggested that PapG induces the release of CpxP from CpxA resulting in the activation of the Cpx-TCS (Fig. 3d; Isaac et al., 2005). An elongated hydrophobic cleft on the convex surface of the CpxP dimer might act as a sensory part for pilus subunits (Zhou et al., 2011). However, it remains mysterious which region of pilus subunits is recognized by CpxP. Only two pilus subunits are known to activate the Cpx-TCS: the PapG adhesin and the fibrillum subunit PapE (Jones et al., 1997). It has been suggested that the N-terminal extension of PapE, which is essential for the assembly of pilus subunits, is crucial for recognition of PapE by CpxP (Lee et al., 2004; Isaac et al., 2005). However, PapG is missing an N-terminal extension that is present in the other subunits which are not recognized by the Cpx-TCS (Lee et al., 2004). Very recently, a crucial role of the Cpx system in inter-kingdom signalling between host and bacteria has been discovered (Karavolos et al., 2011). In S. Typhi, exposure to host stress neuroendocrine hormones leads to increased haemolytic activity through the secretion of haemolysin HlyE-containing membrane vesicles (Karavolos et al., 2011).

We restored the wild-type fnr allele on the chromosome in this way (replacing fnr∷tmpR) rather than providing it in trans due to concerns that fnr provided in multicopy can show uncharacteristic effects such as gene activation under aerobic conditions (Reyes-Ramirez & Sawers, 2006) and a narrowing of the difference between better and

poorer FNR activation sites (Scott et al., 2003). However, because our V. fischeri-derived allele-replacement constructs were not appropriate (homologous) ITF2357 purchase for exchange into E. coli, we provided the putative fnr of V. fischeri ES114 to E. coli in trans on plasmid pJLB6, which restored anaerobic respiration of E. coli fnr mutant PC2 on nitrate (Fig. 1d). Taken together, our results indicate that the putative V. fischeri FNR is similar in both sequence and function to E. coli FNR. We tested whether FNR regulates lux expression by monitoring the luminescence of strains grown aerobically or anaerobically (Fig. ICG-001 in vivo 2a and b). The luminescence of the fnr mutants was similar to that of their parent strains under aerobic conditions (Fig. 2a). FNR is inactivated by oxygen, and we therefore also assessed lux expression anaerobically. Luciferase uses oxygen as a substrate, and so anaerobic cultures do not luminesce; however, as with all luminescence measurements, samples removed from anaerobic bottles were shaken for ∼10 s to saturate luciferase with oxygen

before measuring luminescence. When grown anaerobically, luminescence was higher in fnr mutant EVS601 than in MJ1 (Fig. 2b). The magnitude of this difference varied between STK38 1.5- and 20-fold, and averaged eightfold, in five experiments. The luminescence of ES114 and fnr mutant JB1 was below the background, appearing the same as a dark ΔluxCDABEG strain (data not shown), which raised the possibility that FNR regulates lux in ES114, but that the overall luminescence is below detection. To test this possibility, we added the luminescence-stimulating autoinducer 3-oxo-C6-HSL to anaerobic cultures of ES114 and its fnr mutant JB1. 3-oxo-C6-HSL stimulated the luminescence of ES114 and JB1, and under

these conditions, JB1 was brighter than ES114 (Fig. 2c). We considered the possibility that increased luminescence in V. fischeri fnr mutants could result from increased availability of luciferase’s substrates due to the physiological effects of this global regulator. To test this possibility, we disrupted fnr in a background where the luxCDABEG genes are under the control of LacIq and a non-native promoter. In this background, FNR had no significant effect (P>0.05) on luminescence (Fig. 2c). Thus, the repressive effect of FNR on luminescence is dependent on the native lux promoter. The luxICDABEG operon can be subject to positive feedback regulation, because the autoinducer synthase LuxI generates 3-oxo-C6-HSL, which, in combination with LuxR, stimulates luxICDABEG transcription. Given the amount of 3-oxo-C6-HSL added exogenously to the cultures (Fig.

In a C. elegans infection model, worms that were fed P. aeruginosa lawns died from the production of multiple phosphate-regulated virulence factors that resulted in the ‘red death’ phenotype (Zaborin et al., 2009). We tested the role of olsA in killing C. elegans by comparing the killing efficiency of worms fed with the wild-type PAO1 and olsA∷lux strains. The olsA mutant was not impaired for killing C. elegans after 7 days postinfection (Fig. 5c). In this study we report the identification of the OL biosynthesis genes olsBA in P. aeruginosa.

These genes are widely conserved among the genomes of the other Pseudomonas genomes annotated in the Pseudomonas Genome Database (Winsor et al., 2009) and other Gram-negative bacteria (Geiger et al., 2010), but have been studied only in two species of bacteria to date. The P. aeruginosa olsBA genes were strongly induced

by phosphate limitation and are required for OL see more production. The production of a phosphate-free membrane PD0332991 price lipid is a mechanism to adapt to phosphate-limiting conditions; however, we could not detect any major physiological consequence in a mutant unable to produce OLs. Despite limiting phosphate, the olsA mutant showed no significant growth defect, which is consistent with a report showing that only double mutants lacking OLs and diacylglyceryl-N,N,N-trimethylhomoserines have significant growth defects under these conditions (Lopez-Lara et al., 2005). We provide evidence that OLs do not contribute to antimicrobial peptide resistance, which contradicts the conclusions of an earlier study in P. fluorescens that showed a correlation between OL production and peptide resistance under phosphate-limiting conditions (Dorrer & Teuber, 1977). It was proposed that the positive charge of ornithine may prevent binding of cationic peptides to membranes (Dorrer & Teuber, 1977), but at neutral pH, OLs are zwitterionic, with a net neutral charge similar to phosphatidylethanolamine.

However, we did observe increased resistance of cells grown in limiting phosphate and this is likely due to the remodeling of the outer membrane under phosphate limitation because the outer membrane was more impermeable to NPN incorporation after polymyxin B treatment RVX-208 (Fig. 5b). The most likely explanation, given that the NPN assay reflects self-promoted uptake across the outer membrane, is that cells grown in limiting phosphate incorporate less phosphate into the lipopolysaccharide in the outer membrane, and this may reduce peptide binding to the outer membrane. It is worth noting that under normal growth conditions, P. aeruginosa lipopolysaccharide contains 12–13 phosphate residues (Peterson et al., 1985). Ingram et al. (2010) recently described a phosphatase that cleaves 1- and 4-phosphates from lipid A in Rhizobium etli and contributes to antimicrobial peptide resistance.

Starting ART early in severely immunosuppressed HIV-positive patients presenting with TB is associated with decreased CHIR-99021 supplier mortality and a lowering of the rates of disease progression but rates of IRD are high. Patients with HIV and a CD4 cell count >350 cells/μL have a low risk of HIV disease progression or death during the subsequent 6 months of TB treatment, depending on age and VL [6]. They should have their CD4 cell count monitored regularly and ART can be

withheld during the short-course of TB treatment. One study performed in HIV-associated TB meningitis in the developing world, where 90% of the patients were male, the majority drug users, many with advanced disease and the PD0332991 in vitro diagnosis being made clinically, showed no difference in mortality starting ART early or late [7]. We recommend EFV in combination with TDF and FTC as first-line ART in TB/HIV coinfection 1B We recommend that when rifampicin is used with EFV in patients over 60 kg, the EFV dose is increased to 800 mg daily. Standard doses of EFV are recommended if the patient weighs <60 kg 1C We recommend that rifampicin is not used with either NVP or PI/r 1C We recommend that where effective ART necessitates the use

of PI/r, that rifabutin is used instead of rifampicin 1C Proportion of patients with active TB on anti-TB therapy started on ART containing EFV, TDF and FTC. HIV-related TB should be treated with a regimen, including rifamycin for the full course of TB treatment, unless there is rifamycin resistance or intolerance. Rifamycins frequently interact with ARV medications and can lead to similar toxicities, notably rash and hepatitis. We recommend EFV as the preferred therapy for ART 4��8C because of its confirmed potency when used in TB/HIV coinfection [8-10], and its efficacy in RCT. We recommend that EFV be given with TDF and FTC due to the availability

of a once-daily co-formulation, a reduced risk of rash compared with NVP and improved efficacy at higher HIV VLs (commonly occurring in this setting). ABC-3TC is an alternative acceptable NRTI backbone in patients with lower HIV VLs and that are HLA-B*57:01 negative (see Section 5.3 Which NRTI backbone). There is significant variability in the effect that rifampicin has on EFV concentrations because of liver enzyme induction, especially of CYP450 3A4 [8,11–13]. Subtherapeutic EFV concentrations may occur among patients who weigh more than 60 kg who are taking standard dose EFV together with rifampicin, and increasing the dose of EFV from 600 mg daily to 800 mg daily may be necessary; however, there is a risk of increasing adverse effects.

Several literature studies have reported the effect of sulfate on desulfurization activity. Li et al. (1996) reported that although sulfate represses the dsz genes, it does not inhibit the activity of desulfurizing enzymes (Wang & Krawiec, 1996). They observed that the desulfurizing activity increased with decreasing amount of sulfate in the medium. Similarly, Omori et al. (1995) also observed enhanced desulfurizing rates arising from the removal of byproduct sulfate from a succinate-based medium. To understand this phenotype using our in silico model, we analyzed PI3K inhibitor fluxes for three scenarios (Table 2) with a succinate uptake at 20 mg g−1 dcw h−1. In run 6, we

allowed unlimited DBT as the sole sulfur source and obtained the maximum desulfurizing rate of 0.07 mmol g−1 dcw h−1. In run 7, we allowed unlimited sulfate as the sole sulfur source, and obtained the maximum sulfate uptake of 10.80 mg g−1 dcw h−1. Then,

in subsequent runs, we allowed progressively increasing amounts of sulfate (from 0% to 100% find more of the maximum sulfate uptake of 10.80 mg g−1 dcw h−1 from run 7) with unlimited DBT. From Fig. 3, we see that the desulfurizing activity clearly decreases with increasing amount of sulfate. Thus, our model successfully explains the observations of Omori et al. (1995) and Li et al. (1996). Our earlier comment on energy needs again readily explains this effect. When the desulfurizing Adenosine enzymes are already present, then the organism is able to utilize (desulfurize)

DBT. However, sulfate promotes higher growth at lower energy, and so the organism prefers sulfate consumption over DBT conversion. Only when sulfate is limited, it desulfurizes DBT. In other words, no desulfurization is possible even in the presence of desulfurizing enzymes if the medium has sufficiently high concentration of sulfate to meet the sulfur needs of R. erythropolis. To our knowledge, no previous experimental work has elucidated this phenotype, which our model made possible. Yan et al. (2000) studied the relative efficacy of ethanol, glucose, and glycerol as sole carbon sources for the growth and desulfurizing activity of R. erythropolis. They reported ethanol to yield the highest growth and desulfurizing rates, followed by glucose, and then glycerol. To simulate this phenotype, we considered three separate scenarios with unlimited DBT and one carbon source. In each scenario, we fixed the uptake of the respective sole carbon source at 20 mg g−1 dcw h−1 and used maximum biomass as the cellular objective. Our model gave the highest growth rate of 1.39 h−1 and the highest desulfurizing rate of 0.18 mmol HBP g−1 dcw h−1 for ethanol. In contrast, the rates were 0.60 h−1 and 0.08 mmol HBP g− 1dcw h−1 for glucose, and 0.59 h−1 and 0.07 mmol HBP g−1 dcw h−1 for glycerol. Thus, our model qualitatively confirms the experimental results of Yan et al. (2000).

, 2009). Mesorhizobium loti induced small white ‘tumor-like’ growth on Leucaena leucocephala, but a mutant in a conserved structural component of T3SS (the NVP-BGJ398 datasheet M. loti rhcJ mutant strain) formed large pink nodules (Hubber et al., 2004). Little is known about the role of each of the putative M. loti T3SS effectors. The protein encoded by mlr6316 has been described to have a partial negative

effect on Le. leucocephala nodulation (Hubber et al., 2004), whereas the protein encoded by mlr6361 has been described to have a negative role in Lo. halophilus nodulation (Okazaki et al., 2010). However, it has not yet been determined which of the proteins secreted by the M. loti T3SS are involved in the positive nodulation effects observed in some Lotus spp. The aim of this work

was to determine whether the N-terminal regions of proteins encoded by mlr6316 and mlr6331 are able to direct the protein secretion via M. loti T3SS and to determine the involvement of the different T3SS putative effectors in the symbiosis with two different Lotus species. Bacterial strains and plasmids used in this study are listed in Supporting Information, Table S1. MAFF303099 strains were grown at 28 °C in AB minimal medium (Douglas et al., 1985) supplemented with sucrose (0.5% w/v). When necessary, antibiotics were added to the following final concentrations (μg mL−1): gentamicin (Gm), 30; ampicillin (Amp), 100; neomycin selleck chemicals (Nm), 100; spectinomycin (Sp), 100; and tetracycline (Tc), 10 for Escherichia coli or 1 for M. loti. For T3SS induction, naringenin was added to cultures at an optical density 600 nm (OD600 nm) of 0.1 to a final concentration of 1 μM. pBAD-mlr6331 was constructed as previously described (Sánchez et al., 2009). The oligonucleotide primer pairs used are described in Table S1. Sequences encoding the N-terminal portion of the protein, together with the upstream

promoter region, were cut from pBAD-mlr6361 (59 aa), pBAD-mlr6316 (160 aa), pBAD-mlr6358 (160 aa) (Sánchez et al., 2009), and pBAD-mlr6331 (177 aa), respectively, and cloned into the pK18mobTc vector (Sánchez et al., 2009). The same plasmids were also triclocarban introduced by biparental mating into an M. loti rhcN pMP2112 mutant strain. Supernatant protein extractions were carried out by direct TCA precipitation as previously described (Sánchez et al., 2009). Supernatant was concentrated approximately 2000 times. For total (intracellular) bacterial protein extractions, 1 ml of the bacterial cultures used above was centrifuged and the pellets dissolved in cracking buffer. Proteins were separated using SDS-PAGE and then stained using silver nitrate. For immunoblotting, anti-NGR234 strain NopA (Marie et al., 2004) or a commercially available anti-FLAG M2 monoclonal antibody (Sigma) was used. Analyzed mutants and oligonucleotides pairs used for their construction are described in Table S1.