This study

investigated the effect of trace iron conditio

This study

investigated the effect of trace iron conditions on the growth of these two species, as well as their response to iron sequestration by chelators. Metal analysis by ICP-MS revealed high residual concentrations (0.12 μM) of iron in the chemically defined medium used in spite of the absence of added iron and demonstrated the requirement for deferration and confirmatory trace Fe analysis. The iron contamination from other medium constituents could be successfully reduced to <0.02 μM in batch processes using an insoluble chelating resin. Cu concentrations were also significantly reduced in the extracted chemically defined medium, but significant recovery of growth could be see more achieved by supplementation of the extracted medium with Fe only. Both C. albicans and C. vini were found to require Cytoskeletal Signaling inhibitor approximately 0.5 μM added iron for complete unrestricted growth in the extracted chemically defined medium, but differed in their abilities to grow at reduced iron concentrations. The observed differences between C. albicans and C. vini were consistent with the different environments these

respective yeast species typically colonize or invade. Grape musts and wines, in which C. vini typically appears as a spoilage yeast, generally have high Fe concentrations of between 30 and 200 μM (Ough et al., 1982). The predominantly reducing environment and the low pH of grape musts and wines also favour the formation of the more soluble free ferrous species, and this Fe would be expected to have a higher bioavailability (Howard,

1999). In sharp contrast, the ecological niches that C. albicans can colonize or invade in relation to human pathogenesis are highly limiting for Fe (Weinberg, 1999). Desferrioxamine and deferiprone are two chelators used clinically very to relieve the Fe overload associated with certain human haematological disorders such as thalassaemia (Chaston & Richardson, 2003; Franchini, 2006). Desferrioxamine failed to inhibit both C. albicans and C. vini. Deferiprone did not inhibit C. vini while leading to a slightly increased lag phase in C. albicans. However, the observed differences between C. albicans and C. vini persisted in their growth response in the presence of lactoferrin. Lactoferrin is a major component of the mammalian innate immune system (Actor et al., 2009) and one of the vertebrate host defence Fe chelators, which is present in mucosal secretions (Gonzalez-Chavez et al., 2009). Lactoferrin, at the physiologically relevant concentration of 0.25 mg mL−1 and at pH 4.5, and thus, representative of the vaginal environment (Novak et al., 2007), only led to a transient inhibition of C. albicans, but inhibited the growth of C. vini over the incubation period. The results are in agreement with the lack of observed pathogenicity of C. vini and its greater susceptibility to iron restriction, while the pathogenicity of C.

This study

investigated the effect of trace iron conditio

This study

investigated the effect of trace iron conditions on the growth of these two species, as well as their response to iron sequestration by chelators. Metal analysis by ICP-MS revealed high residual concentrations (0.12 μM) of iron in the chemically defined medium used in spite of the absence of added iron and demonstrated the requirement for deferration and confirmatory trace Fe analysis. The iron contamination from other medium constituents could be successfully reduced to <0.02 μM in batch processes using an insoluble chelating resin. Cu concentrations were also significantly reduced in the extracted chemically defined medium, but significant recovery of growth could be selleck chemical achieved by supplementation of the extracted medium with Fe only. Both C. albicans and C. vini were found to require DZNeP ic50 approximately 0.5 μM added iron for complete unrestricted growth in the extracted chemically defined medium, but differed in their abilities to grow at reduced iron concentrations. The observed differences between C. albicans and C. vini were consistent with the different environments these

respective yeast species typically colonize or invade. Grape musts and wines, in which C. vini typically appears as a spoilage yeast, generally have high Fe concentrations of between 30 and 200 μM (Ough et al., 1982). The predominantly reducing environment and the low pH of grape musts and wines also favour the formation of the more soluble free ferrous species, and this Fe would be expected to have a higher bioavailability (Howard,

1999). In sharp contrast, the ecological niches that C. albicans can colonize or invade in relation to human pathogenesis are highly limiting for Fe (Weinberg, 1999). Desferrioxamine and deferiprone are two chelators used clinically Urease to relieve the Fe overload associated with certain human haematological disorders such as thalassaemia (Chaston & Richardson, 2003; Franchini, 2006). Desferrioxamine failed to inhibit both C. albicans and C. vini. Deferiprone did not inhibit C. vini while leading to a slightly increased lag phase in C. albicans. However, the observed differences between C. albicans and C. vini persisted in their growth response in the presence of lactoferrin. Lactoferrin is a major component of the mammalian innate immune system (Actor et al., 2009) and one of the vertebrate host defence Fe chelators, which is present in mucosal secretions (Gonzalez-Chavez et al., 2009). Lactoferrin, at the physiologically relevant concentration of 0.25 mg mL−1 and at pH 4.5, and thus, representative of the vaginal environment (Novak et al., 2007), only led to a transient inhibition of C. albicans, but inhibited the growth of C. vini over the incubation period. The results are in agreement with the lack of observed pathogenicity of C. vini and its greater susceptibility to iron restriction, while the pathogenicity of C.

The regulatory mechanism for PHB biosynthesis in B thuringiensis

The regulatory mechanism for PHB biosynthesis in B. thuringiensis and diverse Bacillus species is still poorly understood. We now report that disruption of the sigH gene or the gene encoding the master sporulation transcription factor Spo0A severely impaired PHB accumulation in B. thuringiensis. Complementation

of the spo0A mutation with the spo0A gene restored PHB accumulation. We have found that the requirement of Spo0A for PHB accumulation is independent of the transition state regulator AbrB and of loss of sporulation ability. We also show that Spo0A is required for the expression of three genes involved in PHB biosynthesis. These findings have uncovered a new role of Spo0A in the regulation of stationary-phase-associated cellular events. Poly(3-hydroxybutyrate) (PHB) is a polyester accumulated by numerous bacteria as an intracellular carbon and energy storage Selleckchem GDC-0068 material in response to nutritional stress (for recent reviews, see Waltermann & Steinbuchel, 2005; Jendrossek, 2009). A cluster of five PHB-related genes, that is phaP (encoding a phasin protein),

phaQ (a repressor of phaP expression), phaR (a subunit of PHB synthase), phaB (acetoacetyl-CoA reductase), and phaC (a subunit of PHB synthase), was previously identified and characterized from the PHB-producing Bacillus megaterium (McCool & Cannon, 1999; Lee et al., 2004). The PHB synthase of B. megaterium requires both the AP24534 clinical trial PhaR subunit and the PhaC subunit for activity (McCool & Cannon, 2001). The genetic organization of Bacillus thuringiensis counterparts of these five PHB-related genes is the same as that of B. megaterium. However, the regulatory mechanism for PHB biosynthesis

in diverse Bacillus species is poorly understood. The master transcription factor Spo0A is a response regulator that plays a central role in the initiation of sporulation of Bacillus subtilis and other Bacillus species (for recent reviews, see Piggot Adenosine & Hilbert, 2004; Stephenson & Lewis, 2005). Bacillus subtilis Spo0A is also known to be involved in controlling other cellular events, such as biofilm formation (Hamon & Lazazzera, 2001), development of competence for DNA uptake (Hahn et al., 1995), cannibalism (Gonzalez-Pastor et al., 2003), cold and heat resistance (Mendez et al., 2004), bacilysin biosynthesis (Karatas et al., 2003), and extracellular-degradative enzyme production (Kodama et al., 2007). Phosphorylated Spo0A is capable of binding to a specific DNA sequence, called the 0A box (5′-TGNCGAA-3′), found in promoter regions of its target genes to repress or stimulate transcription (Molle et al., 2003). The pathway to Spo0A phosphorylation involves a multicomponent phosphorelay, in which the phosphate group is initially transferred from multiple sensor histidine kinases to Spo0F, then to Spo0B, and eventually to Spo0A.

While overall tone-evoked response magnitudes were comparable bet

While overall tone-evoked response magnitudes were comparable between the two structures, tone signal : noise was significantly greater within the OT than in the PCX. Panobinostat research buy No effect of tone frequency (1–55 kHz) was found within either structure, with most units being narrowly tuned to a single frequency. These results suggest that a major portion of odor-evoked output from the olfactory bulb (i.e. that entering the OT and PCX) is subject to auditory sensory input in a manner that may modulate odor information processing,

odor-guided behaviors and perception. “
“Behavioral rhythms induced by methamphetamine (MAP) and daily restricted feeding (RF) in rats are independent of the circadian pacemaker in the suprachiasmatic nucleus (SCN), and have been regarded to share a common oscillatory mechanism. In the present study, in order to examine the responses of brain oscillatory systems to MAP and RF, circadian rhythms in clock gene, Period2, expression were measured in several brain ALK inhibitor areas in rats. Transgenic rats carrying a bioluminescence reporter of Period2-dLuciferase were subjected to either daily injection

of MAP or RF of 2 h at a fixed time of day for 14 days. As a result, spontaneous movement and wheel-running activity were greatly enhanced following MAP injection and prior to daily meal under RF. Circadian Per2 rhythms were measured in the cultured brain tissues containing one of the following structures: the olfactory bulb; caudate-putamen; parietal cortex; substantia nigra; and SCN. Except for the SCN, the circadian Per2 rhythms in the brain tissues were significantly phase-delayed by 1.9 h on average in MAP-injected rats as compared with the saline-controls. On the other hand, the circadian rhythms outside the SCN were significantly phase-advanced by 6.3 h on average in rats under RF as compared with those under ad libitum feeding. These findings indicate that the circadian rhythms in specific brain areas of the central dopaminergic system respond differentially to MAP

injection and RF, suggesting that different oscillatory mechanisms in the brain underlie the MAP-induced behavior and pre-feeding activity under RF. “
“Glutamate is the major excitatory neurotransmitter of the central nervous system in vertebrates. Excitotoxicity, caused by over-stimulation why of the glutamate receptors, is a major cause of neuron death in several brain diseases, including epilepsy. We describe here how behavioural seizures can be triggered in adult zebrafish by the administration of kainate and are very similar to those observed in rodent models. Kainate induced a dose-dependent sequence of behavioural changes culminating in clonus-like convulsions. Behavioural seizures were suppressed by DNQX (6,7-dinitroquinoxaline-2,3-dione) dose-dependently, whilst MK-801 (a non-competitive NMDA receptor antagonist) had a lesser effect.

In addition, we performed receiver operating characteristic (ROC)

In addition, we performed receiver operating characteristic (ROC) analysis to assess the accuracy of EBV DNA load as a predictive marker of lymphoma [as estimated by the area under the curve (AUC)]. The optimal cut-off value of EBV DNA load for differentiating patients at risk of lymphoma from other patients was determined as the point of the ROC curve with the shortest distance to the 100/100% sensitivity/specificity angle (upper left corner) [i.e. lowest value for the term (1 – sensitivity)2 + (1 – specificity)2, assuming equal costs of false positive and false negative results]. The sensitivity, specificity and OR for developing lymphoma were then provided for the identified

cut-off point. All statistical analyses were performed using sas 9.2 (SAS Institute Inc., VEGFR inhibitor Cary, NC). EBV DNA was positive in PBMC samples from all lymphoma cases collected over the 3 years preceding the Obeticholic Acid diagnosis, while it was positive in 78 to 81% of samples from controls collected during the same period of time (Fig. 1a). Interestingly, eight of the 37 controls had undetectable EBV loads in PBMC1 while none of the 20 cases had undetectable EBV loads in PBMC1 (P = 0.04) (Fig. 1a). EBV load in PBMCs measured a median of 10 months before diagnosis was associated with an increased risk of B lymphoma [OR 2.48 (95% CI 1.16; 5.32) per increase in EBV

load of 1 log copies/106 PBMCs] (Table 2). Similar results were obtained when the OR was adjusted for CD4 cell count nadir instead of CD4 cell count at sample date (OR 2.33; 95% CI 1.12; 4.81). The OR associated with EBV load quantified in a sample collected earlier (median of 24 months before diagnosis) was of borderline significance, probably because of a smaller number of PBMC samples available for that period. When we restricted the analysis to the patients with a CD4 cell count > 300 cells/μL, the median EBV load was still lower in controls (median 2.69) than in cases (median 3.63), mainly because four out of 14 controls had undetectable EBV load vs. none of

seven cases. EBV DNA was more often detectable (> to EBV PCR threshold value or detectable but < to EBV PCR threshold value) in sera from cases than in sera from controls (with 24 to 25% positive detection in the last 3 years for cases vs. 8 to 10.5% for Unoprostone controls) (Fig. 1b); however, this difference was significant only for serum 2 samples (collected a median of 15.3 months before the diagnosis of lymphoma) (Table 2). EBV DNA was positive in PBMC samples from all tested cases during the 3 years preceding the diagnosis of cerebral lymphoma, but it was also positive in 87 to 94% of controls during the same period (Fig. 2a). EBV DNA was not more often detectable (> threshold or detectable < threshold) in sera from cases than in sera from controls (with 0 to 23.1% positive detection in the last 3 years for cases vs. 4.8 to 12% for controls) (Fig. 2b).

1b) A terminator was predicted by webgester downstream of yaaH o

1b). A terminator was predicted by webgester downstream of yaaH or, in antisense at the same position, downstream of mog. This suggests that yaaW is most likely organized as operon yaaIWH in EHEC and transcribed from the yaaI-promoter and terminated downstream of yaaH.

Interestingly, data from genexpdb indicate that htgA and yaaW are expressed differentially in E. coli strains under certain experimental conditions (see Table 1), clearly prohibiting htgA synonymizing with yaaW, which has been performed in some databases. HtgA and YaaW were expressed in EDL933 using a plasmid that generates concomitant myc and His-tag fusions. Proteins were prepurified using the his-tag and detected on Western blots using the myc-tag. YaaW (30 kDa) was detectable, but no band for HtgA was found (Fig. 2), which is in accordance with Narra et al. Alpelisib nmr (2008). Thus, the protein might be unstable Selleck Gefitinib and difficult to discover. Missiakas et al. (1993) presented a 21-kDa gene product by 35S-labeling, which is a more sensitive approach. Previous work always used a double knockout mutant. We created strand-specific deletion mutants for the first time, in which only htgA or yaaW was interrupted (Fig. 3). The annotated htgA-start codon is CTG, which is quite rare for bacteria. The next GTG is more likely to be the start codon. Counting from there, htgA has 525 bp (or 174 amino acids); our htgA-knock out terminates either product. By

introducing a single-point mutation to create a stop in one frame, we minimized the disturbance of the other, as the mutations are synonymous in the latter (Tunca et al., 2009). For the first time, it was possible to distinguish

effects of ΔhtgA from ΔyaaW. Both mutants showed no difference in their growth compared with wild type at 37 °C or after temperature shift from 30 °C to 45 °C (Fig. 4a). As no heat shock phenotype of ΔhtgA could be confirmed (as found before, Nonaka et al., 2006), htgA should no longer be annotated as heat shock gene. In minimal medium, biofilm formation of ΔhtgA or ΔyaaW was reliably increased when incubated for 48 h at 37 °C (Fig. 4b). This is in accordance with Domka et al. (2007), who found a threefold increase in biofilm formation for E. coli K12 in a htgA/yaaW double mutant. We speculate ALOX15 that the higher increase compared with our experiments might be due to additive effects of both genes in the double mutant compared with each single one. We therefore suggest to rename htgA to mbiA (modifier of biofilm). As no difference in growth could be found, we measured the metabotypes. Metabolite changes could still be detectable even though they may not manifest in growth (Raamsdonk et al., 2001). ΔhtgA, ΔyaaW, and wild type were subjected to nontargeted metabolomics using ICR-FT/MS. Indeed, twenty-two different metabolites (putatively annotated, see Table S3) between the strains were found significantly changed (P ≤ 0.01).

We demonstrate that tet(S), identical to tet(S)

We demonstrate that tet(S), identical to tet(S) DNA Damage inhibitor found on the enterococcal conjugative transposon Tn6000, is responsible for the observed resistance. The gene is located on a small, low copy number plasmid and is flanked by IS1216 elements. The tet(S) gene is capable of excising from the plasmid together with one of the IS1216 elements. The plasmid contains a putative toxin/antitoxin system related to relBE. Deletion of the toxin, relE, did not result in plasmid instability but did increase the fitness of the mutant compared to the wild-type

strain. “
“In the presence of vaporized p-cresol, Pseudomonas alkylphenolia KL28 forms specialized aerial structures (SAS). A transposon mutant of strain KL28 (C23) incapable of forming mature SAS was isolated. Genetic analysis of the C23 mutant revealed the transposon insertion in a gene (ssg) encoding a putative glycosyltransferase, which is homologous to the Pseudomonas aeruginosa PAO1 PA5001 gene. Deletion of ssg in KL28 caused the loss of lipopolysaccharide O antigen and altered the composition of the exopolysaccharide. Wild-type KL28 produced a fucose-, glucose- and mannose-rich exopolysaccharide, while the mutant exopolysaccharide completely lacked fucose and mannose, resulting in an exopolysaccharide with glucose as the major component. The mutant

strain showed reduced surface spreading, pellicle and biofilm formation, probably due to the cumulative effect of lipopolysaccharide truncation and altered exopolysaccharide composition. ABT-199 concentration Our results show that the ssg gene of KL28 is involved in both lipopolysaccharide and exopolysaccharide biosynthesis and thus plays an important role in cell surface properties and cell–cell interactions of P. alkylphenolia. Pseudomonas is a genus

of Gammaproteobacteria, capable of thriving in diverse environments ranging from hydrocarbon-contaminated water and soil to plant Reverse transcriptase and animal tissues (Rocchetta et al., 1999; Gibson & Parales, 2000; Stover et al., 2000; Ramos et al., 2001). Its ecological success stems in part from the outer cell membrane, which mainly consists of lipopolysaccharide. Lipopolysaccharide mediates interactions with the environment, reduces outer membrane permeability thereby increasing resistance to agents such as antibiotics and plays a critical role in cell motility, adhesion and attachment to a substratum/surface (Nikaido & Vaara, 1985; King et al., 2009; Lindhout et al., 2009). In addition to lipopolysaccharide, the exopolysaccharide that is secreted by bacteria also plays a physical role in cell–cell and cell–substratum attachment, thereby aiding the establishment of multicellular communities such as biofilms (Sutherland, 2001).

5, first row) An analogous pattern was seen for CagA-∆N-transfec

5, first row). An analogous pattern was seen for CagA-∆N-transfected cells (Fig. 5, second row). In cells transfected with CagA-∆C, an evident cytoplasmic distribution of CagA (red) was seen. On the other hand, hardly any GM1 co-localized signal was detected in the plasma membrane (Fig. 5, third row). These observations support that CagA CTD

containing the EPIYA repeats is important for CagA tethering to the membrane raft microdomains. Several lines of evidence suggest that tethering of CagA to membrane-associated components is crucial for its subsequent functions: (i) following H. pylori infection, translocated CagA binds to raft-associated SFKs and undergoes tyrosine phosphorylation in the EPIYA motifs (Stein et al., 2002); (ii) CagA associates with the epithelial tight-junction scaffolding protein ZO-1 (Amieva selleck chemicals llc et al.,

2003); (iii) CagA interacts with membrane-externalized phosphatidylserine (PS) to initiate its entry into cells in epithelial cells (Murata-Kamiya et al., 2010); and (iv) depletion of cellular cholesterol blocks internalization of CagA into host cells (Lai et al., 2008). Of note, those identified CagA partners including c-Src (Lai et al., 2008), ZO-1 (Nusrat et al., 2000), and PS (Pike et al., 2002) have been click here shown to associate with DRMs. In addition to CagA, the H. pylori TFSS component CagL was found to bind and activate α5β1 integrin (Kwok et al., 2007), which is abundantly localized in cholesterol-rich microdomains (Leitinger & Hogg, 2002). This interaction was further demonstrated to trigger the delivery of peptidoglycans

across the cell membrane, resulting in the induction of NF-κB and IL-8 responses in the epithelial cells (Hutton et al., 2010). Collectively, these results suggest that TFSS, as well as internalized CagA, can reside primarily in cholesterol-enriched microdomains, where they interact old with various signaling molecules, inducing multiple cellular responses, including IL-8 secretion, cell motility, proliferation, and polarity. Our study shows that the CTD of CagA containing EPIYA repeats, either ABC-type (Western type) or AABD-type (East Asian type), is important for raft tethering and for IL-8 induction in AGS cells. Mutants that lacked the CTD lost their normal ability to associate with membrane rafts, in accord with the finding from Higashi et al. (Higashi et al., 2005). In polarized madin-darby kidney cells (MDCK), however, the N-terminal rather than the C-terminal region of CagA tethered to the cell–cell junctions (Bagnoli et al., 2005). Of note, a recent report using polarized and non-polarized cells to demonstrate that CagA utilized at least two distinct mechanisms for membrane association, relying on the status of epithelial polarity (Murata-Kamiya et al., 2010).

Stocks are placed in these hospitals and consumption and expirati

Stocks are placed in these hospitals and consumption and expiration GDC-0068 order dates are checked twice a year by WHO. WHO keeps an emergency stock of drugs at its headquarters in

Geneva, whereas for regular distribution to major DECs in need of large amounts, WHO has the collaboration of Médecins Sans Frontières Logistique (Bordeaux, France), which provides storage facilities and shipment services. Drugs are shipped either by express courier, by air or boat depending on the urgency and circumstances. During the period 2000 to 2010, 94 HAT cases diagnosed in non-DECs were reported. Seventy-two percent of them corresponded to the Rhodesiense form of the disease (Table 2), whereas 28% corresponded to the Gambiense form (Table 3). Among Rhodesiense HAT cases, 82% were diagnosed in first stage and 18% were diagnosed in second stage. Among Gambiense HAT cases, 23% cases were diagnosed in first stage,

while 77% were diagnosed in second stage. Ninety-three percent of the HAT Rhodesiense cases diagnosed were foreigners traveling to endemic areas for a short period of time. This category includes tourists (60) and soldiers (2). Rangers working in wildlife areas make up the remaining 7%. Forty-two percent of the HAT Gambiense cases diagnosed were expatriates living in endemic phosphatase inhibitor library countries for extended periods, mostly for business, including forest activities (9), but also as staff of the United Nations (1) or as religious missionaries (1). Fifty-eight percent were nationals from DECs, living in the non-DEC of diagnosis for political reasons [ie, refugees from Democratic Republic of Congo (DRC) and from Sudan although based Acyl CoA dehydrogenase in Uganda (5)] or for economic reasons [ie, migrants from DRC (3), Cameroon (3), Angola (2), and Equatorial Guinea (2)]. HAT cases were diagnosed in non-DECs

in the five continents (Figure 1). Forty-three percent of the cases were diagnosed in Europe and 23% in North America. South Africa is the non-DEC diagnosing the highest number of Rhodesiense HAT imported cases, 37% of the total. This is probably due to its proximity to DECs with famed protected areas and game reserves (GR), but also because it often represents the first step in health care seeking for acute health problems in south and east African countries. In the second line are countries that hold historical or economic links with DECs and whose citizens travel more often to DECs for tourism. These include the United States (25% of cases) and the UK (15% of cases). Other European countries account for 18% of cases [The Netherlands (5), Belgium (2), Italy (2), Sweden (1), Norway (1), Germany (1), Poland (1)]. Finally, 5% of the remaining cases were diagnosed in India, Brazil, and Israel.

, 2007) First identified in the phytopathogen Agrobacterium tume

, 2007). First identified in the phytopathogen Agrobacterium tumefaciens,

these polysaccharides are essential for survival and infection in several Eukaryote – microbe interactions including legume-rhizobia symbioses between Sinorhizobium meliloti, Sinorhizobium fredii, Mesorhizobium loti, and their respective host legumes (Dylan et al., 1986; Geremia et al., 1987; Ielpi et al., 1990; Bhagwat et al., 1992; Breedveld & Miller, 1994; D’Antuono et al., 2005; Crespo-Rivas et al., 2009). CβG of Brucella abortus are essential for intracellular survival and replication by preventing phagosome–lysosome fusions (Arellano-Reynoso et al., 2005). In a similar fashion, CβG produced by the phytopathogen Xanthomonas campestris pv. campestris (Xcc) are necessary for bacterial survival on tobacco leaves where they suppress systemic learn more plant immune responses (Rigano et al., 2007). In S. meliloti, NdvB and NdvA are responsible for CβG synthesis and translocation to the periplasmic

space, respectively, roles that are essential for nodulation (Breedveld & Miller, 1994). The effects of mutated ndvA and ndvB may not be direct however and could be related to a combination of pleiotropic disturbances associated with the absence of CβG, hypo-osmotic adaptation, motility, attachment BTK inhibitor in vitro and infection (Dylan et al., 1990). As CβG are present in bacteroids (Gore & Miller, 1993) of Bradyrhizobium japonicum, CβG might also be important within functional nodules. Rhizobium (Sinorhizobium) sp. strain NGR234 (hereafter

NGR234) has the largest known host range of legumes (Pueppke & Broughton, 1999). NGR234 synthesizes cyclic β-1,2-glucans, and previous chemical analyses showed that more than 90% of CβG are substituted with anionic sn-1-phosphoglycerol residues (Batley et al., 1987). In this study, the NGR234 cyclic glucan synthase encoded by ndvB was identified and functionally characterized by mutational analysis to observe its role on nodule formation.. The strains and plasmids used in this study are listed in Table 1. Escherichia coli strains were grown at 37 °C in Luria–Bertani medium (Sambrook et al., 1989). NGR234 and derivative strains were grown at 27 °C in tryptone/yeast medium (TY) (Beringer, 1974) or in the hypo-osmotic minimal GYM medium (Dylan et al., 1986) to which NaCl was added at final concentrations of 25, 50, or 100 mM. If necessary, antibiotics were added to the media at the following Cediranib (AZD2171) concentrations: gentamycin (Gm) and tetracycline (Tc), 20 μg mL−1; kanamycin (Km) and spectinomycin (Sp), 50 μg mL−1; rifampicin (Rif), 100 μg mL−1. To generate the ndvB mutant, a fragment of 2779 bp was amplified by PCR using the specific primers (5′-CTGCCGCATACCAGGAAGGG-3′ and 5′-TCGTCAGGCTGAAGATGTAAGG-3′) and cloned into the SmaI site of pBluescript KS(+), creating pGF01. The fragment cloned included 290 bp of the upstream intergenic space and 2489 bp of the 5′ end of ndvB. An Ω interposon conferring spectinomycin resistance was excised from pHP45Ω (Fellay et al.