Microbial Ecol 1986, 12:65–78.CrossRef 56. Selvam K, Vishnupriya B, Subash Chandra Bose V: Screening and quantification of marine actinomycetes producing industrial enzymes

amylase, cellulase and lipase from South cost of India. Int J Pharma Biol 2011, MAPK inhibitor 2:1481–1487. 57. Jang H-D, Chen K-S: Production and purification of thermostable cellulases from Streptomyces transformant T3–1. World J Microbiol Biotechnol 2003, 19:263–268.CrossRef Competing interests The authors declare that they have no competing interest. Authors’ contribution Research concept and the experiments were performed by BM and LAR, NVV and RK analyzed the data and reviewed the manuscript. All authors approved the final manuscript.”
“Background Pseudomonas aeruginosa is the major pathogen involved in the decline of lung

function in patients with cystic fibrosis (CF) [1–5]. Its presence in the lungs is associated with an increased mortality and morbidity of Selleck VS-4718 CF patients [6]. Early detection of this bacterium from respiratory tract is determinant because it ensures effective patient management [5, 7, 8]. Indeed, after intermittent colonization by different strains, once acquired, chronic P. aeruginosa colonization by mucoid and biofilm-growing isolates is difficult to eradicate [2, 4, 9, 10]. Thus, the earlier the treatment toward P. aeruginosa onset, the higher the chance to efficiently control P. aeruginosa [5, 7, 8]. However, accurate identification of this bacterium in CF sputum by selleck conventional microbiology techniques is known

to be limited. This can be explained by a large phenotypic diversity of P. aeruginosa isolates recovered from CF patients such as loss of pigment production or exopolysaccharide production. Moreover, Singh et al. demonstrated that P. aeruginosa can form biofilms in the airways of CF patients [11]. Biofilms contain bacterial cells that are in a wide range of physiological states. One of the mechanisms 17-DMAG (Alvespimycin) HCl contributing to this physiological heterogeneity includes the adaptation to the local environmental conditions. For instance, bacterial cells from the deep layers of biofilm depleted of oxygen [12] can grow in anaerobic conditions. Therefore, the CF patients isolates obtained from biofilms, i.e. in anaerobic conditions, grow hardly in aerobic conditions on a conventional culture medium [13]. Another limitation of conventional culture is that P. aeruginosa can be easily misidentified with closely related Gram-negative bacilli in CF sputum [14–19]. The use of molecular techniques such as PCR could improve accurate identification of P. aeruginosa [14–19], and consequently, its early detection in CF sputum patients [20–24]. To date, there is no consensus for a universal protocol for the molecular detection of P. aeruginosa. Indeed, its genome is known to be highly polymorphic. Changes that can occur at the genetic level could compromise the reliability of molecular identification techniques.

Four different intimin types were identified: θ2 (theta), σ (sigma), τ (tau) and upsilon (Table 1). We have detected in aEPEC strains 4281-7 and 1632-7 (serotypes O104:H- and O26:H-, respectively)

two new intimin genes eae-τ and eae-ν that showed less than 95% nucleotide sequence identity with existing intimin genes. Furthermore, a third new variant of the eae gene (theta 2 – θ2) was identified selleck in the aEPEC strain 1871-1 (serotype O34:H-). The complete nucleotide sequences of the new eae-θ2 (FM872418), eae-τ (tau) (FM872416) and eae-upsilon; (FM872417) variant genes were determined. By using CLUSTAL W [41] for optimal sequence alignment, we determined the genetic relationship of the three new intimin genes and the remaining 27 eae variants. A genetic identity of 90% was calculated between the new eae-τ (tau) variant and eae-γ2 (gama2),

eae-θ (theta) and eae-σ (sigma) genes. The eae-upsilon; showed a 94% of identity with eae-ι1. The eae-θ2 (theta-2) gene is very similar (99%) to eae-θ of Tarr & Whittam [20] and to eae-γ2 of Oswald et al. [19]. Table 1 Characteristics of the aEPEC strains studied. Strain Serotype Intimin Type Tozasertib adherence pattern FAS test         HeLa cells T84 cells 0621-6 ONT:H- σ * LA + + 1551-2 ONT:H- ο LA + + 1632-7 O26:H- upsilon; ** DA + + 1871-1 O34:H- θ2 ** LAL + + 4051-6 O104:H2 ο AA + + 4281-7 O104:H-

τ** LAL + + E2348/69 O127:H6 α1 LA + Milciclib chemical structure + Adhesion pattern detected on HeLa cells: localized adherence (LA), localized adherence like (LAL), aggregative adherence (AA) and diffuse adherence (DA) (Vieira et al., 2001). (*) Strains that had eae gene sequenced in this study and (**) strains that carry new intimin subtypes (GenBank accession numbers: 1871-1 (FM872418); 4281-7 (FM872416) and 1632-7 (FM872417). Quantitative assessment of bacterial invasion Farnesyltransferase was performed with all strains, but different incubation-periods were used to test aEPEC strains (6 h) and tEPEC E2348/69 (3 h), because the latter colonizes more efficiently (establishes the LA pattern in 3 h) than aEPEC strains [3] and induce cell-detachment after 6 h of incubation (not shown). The quantitative gentamicin protection assay confirmed the invasive ability of aEPEC 1551-2 in HeLa cells and showed that 4 of the other 5 aEPEC strains studied were also significantly more invasive than tEPEC E2348/69 (Fig. 1A). The percentages of invasion found varied between 13.3% (SE ± 3.0) and 20.9% (SE ± 2.4), respectively, for aEPEC strains 4051-6 (intimin omicron) and 0621-6 (intimin sigma). When compared to tEPEC E2348/69 (intimin alpha 1) (1.4% ± 0.3), the invasion indexes of all strains were significantly higher (p < 0.05), except for aEPEC strain 4281-7 (intimin tau, 2.4% ± 0.3).

In contrast, the T. brucei TRF protein

(TbTRF) appears to co-localize with most telomeres at all stages of the cell cycle in both bloodstream and procyclic forms [24]. Whether LaTRF also has other cellular roles or if its association with telomeres occurs in a cell cycle dependent manner is not clear at this stage. Figure 3 LaTRF partially co-localizes with L. amazonensis telomeres. LaTRF (red), using anti-LaTRF serum, was combined with FISH (green) using a PNA-telomere probe specific for TTAGGG repeats. DAPI (blue) was used to stain DNA in the nucleus (N) and in the kinetoplast see more (K). Images were organized in panels p1-p4 showing the co-localization patterns in merged (a): telomeres and LaTRF, and in merged (b): DAPI, telomeres and LaTRF. Merged images were done using NIS elements software (v. Br 2.30). LaTRF interacts in vitro and in vivo with L. amazonensis telomeres using a Myb-like DNA binding domain EMSA assays were done with renatured protein extracts containing full length LaTRF, the Myb-like DNA binding domain (LaTRFMyb) (Figs 4 and 5, see Selleckchem WZB117 additional file 1) and with L. amazonensis nuclear extracts (Fig 6), to investigate whether LaTRF, like its vertebrate and trypanosome counterparts [18, 24], was able to bind double-stranded telomeric DNA in vitro. Figure 4 Recombinant LaTRF and the mutant bearing

the C-terminal Myb domain bind in vitro double-stranded telomeric DNA. Electrophoretic selleck kinase inhibitor mobility shift assays (EMSA) were done using radiolabeled double-stranded telomeric DNA (LaTEL) as probe. Protein:DNA complexes were separated in a 4% PAGE in 1X TBE. EMSA was done with E. coli BL21 protein extract (lane 2), recombinant full length LaTRF (lanes 3-6) and a mutant bearing the C-terminal Myb domain (lanes 7-9). A supershift assay many was done with anti-LaTRF serum (lane 6). Assays were also done in the presence of 20 fold excess of non-labeled LaTEL as specific competitor (lanes 4 and 8) or 100 fold excess of double-stranded non-specific poly [dI-dC] [dI-dC] DNA (lanes 5 and 9). In lane 1, no protein was

added to the binding reaction. The original gel image and its content are shown as additional file 1: Figure S1. Figure 5 Supershift and competition assays confirm that recombinant full length LaTRF bind in vitro double-stranded telomeric DNA. Electrophoretic mobility shift assays (EMSA) were done using radiolabeled double-stranded telomeric DNA (LaTEL) as probe. Protein:DNA complexes were separated in a 4% PAGE in 1X TBE. EMSA was done with recombinant full length LaTRF and anti-LaTRF serum in the absence (lane 2) and in the presence of 20 fold excess of non-labeled LaTEL as specific competitor (lane 3) or 100 fold excess of double-stranded non-specific DNA (poly [dI-dC] [dI-dC]) as non specific competitor (lane 4).

In mammals various α-CA isoforms with different subcellular localization and tissue distribution are implicated in many physiological processes such as carboxylation/decarboxylation GDC 941 reactions, transport of CO2 and/or HCO3-, pH regulation, ion exchange, calcification, see more metabolism of urea, glucose and lipids, tumorigenicity, bone resorption and many other physiological and pathological processes [5]. Members of β-CAs are predominant in

plants, algae, archaea and bacteria. In photosynthetic organisms β-CAs play an important role in transport and autotrophic fixation of CO2 while in prokaryotes β-CAs are involved in wide range of cellular functions including provision of HCO3 – for carboxylating enzymes which catalyze key steps in biosynthetic pathways for essential metabolites, such as amino acids, nucleotides, fatty acids [6, 7]. The γ-CAs are predominant in bacteria and archaea domains. In eukaryotes, they have so far been described only in photosynthetic organisms. While the physiological role of α-CAs in mammals and β-CAs in plants and prokaryotes, have been extensively studied, the role of γ-CAs remain elusive. To date, the only γ-CA that has been extensively characterized is “”Cam”" from the methanogenic CHIR-99021 ic50 archaeon Methanosarcina thermophila [8, 9]. In the

cyanobacterium Synechocystis, the bifunctional CcmM protein localized in carboxysome (structure involved in CO2 concentration) shows an N-terminal γ-CA like domain which has been proposed to bind HCO3 Palmatine -/CO2 [10]. However, no carbonic anhydrase activity could be detected for the recombinant CcmM expressed in E. coli. Recently, a similar role for binding and transporting bicarbonate has been proposed for γ-CA subunits of plant mitochondrial complex, suggesting that the so-called γ-CAs in photosynthetic

eukaryotic organisms do not act as carbonic anhydrases but may have related activity contributing to CO2 recycling in photorespiration, or play a role in the carbon transport between mitochondria and chloroplasts to increase the efficiency of photosynthetic CO2 fixation [11]. Unraveling of microbial genome sequences has shown that γ-CAs are widespread in prokaryotes, and it is likely that these enzymes play diverse roles in microorganisms. Investigations into the ways in which archaea and bacteria domains use γ-carbonic anhydrase may reveal novel aspects of prokaryotic physiology. We are analyzing the role of carbonic anhydrases in a nonphotosynthetic, Gram-negative, plant growth promoting α-proteobacterium, Azospirillum brasilense that lives in close association with the roots of several important crop plants and grasses and stimulates the growth of its host plant by producing phytohormones and siderophores [12]. Earlier, we have cloned the gene encoding β-CA from A. brasilense, overexpresed, purified and characterized β-CA. We also showed that the transcription of bca gene was down regulated by stationary phase, elevated CO2 and acidic pH [13].

Each sample was repeated three times using 105 cells per test.

The cells treated with PBS were set as the control. Results and discussion Preparation and characterization of BLPs Formulation variables selleck screening library influence the physicochemical properties of insulin-loaded liposomes such as entrapment efficiency and particle size [32, 33]. It is of high importance to effectively entrap insulin into liposomes so as to reduce the bulk dosage and avoid waste of drug. The main variables including lipid/cholesterol ratio, drug/lipid ratio, the buffer pH upon hydration, and phase ratio in preparing W/O emulsion were optimized to obtain liposomes with high insulin entrapment efficiency and suitable particle size. Figure 1 shows the effects of preparative variables on the entrapment efficiency and particle size. The presence of cholesterol exerts significant influence on the properties of the lipid bilayers of the liposomes. It is known that the addition of cholesterol to lipid bilayers decreases its permeability to water [34]. Suitable lipid/cholesterol ratio will accommodate more insulin molecules and generate liposomes with desirable membrane fluidity, which are helpful to prevent the leakage of insulin from the internal aqueous compartments. The liposomes with a lipid/cholesterol ratio of

3/1 or 4/1 produced higher insulin entrapment (Figure 1A). Considering the factors that influenced drug BVD-523 supplier entrapment and 3-deazaneplanocin A resistant permeability to water, a lipid/cholesterol ratio of 3/1 seemed to be more promising. The effect of drug/lipid ratio on the entrapment efficiency Ponatinib molecular weight is shown in Figure 1B, from which we could see that the entrapment efficiency increased as the lipid content increased. Generally,

high proportion of lipid in liposomes can generate more space to host more insulin molecules. Figure 1C showed that the buffer solution of pH 3.8 used for hydration was most suitable to prepare liposomes. Lower entrapment efficiencies were obtained around the isoelectric point of insulin (pH 5.3 to 5.4), which may be attributed to the loss of insulin because of reduced solubility. The particle size of liposomes increased as the pH increased owing to the change of surface charge. In general, natural phospholipids such as soybean or egg lecithins are negatively charged. When pH goes up, the charge density of phospholipids will raise correspondingly, which results in more electrostatic repulsion that is unfavorable to form small liposomes. Higher organic-aqueous phase ratio resulted in higher entrapment efficiency as observed from Figure 1D because increasing the organic phase was beneficial to the formation of fine emulsions, which would lead to fine insulin dispersion in the mixed lipids. Figure 1 The effects of formulation variables on entrapment efficiency and particle size. (A) Ratios of lipid/cholesterol and (B) drug/lipid, (C) pH upon hydration, and (D) organic/aqueous ratio of phase.

The high quality of the AAO obtained makes it very promising for nanofabrication. Silicon nanowires Silicon nanowire (NW) arrays are widely studied nowadays because of their potential applications in microelectronics or detectors. Among the fabrication techniques, CVD is favoured. However, conventional techniques do not allow a good control on the position nor the homogeneity of the wires. Highly organised porous ITF2357 molecular weight alumina has been successfully used

as a template for the catalytic CVD growth of defect-free array of Si NW. For this, alumina is build on a <100> Si conductive wafer as described previously. Mould and anodization characteristics are adapted to the desired diameters, period and thickness of the future Si NW arrays. Energy dispersive X-ray analysis was performed on the cross section of the NW array before removal of the alumina template. High voltage of the electron

beam of an GDC-0449 molecular weight ultra-Zeiss SEM was settled at 5 kV, and the sample was positioned at a working distance (WD) of 7 mm. Atoms of aluminium, oxygen, gold and silicon were mapped. Figure 3a,b,c,d,e shows the map of these atoms, and an intensity profile of Si, Al and O atoms is presented in Figure 3f. As expected, silicon is present in the template’s pores, the template is composed of aluminium and oxygen, and gold is present at the upper end of the silicon wires. click here Figure 3 Energy dispersive X-ray (EDX) analysis of Si NW. (a) SEM image of the cross section, (b) aluminium cartography, (c) oxygen cartography, nearly (d) silicon cartography, (e) gold cartography and (f) profile counts of oxygen, aluminium and silicon, along the arrow of (a). The EDX analyses were conducted at 5-kV high voltage

and for a 7-mm WD. Top view of silicon wires are reported in Figure 4a, showing a good filling rate around 80%. Different periods and diameters for the NWs are shown in Figure 4b,c,d,e, before or after the removal of the catalyst. One can notice the very good quality of the triangular lattice as well as the smooth cylindrical surface of the wires. On the foreground of Figure 4b, a few disordered wires have grown above the hexagonal array. Those wires are due to gold droplet coalescence above the alumina array. Indeed, when the wires reach the top surface of the alumina template, the gold droplets coalesce and nanowires with a bigger diameter grow above the array. As the <111> direction is the prefer orientation for NW growth [35] and because the growing conditions widely change outside the alumina, these nanowire kink with an angle of 54.7°. Besides, according to the homogeneity of the catalyst deposition, a difference in the speed of growth of the wires can be observed over the substrate between wires. It leads to small differences in the wires’ height, as shown in Figure 4d.

However, profiles AMP-CIP-NAL and CIP-NAL were observed in five out of seven zones (Table 1). Phage typing Among the 40 isolates, 11 different phage types were observed: 6a (n = 19), 1 (n = 8), 14c (n = 2), 21 (n = 2), 4b (n = 1), 13 (n = 1), 35 (n = 1), 37 (n = 1), 911 (n = 1), three atypical lytic patterns, and one untypable (Figure 1). Significant variation in phage susceptibility was observed. Susceptibility to 11 typing phages differentiated the two most common phage types (6a and 11). Phage types 21, 35, & 37 differed by their

susceptibility to four to six of the typing phages. Pulsed-field gel electrophoresis typing NCT-501 Seven different previously known XbaI PFGE patterns [JEGX01.0158 (n = 16), JEGX01.0002 (n = 7), JEGX01.0019 (n = 6), JEGX01.0167 (n = 2), JEGX01.0008 (n = 1), JEGX01.0325 (n = 1), JEGX01.0653 (n = 1)] were identified among the 40 isolates in addition to six patterns which were new to the PulseNet USA database. The isolates were further subtyped using a second enzyme, BlnI, which revealed seven different previously known BlnI PFGE patterns [JEGA26.0010 GM6001 order (n = 31), JEGA26.0017 (n = 1), JEGA26.0058 (n = 1), JEGA26.0067 (n = 1), JEGA26.0068 (n = 1), JEGA26.0120 (n = 1), JEGA26.0155 (n = 1)] and two additional patterns which were new to the PulseNet USA database. In total 14 XbaI/BlnI PFGE pattern combinations were detected (Figure 1). Multiple-locus variable-number

tandem repeat analysis The 40 strains generated seven different MLVA types. Variation was observed at loci VNTR-1 (n = 4), VNTR-2 (n = 2), VNTR-5 (n = 8) and VNTR-9 (n = 2).

The most common profile (5-5-1-10-3-3-11) contained 20 isolates. (Figure 1). Three isolates displayed variation both at loci VNTR-1 and VNTR-5 (allelic profile: 4-5-1-10-3-3-10), one isolate displayed variation in three loci VNTR-1, VNTR-5 and VNTR-9 (allelic profile: 8-5-1-10-2-3-7), one isolated showed variation in four loci VNTR-1, VNTR-2, VNTR-5 and VNTR-9 (allelic profile: 6-6-1-10-2-3-6), before and the remaining 15 isolates exhibited variation only at locus VNTR-5 (Figure 1). Analysis of the composite data set Composite analysis based on PFGE and MLVA data grouped the 40 isolates into 22 genotypes. Seven genotypes contained multiple isolates; 15 genotypes were comprised of a single isolate. No single genotype was responsible for either gastroenteritis or bacteremia among Thai patients. In Five instances, the same genotype was isolated from both stool and blood in different zones and time periods (Figure 1). Discussion Previous studies selleck screening library indicated that infection with Salmonella serovar Enteritidis was a statistically significant risk factor for bacteremia among Thai patients [7, 17, 18]. The goal of this study was to characterize Salmonella serovar Enteritidis isolates obtained from blood and stool specimens in Thailand in a spatial and temporal context and determine if a particular clone is associated with bacteremia based on the information described by Hendriksen et al.[7].

For each PAH species three samples were prepared and each sample was measured three times. Results Microscopy and DLS of Vesicle Solutions Phase-contrast and fluorescence microscopy of vesicle preparations with a 1:200 ratio of pyrene/decanoic acid are shown in Fig. 1. PAHs are fluorescent under UV light and incorporation LB-100 can therefore be determined by fluorescence microscopy. The vesicles were heterogeneous, ranging from 100 nm to 5 μm with a mean of 200 nm. Vesicles were largely spherical at first, but tubular vesicles dominated a few minutes later after attaching to the surface of the glass slide

or coverslip (Apel et al., 2002). Incorporation of PAHs did not influence mean vesicle sizes or the size distribution. Vesicles of pure decanoic acid disappeared at pH 7.6, but incorporation of 1-hydroxypyrene had a modest stabilizing effect, with vesicles still apparent at pH 8.1. Fig. 1 Phase-contrast a and fluorescence b microscopy of 0.3 mM pyrene + 59.7 mM DA (200:1) + FA mix. Tubular structures are formed by vesicles adhering to the coverslip or glass slide. Pyrene fluorescence is clearly localized to the membrane PAH Incorporation UV Fluorescence microscopy showed that PAH derivatives could be incorporated into the membrane in different concentrations. Pyrene could be incorporated in a 1:200 NU7026 supplier mole ratio with decanoic acid while 1-hydroxypyrene (Fig. 2-a) and

9-anthracene carboxylic acid (Fig. 2-b) were incorporated up to 1:10 ratios. Only 1:50 ratios of 9-fluorenone and 1-pyrene carboxaldehyde Roflumilast could be incorporated before macroscopic aggregates formed or PAHs precipitated. In some cases (1-pyrene carboxaldehyde, 9-fluorenone), 10 freeze-thaw click here cycles using liquid nitrogen to homogenize the bilayers prevented the formation of macroscopic aggregates. Fig. 2 Fluorescence microscopy of a

5.5 mM 1-hydroxypyrene + 54.5 mM DA (1:10) + FA mix and b 5.5 mM 9-anthracene carboxylic acid + 54.5 mM DA (1:10) + FA mix samples. Fluorescence is clearly localized to the membrane boundary CVC Measurements Conductimetric titration was performed on vesicle preparations to determine CVC values. Figure 3 shows CVC measurements for a 1:10 1-hydroxypyrene / decanoic acid sample, the measured CVC values (Fig. 4) are in the range of previously published values (Monnard and Deamer 2003; Cape et al. 2011). Of the PAH derivatives that were tested, only 1-hydroxypyrene showed a significant reduction in CVC, forming fluffy macroscopic aggregates around the measured CVC value. All other samples became completely clear when diluted below the measured CVC values. Fig. 3 Conductimetric titration of a 5.5 mM 1-hydroxypyrene + 54.5 mM DA (1:10) + FA mix sample. The measured CVC is 21.6 mM and this coincides with the formation of macroscopic fluffy aggregates Fig. 4 CVC values determined by conductimetric titration. CVC’s are: 24.00 ± 0.7 mM for 60 mM DA + FA mix samples, 24.3 ± 2.

2002; Yonkers et al. 2003; Robinson and Sahakian 2008; Burcusa and Iacono 2007; Hardeveld et al. 2010), and this was confirmed by our results. Sickness absence due to adjustment disorders

and distress symptoms were the most frequently diagnosed recurrent disorders, which makes the MEK162 clinical trial social and economic burden of these disorders considerable despite their shorter duration. Recurrence of major depressive disorder in specialized mental healthcare settings is high (60% after 5 years, 67% after 10 years and 85% after 15 years) and seems lower in the general selleckchem population (35% after 15 years) (Hardeveld et al. 2010). The RD of sickness absence due to anxiety and depressive symptoms was high, amounting to 37.9 and 43.6, respectively, per 1,000 person-years. Recurrent sickness absence due to other mental disorders Our results show that sickness absence due to CMDs predisposes to sickness absence due to other mental disorders.

After sick leave with depressive symptoms, the RD of sickness absence due to other mental disorders was 68.7 per 1,000 person-years, and after anxiety disorders it was 56.2 per 1,000 person-years. Depression is associated with a high risk of long-term sickness absence and work disability (Bültmann et al. 2006, 2008; Lerner and Henke 2008). Our results add that after return to work, the risk of recurrent sickness absence due to CMDs has also increased. After an initial episode of sickness absence due to distress, the RD of recurrent sickness absence due to other mental disorders Tariquidar research buy was 48.0 per 1,000 person-years, and after an initial episode with adjustment disorders, it was 45.0 per 1,000 person-years. Determinants of recurrent sickness absence due to CMDs The number of previous episodes and subclinical residual symptoms appears to be the most important predictors of recurrence of major depressive disorder (MDD). Gender, civil status and socioeconomic status seem not related to the recurrence of MDD (Burcusa and Iacono 2007; Hardeveld et al. 2010). We investigated the risk of recurrent sickness absence due to CMDs (same or another mental disorder)

by gender, age, marital status and salary scale. Sickness absence due to CMDs occurred more often in women, and this has been reported earlier (Bijl et al. 2002; Hensing and Arachidonate 15-lipoxygenase Wahlstrom 2004). Mueller et al. (1999) reported that women had a higher recurrence of a major depressive disorder than men. It is interesting to note that this gender difference seems to disappear after an initial episode of sickness absence due to CMDs. This finding might be biased by the longer episodes of sickness absence found in women than in men (Blank et al. 2008), but this merits further investigation. In men, depressive symptoms were related to higher recurrence of sickness absence due to CMDs than distress symptoms and adjustment disorders.

The plasmid pRmM57 (nodC::lacZ fusion) [14] was used to test the expression of the nodC gene and pGD499 (npt::lacZ fusion) [15] to test the expression of the constitutive kanamycin resistance gene. The pMPTR4 plasmid is a pMP220 [24] derivative CHIR-99021 in vivo in which an EcoRI fragment of 0.6 kb harbouring

the intergenic fadD-tep1 region was cloned to create a tep1::lacZ transcriptional fusion. The pGUS3 plasmid containing an nfeD::gusA fusion was used in competition assays [25]. Triparental bacterial matings were performed using pRK2013 as helper plasmid [26]. E. coli was grown routinely at 37°C in Luria-Bertani medium (LB) [27]. S. meliloti strains were grown at 30°C in TY complex medium [28] or in defined minimal medium (MM) [29]. Growth was click here determined regularly in a spectrophotometer measuring the absorbance at 600 nm. Glucosamine and N-acetyl glucosamine were obtained from Sigma-Aldrich. Construction of a S. meliloti tep1 mutant A null-mutant in ORF SMc02161 was obtained by allelic exchange. Firstly, a 3.6 kb SacI fragment

containing this ORF was subcloned from the fadD containing cosmid pRmersf442 [2] into pUC18 [30] to give pTrans1. To disrupt the ORF SMc02161 in pTrans1, a 2 kb SmaI fragment containing the streptomycin/spectinomycin Torin 2 price resistance cassette from pHP45Ω [31] was inserted into a unique EcoRV site to give pTrans2. Next, the SacI fragment containing the disrupted ORF was treated with T4 DNA polymerase (Roche Biochemicals) to make blunt ends and then cloned into the SmaI site of the suicide vector pK18mobsac [32] to give pTrans3. This vector was then used for allelic exchange by introducing it into the S. meliloti strains GR4, and the fadD mutant QS77 via triparental mating, and selecting putative mutants by streptomycin/spectinomycin resistance and sensitivity to sucrose. The resulting SMc02161 mutant GR4T1, and double fadD, SMc02161 mutant QSTR1 were confirmed by Southern hybridization with a specific probe. Construction of a S. meliloti nodC mutant To obtain a nodC mutant in S. meliloti, a fragment was amplified from the chromosomal DNA of S. meliloti GR4 by PCR using 5′-CAGATTCAAGGTCACGAAGTGGCTAAC-3′

Digestive enzyme and 5′-ATAAGCTTGTGACAGCCAGTCGCTATTG-3′ as forward and reverse primers respectively. An EcoRI-PstI fragment of 1.5 kb derived from the PCR product and containing half of the nodB gene and most of the nodC gene was subcloned into pUC18 [30] to obtain pGRC8. To disrupt nodC, pGRC8 was digested with SalI and treated with Klenow (Roche Biochemicals) to create blunt ends. Next, the 2 kb SmaI fragment containing the streptomycin/spectinomycin resistance cassette from pHP45Ω [31] was introduced to give pNC150. The 3.5 kb EcoRI-PstI fragment from pNC150 containing the disrupted nodC gene was inserted into EcoRI-PstI digested pK18mobsac [32] to give pNC200. This suicide vector was then used to obtain the S. meliloti nodC mutant GR4C5, which was confirmed by Southern hybridization.