coli OP50 was significantly reduced (Figure 2). Under the other H2O2 conditions, treatment Ka4 in association with OP50 was almost similar to Ka4 alone. In non-stress conditions, all treatments were statistically equal, indicating that the bacteria Pembrolizumab cost used were not harmful to the nematodes. Figure 2 Mortality percentages of Bursaphelenchus xylophilus virulent (Ka4) and avirulent (C14-5), with and without bacteria ( Serratia spp. LCN-4, LCN-16 and PWN-146, and E. coli OP50) under oxidative stress conditions. For each H2O2 condition, columns with different letters reflect statistical differences (p < 0.05). In control conditions

(0 mM H2O2), no statistical differences were found between all treatments. Observation of the nematode-bacteria association After 1 h contact between

B. xylophilus and its associated bacteria, microcolonies were found along the nematode body (Figure 3A). After extensive washing, bacteria were still present in lesser amounts, and scarcely attached to the nematode cuticle (Figure 3B). In order to test if the bacterial adhesion to the nematode became stronger, and if the nematode could uptake bacteria into its body, we performed co-culturing of the nematodes with the GFP-labelled bacteria on the same plate for 24 h. Successful GFP-labelling of B. xylophilus-associated bacteria was only obtained for Serratia spp. LCN-4 and Serratia spp. LCN-16. Serratia spp. PWN-146 were previously found to be multi-drug resistant to the antibiotics available to select for GFP-containing minitransposons https://www.selleckchem.com/products/PD-0332991.html [8]. After 24 h contact with Serratia spp. LCN-16, the density of nematode-attached bacteria was sparse (Figure 3C-F), and also no GFP fluorescence signal was detected in the nematode (Figure 3C-F). Taken together, the adhesion of these bacteria to the nematode surface and organs seems to be weak and non-specific. Figure

3 Observation of Serratia sp. LCN-16 in association with Bursaphelenchus xylophilus after 1 h and 24 h contact. (A, B) Differential interference contrast (DIC) microscope images of B. xylophilus, treated by 1 h contact of bacteria before (A) and after (B) washing with sterile PAK5 DW. (C-F) DIC and fluorescence-merged images of B. xylophilus, treated by 24 h contact of bacteria and washed with sterile DW. The images of the head (C) and tail (D) region were captured in a single focal plane . Serial-section images were acquired and stacked, showing surfaces of the head (E) and tail (F) region. Scale bars, (A), (B), 30 μm; (C)-(F), 20 μm. Relative gene expression of Bxy-ctl-1 and Bxy-ctl-2 Using the C. elegans catalases (Ce-CTL-1, Ce-CTL-2 and Ce-CTL-3) as the search queries, only two catalases were predicted in the B. xylophilus genome, Bxy-CTL-1 (BUX.s00579.159) and Bxy-CTL-2 (BUX.s01109.377) [30]. Both cDNA sequences presented open reading frames (ORF). The longest ORF for Bxy-ctl-1 encodes a 513 aa protein with the molecular weight of ~59kDa.

Moreover, this segment in pGP704 has flanking EcoRI and BamHI sequences that prevent the cognate restriction enzymes being used for cloning. For pBAM1, the whole oriV region was streamlined to a minimum (392 bp) and the selleck chemicals internal HindIII removed (while keeping a sequence in the former site with similarity to the functional repeats). Finally, the termini of the segment were furnished by the infrequent restriction site AscI to create the origin of replication module. These changes did not affect any of the properties described for the natural R6KoriV

sequences [9]. pBAM1 and its derivatives are maintained in the specialized strain E. coli CC118λpir, which expresses the π protein from a lysogenic phage Selleckchem AZD1152 HQPA [4]. The next module of the plasmid frame was the sequence that contains the origin of transfer oriT (Figure 1) and enables transfer of pBAM1 from the host strain to a new recipient, when recognized by the conjugative machinery encoded by the broad host range plasmid RK2, also called RP4 [11]. Since the RP4/RK2 conjugal transfer system

is the most promiscuous of all DNA mobilization device known, the presence of oriT allows mobilization of pBAM1 into virtually any Gram-negative or Gram-positive bacteria [12] and can even be passed into fungi [13] and eukaryotic cells [14], provided that the construct is exposed to the action of the Tra proteins of RP4 [8]. This transfer can be made by either setting up a tri-parental mating mixture with a donor strain (e.g. E. coli CC118λpir) bearing the R6KoriV/RP4oriT plasmid, a recipient bacterium and helper cells bearing the mob/tra region of RP4 cloned in a plasmid which does not replicate in the recipient [8]. As an alternative,

Oxalosuccinic acid the donor λpir + strain may have the tra/mob functions integrated in its chromosome (for instance, E. coli S17-1λpir) allowing bi-parental mating [15]. Other λpir + E. coli donor strains such as E. coli RH03, which have been engineered to facilitate counter-selection, are also eligible to this end [16]. The oriT region employed in most plasmid vectors designed for mobilization purposes is exceedingly large (1728 bp) and flanked by BamHI sites [8]. As before, we trimmed down the oriT to the minimum of 244 bp required for functionality [11], eradicated one SfiI site present within the core oriT sequence (to allow its inclusion in the polylinker of the vector) and the streamlined module was flanked by the two rare enzyme sites FseI and PshAI. Note, however, that in some cases the plasmid can just be electroporated into target cells and conjugation may not be necessary, although the efficiency is considerably lower.