Comparison of metabolite and gene expression profiles of C. perfringens grown with cystine or homocysteine To obtain new insights into the regulation in response to sulfur availability, we compared the metabolome and the transcriptome of C. perfringens after growth in the presence of 0.5 mM cystine or 1 mM homocysteine. The doubling time was about two-fold higher for C. perfringens strain 13 grown in the presence of homocysteine than in the presence https://www.selleckchem.com/products/rxdx-106-cep-40783.html of cystine. Cystine allows efficient growth while homocysteine is a poor sulfur source for C. perfringens. This suggests that some metabolites are limiting during growth with homocysteine. So, we measured the

intracellular concentration of several sulfur compounds and amino acids by HPLC in crude extracts of strain 13 grown in the presence of cystine or homocysteine

(Fig. 3). The intracellular concentration of methionine remained undetectable Fluorouracil purchase in both growth conditions. This suggests that methionine biosynthesis is not very efficient and/or that methionine requirements are high. Homocysteine can be detected only during growth with this compound suggesting that homocysteine was mainly taken up from outside under these conditions. Cystine, cysteine but also proline pools were below the threshold of detection during growth with homocysteine while their intracellular concentrations ALOX15 were 325 μM, 236 μM and 80 μM, respectively during growth with cystine. This strongly suggests that growth in the presence of homocysteine mimics conditions typically associated with cysteine limitation.

The concentration of alanine, lysine and serine and/or threonine differed to a lesser extent in these two conditions. Figure 3 Intracellular concentration of sulfur compounds (A) and amino acids (B) in strain 13 grown in the presence of cystine or homocysteine. Grey or white boxes indicate the metabolite concentrations extracted from strain 13 grown in the presence of 0.5 mM cystine or 1 mM homocysteine, respectively. The mean value of three independent experiments is presented. # indicates that the metabolite is not detectable. We further compared gene expression profiles of strain 13 grown in the presence of cystine or homocysteine. For this purpose, we designed a microarray containing oligonucleotides representative of 2706 genes of C. perfringens. For each condition, eight data sets generated with RNAs extracted from four independent cultures were used to perform statistical analysis (see Methods). A total number of 177 genes were differentially expressed in these two conditions. Most of them (122 out of 177) were up-regulated in the presence of homocysteine. Some of the controlled genes including those associated with sulfur metabolism, redox functions, carbon metabolism and virulence are presented in Table 1.

In order to reduce the roughness, the pulse

time of TMI is reduced to 8 s for sample B. The PS-341 order obtained InN film shows better flatness (rms = 20) and dark holes have been well removed (Figure 3B2). According to the theoretical simulation of the kinetics of InN formation [17], if the thickness of indium film is larger than two atomic layers, the nitridation of this In film could not well form a InN epilayers in correct stoichiometric ratio (1:1) and the excessive In will lead to roughness. Thus, the TMI pulse time was further decreased down to 4 s. As shown in Figure 3C1, the islands of sample C begin to show regular shape relatively and the surface becomes more flat (rms = 14). Meanwhile, it can

be observed that there are some islands in larger size, as indicated by arrow. The number of these types of large islands further increases in sample D (Figure 3D1), in which the TMI pulse time was set to 3 s. This trend of quality deterioration implies that the indium film deposited during the TMI period turns to be less than one atomic layer and fail to construct indium bilayer. This insufficient coverage of indium layer could not provide the advantage of nitridation of indium bi-layer structure. On the contrary, over-nitridation under N-rich condition leads to the deterioration of the InN film quality of sample D. Therefore, Crizotinib order it could be determined that 4-s pulsed supply of TMI in sample C is the optimal setting. To investigate the optical property of these samples, absorption spectra were recorded to determine the band gap of InN film and the results are shown in Figure 4. Although all four samples’ absorption curves show limited differences due to the small thickness or relatively low crystalline quality of the InN film, their differences of slope’s changes still can be identified. The absorption spectra of sample C and D have a clear slope threshold near the absorption edge. While, for samples A and B such slope threshold is absent and, beyond 1,100 nm, absorptions related

with defect or impurity bands appear. This indicates that sample C has the best film quality due to the optimized pulsed growth with TMI supply. In principle, InN is a direct band semiconductor so that the relationship between its energy band gap and its absorption Adenosine triphosphate coefficient could follow the formula below: (1) where the α is the absorption coefficient and the E g is the band gap. Thus, the E g of our samples could be estimated through the intersectional point of absorption edge’s tangent and horizontal axis. It is found that the E g of sample C and D is about 1.22 and 1.19 eV, respectively. Due to the unclear slope thresholds in samples A and B, the E g is difficult to determine precisely. The range of reasonable E g for samples A and B would be between 0.7 to 0.9 eV, which is lower than those of sample C and D.

aureus in the rats with only the established strain (- pulse). For selleck products S. aureus the bacterial density does not exceed that observed in rats without a pulse and the resident strain has a competitive advantage. Figure 3 Pulse on established populations of same species. Established populations were inoculated into 3-day-old neonatal rats 48 hours prior to pulsing 104 cfu of a marked strain of the same species or PBS. The total bacterial density in nasal epithelium of 6-8 rats with the established

and pulsed population (dark grey) and just the established population (light grey) were tracked over 96 hours after the pulse and expressed as the geometric mean with error bars indicating SE. In addition, the percent of the bacterial density that is pulsed is marked with points with dotted error bars indicating SE. Antibiotic marked strains were switched to be either pulsed or established for H. influenzae (in A and B), S. aureus (in C and D) and S. pneumoniae (in E and F). For both S. pneumoniae and H. influenzae there is an increase in the total density in the rats with the pulse (+ pulse) compared to rats with only the established strain (as shown in representative experiments in Figure 3C-F). We saw the bacterial load increase to varying degrees, more so for H. influenzae than for S. pneumoniae, in each of four replicate experiments (data available upon request). In both of these species, we observe that the pulsed

and resident strains co-exist with the pulse strain becoming 25-90% of the population. For all the species, similar pulse results AZD2014 were obtained in reciprocal experiments (switching pulse and resident strains)

confirming that the results were not due to fitness differences in the antibiotic marked strains. Invasion of Different Species in a Colonized Host Competition between different strains or species can be defined simply as a reduction in the density of one or both strains when both are present. Competition within the same species and particularly in the case of the same strain (as in the above pulse experiment) is usually mediated through a limiting shared resource. Competition between species, in addition to partitioning of a shared resource, can be mediated through inhibitory agents/toxins Leukocyte receptor tyrosine kinase (allelopathy) or predators (in this case components of the immune system [23]). Previous studies suggest that production of hydrogen peroxide by S. pneumoniae may affect the densities of other species [24, 25] and that immune-mediated competition reduces S. pneumoniae density in the presence of H. influenzae [26]. To evaluate the contributions of these different competitive mechanisms we performed invasion experiments (with one strain of each species: Eagan, TIGR4 and PS80) in which one species was resident and a second was introduced (an invader). Evidence for synergistic interactions between H. influenzae and S. pneumoniae or S.

PubMed 40. Fagan PK, Hornitzky MA, Bettelheim KA, Djordjevic SP: Detection of shiga-like toxin (stx1 and stx2), intimin (eaeA), and enterohemorrhagic Escherichia coli (EHEC) hemolysin (EHEC hlyA) genes in animal feces by multiplex PCR. Appl Environ Microbiol 1999, 65:868–872.PubMed 41. Durso LM, Bono JL, Keen JE: Molecular serotyping of Escherichia coli O26:H11. Appl Environ Microbiol 2005, 71:4941–4944.PubMedCrossRef Authors’ contributions MB conceived of the study, carried out the

sequence alignment and drafted the manuscript. SL carried out the PCR reactions. JGM participated in the design and coordination of the study and helped to draft the manuscript. All authors read and approved the final manuscript.”
“Background PLX4032 in vitro Candida parapsilosis is an emerging human pathogen that is currently the second or third most commonly isolated Candida species from blood cultures worldwide [[1–4]]. C. parapsilosis typically is a commensal of human skin and is considered to be of low pathogenicity in the setting of intact host barriers. The species is notorious for its capacity to form biofilms on catheters and other implanted devices, for nosocomial spread by hand Torin 1 carriage, and for persistence in the hospital environment [[1, 3, 5]]. C. parapsilosis is of special

concern in critically ill neonates, causing more than one quarter of all invasive fungal infections in low birth weight infants in the UK [6] and North America [7, 8], and it is a leading cause of neonatal mortality. In low-birth weight neonates, mortality rates are similar between infants with invasive disease due to C. parapsilosis and C. albicans, 39 vs. 42%, respectively [6]. Hence, detailed knowledge of C. parapsilosis interaction with the host has become urgent. However, host immunity to C. parapsilosis infections represents an important, yet understudied area. Recognition and innate immune response against Candida spp. is effected by both professional (eg. macrophages, neutrophils, dendritic cells) [9] as well as semi-professional (eg. epithelial cells) [10] immune cells. The most Reverse transcriptase potent phagocytic cells of the immune

system are neutrophils and macrophages, and they are also considered as the prototypical phagocytic cells of pathogenic Candida [11]. However, the strategic location of antigen-presenting dendritic cells (DC) at epithelial surfaces and in the skin, the primary sites of C. parapsilosis occurrence, places DCs in the first line of defense against invading yeast cells. It has recently been shown that C. parapsilosis induces DC fungipod formation [12], which is associated with immune recognition. Importantly the fungipod response is species specific, since the related fungal pathogens C. tropicalis and C. albicans induce very few and no fungipods, respectively, suggesting significant differences between the response of DCs to different pathogenic Candida species. [12]. At present, the role of DCs in C.

Scatter plots are presented and the regression lines are drawn to visualize relationships. The level of statistical significance was set to 5% and

no multiplicity adjustments were performed. Data were analysed using SAS software© version 9.2. Results Patient disposition and baseline characteristics Of the 174 male patients enrolled in the study, 92 were randomly assigned to receive treatment with teriparatide (n = 45) or risedronate (n = 47). Seventy-seven subjects (83.6 %) completed the 18-month treatment duration (teriparatide, n = 38; risedronate, n = 39), and 28 patients in each treatment group had HRQCT valid measurements. Gefitinib supplier The baseline demographic and clinical characteristics of the patients in the two treatment groups were similar and are reported in full elsewhere [30]. Mean age was 56.3 years (range 25–82 years) and 39.1% had at least one fracture prior to the study. Of the 92 patients, 31 (33.7 %) had a previous osteoporosis

therapy, most commonly bisphosphonates (30 patients). All patients were on GC therapy prior to the study, mainly for musculoskeletal and connective tissue disorders (32.7 %), respiratory, thoracic and YAP-TEAD Inhibitor 1 mediastinal disorders (23.6 %), or for gastrointestinal disorders (15.5 %). The median daily GC dose at baseline was 8.8 mg (interquartile range [IQR] 5.0–15.0 mg/day) and the next median duration of prior GC therapy was 6.4 years (IQR 2.4–13.0 years).

Effects of treatment on bone turnover markers MMRM analysis revealed that the adjusted mean changes from baseline in PINP and CTx at 3, 6 and 18 months of therapy in the teriparatide and risedronate groups (Fig. 1) show significant differences between treatments at each of these time points (p < 0.001) with the exception of CTx at month 18 (p = 0.105). Fig. 1 Mean (SE) changes from baseline for the bone markers a PINP and b CTx at 3, 6 and 18 months in the teriparatide and risedronate treatment groups. *p < 0.001 for between-treatment comparisons, + p < 0.05 for change from baseline within groups. Mixed model repeated measures analysis of changes from baseline including fixed effects for treatment, visit and the interaction between treatment and visit, and random effects for patients nested within treatment, plus the following covariates: age, baseline PINP, fracture <12 months before study, duration of prior bisphosphonate use, screening GC dose, and cumulative GC dose prior to and during study.

PubMedCrossRef 26. Ames P, Studdert CA, Reiser RH, Parkinson JS: Collaborative signaling by mixed chemoreceptor teams in Escherichia coli . Proc Natl Acad Sci USA 2002, 99:7060–7065.PubMedCrossRef 27. Kentner D, Thiem S, Hildenbeutel M, Sourjik V: Determinants of chemoreceptor cluster formation in Escherichia coli . Mol Microbiol 2006, 61:407–417.PubMedCrossRef 28. Skidmore JM, Ellefson DD, McNamara BP, Couto MM, Wolfe AJ, Maddock JR: Polar clustering of the chemoreceptor complex in Escherichia

coli occurs in the absence of complete CheA function. J Bacteriol 2000, 182:967–973.PubMedCrossRef 29. Studdert CA, Parkinson JS: Insights into the organization and dynamics of bacterial chemoreceptor clusters through in vivo crosslinking studies. Proc Natl Acad Sci USA 2005, 102:15623–15628.PubMedCrossRef 30. Gestwicki JE, Kiessling LL: Inter-receptor communication through selleck screening library arrays of bacterial chemoreceptors. Nature 2002, 415:81–84.PubMedCrossRef 31. Lai RZ, Manson JM, Bormans AF, Draheim RR, Nguyen NT, Manson MD: Cooperative signaling among bacterial chemoreceptors. Biochemistry 2005, 44:14298–14307.PubMedCrossRef 32. Sourjik V, Berg HC: Functional interactions between receptors in bacterial chemotaxis. Nature 2004, 428:437–441.PubMedCrossRef 33. Vaknin A, Berg HC: Physical responses of bacterial chemoreceptors. J Mol Biol 2007, 366:1416–1423.PubMedCrossRef 34. Cantwell

BJ, Draheim RR, Weart RB, Nguyen C, Stewart RC, Manson MD: CheZ phosphatase localizes to chemoreceptor patches via CheA-short. J Bacteriol 2003, 185:2354–2361.PubMedCrossRef 35. Banno S, Shiomi D, Homma M, Kawagishi I: Targeting

of the chemotaxis methylesterase/deamidase JAK inhibitor review CheB to the polar receptor-kinase cluster in an Escherichia coli cell. Mol Microbiol 2004, 53:1051–1063.PubMedCrossRef 36. Shiomi D, Zhulin IB, Homma M, Kawagishi I: Dual recognition of the bacterial chemoreceptor by chemotaxis-specific domains of the CheR methyltransferase. J Biol Chem 2002, 277:42325–42333.PubMedCrossRef 37. Schulmeister S, Ruttorf M, NADPH-cytochrome-c2 reductase Thiem S, Kentner D, Lebiedz D, Sourjik V: Protein exchange dynamics at chemoreceptor clusters in Escherichia coli . Proc Natl Acad Sci USA 2008, 105:6403–6408.PubMedCrossRef 38. Barnakov AN, Barnakova LA, Hazelbauer GL: Efficient adaptational demethylation of chemoreceptors requires the same enzyme-docking site as efficient methylation. Proc Natl Acad Sci USA 1999, 96:10667–10672.PubMedCrossRef 39. Wu J, Li J, Li G, Long DG, Weis RM: The receptor binding site for the methyltransferase of bacterial chemotaxis is distinct from the sites of methylation. Biochemistry 1996, 35:4984–4993.PubMedCrossRef 40. Kentner D, Sourjik V: Dynamic map of protein interactions in the Escherichia coli chemotaxis pathway. Mol Syst Biol 2009, 5:238.PubMedCrossRef 41. Li J, Swanson RV, Simon MI, Weis RM: The response regulators CheB and CheY exhibit competitive binding to the kinase CheA. Biochemistry 1995, 34:14626–14636.PubMedCrossRef 42.

This is not what we have observed, since ectopic expression of recU led to a reversal of the phenotypes observed in the absence of RecU, namely the presence of anucleate cells and cells with septa over DNA (Figure  2A-C). This indicates that Daporinad although RecU may have a role in preventing chromosome trapping by the septum, co-regulation of recU and pbp2 expression from the same operon is not required during cell division. Conclusions

We have shown that lack of S. aureus RecU protein has important consequences in the cells, doubling the duplication time, increasing the susceptibility to DNA damage and leading to the appearance of a large population of cells with compact nucleoids, lacking a nucleoid or with septa placed over the chromosome. This shows that the role of RecU in chromosome segregation and DNA repair is crucial for normal growth of S. aureus cells. RecU is encoded in the same operon as the cell wall synthesis protein PBP2 and consequently the two proteins are overexpressed under certain conditions, such as in the presence of cell wall targeting antibiotics [50]. We have buy Palbociclib shown that this genetic organization is not required for correct cell division in rich medium, but it remains to be determined if it becomes advantageous under other, more clinically relevant, conditions. Acknowledgements This work was funded by

grants PTDC/BIA-BCM/66449/2006, PTDC/BIA-BCM/099152/2008 and PEst-OE/EQB/LA0004/2011 from Fundação para a Ciência e Tecnologia. P.R. and H.V. were supported by fellowships SFRH/BPD/23812/2005 and SFRH/BD/38732/2007, respectively. The anti-FtsZ antibody was kindly provided by Dr. E.J. Harry (University of Technology, Sydney, Australia). References 1. Kuzminov A: Instability of inhibited replication forks in E. coli. Bioessays 1995, 17:733–741.PubMedCrossRef

2. Mirkin LY294002 EV, Mirkin SM: Replication fork stalling at natural impediments. Microbiol Mol Biol Rev 2007, 71:13–35.PubMedCrossRef 3. Cox MM, Goodman MF, Kreuzer KN, Sherratt DJ, Sandler SJ, Marians KJ: The importance of repairing stalled replication forks. Nature 2000, 404:37–41.PubMedCrossRef 4. Michel B, Boubakri H, Baharoglu Z, LeMasson M, Lestini R: Recombination proteins and rescue of arrested replication forks. DNA Repair 2007, 6:967–980.PubMedCrossRef 5. Wyman C, Ristic D, Kanaar R: Homologous recombination-mediated double-strand break repair. DNA Repair 2004, 3:827–833.PubMedCrossRef 6. Cromie GA, Connelly JC, Leach DR: Recombination at double-strand breaks and DNA ends: conserved mechanisms from phage to humans. Mol Cell 2001, 8:1163–1174.PubMedCrossRef 7. Ayora S, Carrasco B, Doncel-Perez E, Lurz R, Alonso JC: Bacillus subtilis RecU protein cleaves Holliday junctions and anneals single-stranded DNA. Proc Natl Acad Sci U S A 2004, 101:452–457.PubMedCrossRef 8.

Statistical significance of branching was selleck chemical verified by bootstrapping. The scale bar represents a 5% estimated sequence divergence, and reference sequences were obtained from the GenBank Database. Table 1 Distribution and abundance of 16S rRNA gene sequences in the clone library Organism No. of clones Unclassified Firmicutes 1 Clostridiales   Clostridium 114 Dorea 1 Ruminococcus 2 Subdoligranulum 1    Unclassified Clostridiaceae 34 Unclassified Clostridiales 8 Total 161 Table 2 Polar bear 16S rRNA gene clones

representing 17 valid phylotypes Phylotype Genbank acc. no. Size (bp) No. of clones Nearest valid relative Sequence similarity (%) PBF_d7 FJ375870 1439 67 Clostridium perfringens (CP000246) 99.9 PBF_b25 FJ375795 1466 35 Clostridium sordellii (DQ978216) 99.5 PBF_c44 FJ375859 1438 18 Clostridium sardiniense (AB161368) 98.5 PBM_b9 FJ375922 1427 8 Clostridium hiranonis (AB023971) 98.2 PBF_b17 FJ375788 1402 7 Clostridium colicanis (AJ420008) 99.8 PBM_b1 FJ375916 1433 5 Clostridium glycolicum (X76750) 98.3 PBM_a8 FJ375915 1430 5 Clostridium bartlettii (AY438672) 96.6 PBF_c29 FJ375847 1444 3 Clostridium paraputrificum (AY442815) 99.7 PBF_b21 FJ375792 1452

2 Clostridium perfringens (CP000246) 99.5 PBF_b32 FJ375802 1372 2 Ruminococcus hansenii (M59114) 95.3 Stem Cells antagonist PBF_b47 FJ375816 1464 2 Clostridium sordellii (DQ978216) 98.3 PBM_b18 FJ375928 1459 2 Ruminococcus gnavus (X94967) 99.4 PBM_b10 FJ375923 1460 1 Clostridium sordellii (DQ978215) 99.5 PBF_d3 FJ375866 1436 1 Clostridium perfringens (Y12669) 99.5 PBF_d10 FJ375873 1453 1 Clostridium disporicum (Y18176) 98.3 PBF_b35 FJ375805 1488 1 Firmicutes bacterium (AF157051) 95.1 PBM_a2 FJ375911 1431 1 Unclassified bacterium (DQ057466) 96.6 Total   161     Aerobic heterotrophic cell counts and β-lactamase activity The Astemizole aerobic heterotrophic cell counts ranged from 5.0 × 104 to 1.6 × 106 cfu/ml for the rectum swabs, and from 4.0 × 103 to 1.0 × 105 cfu/g for the faeces samples (Table 3 and 4). The coliform counts for the faeces samples ranged from 3.2 × 103 to 8.0 × 104 cfu/g. There was no growth of ampicillin

resistant bacteria in the faeces samples. For the rectal swabs, the proportion of ampr bacteria ranged between 3% and 44% (Table 3). A total of 144 randomly selected ampr isolates cultivated from rectal swab samples were tested for β-lactamase activity by the nitrocefin test and all isolates showed β-lactamase activity. Table 3 Aerobic heterotrophic, coliform, and ampicillin resistant cells counts (cfu/ml) in rectum swabs from polar bears in Svalbard Polar bear no. Aerobic heterotrophic cellsa Ampr aerobic heterotrophic cellsb % c 1 5.0 × 104 (± 5.0 × 103) 1.6 × 103 (± 6.3 × 102) 3 2 NC 1.0 × 104 (± 1.6 × 103) – 3 NC NC – 5 1.6 × 106(± 2.0 × 105) 8.0 × 105 (± 1.0 × 105) 44 aMean values are based on nine replicates. bMean values are based on three replicates.

Six types of proteins constitute the proteinaceous PHB surface layer in R. eutropha: (i) the PHB synthase (PhaC1) is the key enzyme of PHB synthesis and catalyses the polymerization process of 3-hydroxybutyryl-CoA provided by the central metabolism [9, 17, 18]. The function of a second – catalytically inactive – PHB synthase, PhaC2 [2] is unknown. However, PhaC2 principally has the capacity to bind to PHB granules in vivo [19]; (ii) phasin proteins (PhaPs), in particular PhaP1, cover

most parts of the granule surface and prevent CHIR-99021 datasheet coalescence of granules [20–23]; (iii) PHB depolymerases (PhaZs) are important for reutilization (mobilization) of the polymer during times of starvation [24–28]; (iv) oligomer hydrolases (PhaZb, PhaZc, alternative designation PhaYs) are involved in cleavage of intermediately formed 3-hydroxybutyrate (3HB) oligomers during mobilization

[29]; (v) regulatory proteins (PhaRs) regulate expression of selected phasin genes [30, 31] and (vi) https://www.selleckchem.com/products/Deforolimus.html PhaM represents the prototype of a recently discovered novel type of PHB granule associated protein that has phasin properties but also can bind to DNA [32]. However, despite this considerable amount of knowledge it is still an open question whether PHB granules are formed randomly within the cytoplasm or whether localization of PHB granules is controlled by the bacteria. Several studies using fluorescence microscopy (FM) [33–35] and transmission electron microscopy (TEM) [36, 37] were performed in the last decade Dipeptidyl peptidase to address this question. However, the results of these

studies were inconsistent. While FM analysis of PHB granule formation in different PHB accumulating species suggested a non random localization of “early” PHB granules in the cell periphery of these species [14, 33, 34], investigation of PHB granule formation in R. eutropha by TEM suggested that PHB granules are formed predominantly in the cell centre near dark stained “mediation elements” [36, 37]. Electron cryotomography recently revealed that in R. eutropha PHB granules at different stages of PHB accumulation are localized more or less in the cell center whereas a preferential formation of PHB granules in the cell periphery has not been observed [38]. The reason why FM and TEM resulted in apparently contradicting results remained unclear although the studies were performed with the same wild type strain. In recent studies of our laboratory we showed that PhaM can bind to PHB, to phasin PhaP5, to PHB synthase PhaC1 and to DNA [22, 32]. Consequently, we decided to reinvestigate PHB granule formation and intracellular localization in R. eutropha wild type and in mutants with altered expression of PhaP5 or PhaM.

g., an initiator protein) were provided in trans. To more buy GSK2126458 precisely locate the oriV within repC, we cloned a collection of internal segments of repC into the suicide

vector pDOP (Figure 1). This collection was conjugated into an R. etli strain containing the parental plasmid (CFNX101) as the source of all the trans elements required for replication, but we were unable to obtain transconjugants. To determine if the activation of oriV requires transcription (i.e., the repC mRNA also acts as a replication primer), we constructed a pDOP derivative that contained a repC gene but lacked a SD sequence (pDOP-Cs/SD) (Figure 1). This plasmid was also incapable of replicating in R. etli CFNX101. Similarly, the two plasmids with repC frame-shift mutations, pDOP-CBglII and pDOP-CSphI, were also conjugated into R. etli CFNX101 without success. Overall, these results indicate that RepC exerts its action in cis. RepC as an incompatibility factor Plasmid incompatibility, or the inability of two replicons to coexist in the same cell line, results from the sharing of elements involved in plasmid replication, partitioning or control [30]. The repC open reading frame of p42d, when cloned

in a vector capable of replicating in R. etli, CFNX101, can coexist with p42d [8]. However, all of our attempts to introduce the construct pDOP-C into R. etli CFNX101 www.selleckchem.com/products/idasanutlin-rg-7388.html failed. In contrast, CFNX101 transconjugants carrying a similar construct (pDOP-CsA) that contained the repC gene pSymA of S. meliloti 2011 were easily obtained. The frequencies with which CFNX101/pDOP-CsymA and CFNX107/pDOP-CsymA transconjugants were obtained were similar (average 5 × 10-3). Moreover, the plasmid profiles of the transconjugants showed that pDOP-CsA replicated in these strains as an independent entity. These observations indicate that pDOP-C and

its parental plasmid p42d are incompatible, while that of pDOP-CSymA and p42d are compatible. The RepC protein of S. meliloti 2011 Dynein pSymA shares 54% identity with the p42d RepC protein, and both proteins have very similar secondary structures (Figure 6). To map the RepC regions of p42d that are involved in plasmid incompatibility, a collection of hybrid genes containing fragments of the repC genes from S. meliloti pSymA and R. etli p42d were constructed. A schematic representation of the hybrid genes and their properties is shown in Figure 7. The hybrid genes were designed so that none of the predicted alpha-helix and beta regions of the repC products were disturbed. The hybrid genes were cloned into pDOP under the Plac promoter and transferred by conjugation into R. etli CFNX107 to determine their ability to replicate autonomously and into R. etli CFNX101 to test if they were able to replicate without the interference of p42d.