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  • Bacterial Strains, Plasmids, and Growth Conditions: The bacterial strains and plasmids used in this study are described in Table 1. LB and TSB broths and agar were used for the growth of P. aeruginosa and Escherichia coli strains at 37°C. Cultures were inoculated at an optical density at 600 nm (OD600) of 0.1 with overnight cultures, and strains were grown at 30, 37 or 42°C with aeration in TSB. Recombinant plasmids were introduced into P. aeruginosa using the conjugative properties of pRK2013 (Table 1) or by electroporation. Pseudomonas transconjugants were selected on Pseudomonas isolation agar (PIA, Difco Laboratories) supplemented with appropriate antibiotics. The antibiotic concentrations were as follows: for E. coli, ampicillin (50 µg ml–1), kanamycin (25 µg ml–1), tetracycline (15 µg ml–1), gentamicin (10 µg ml–1); for P. aeruginosa, tetracycline (200 µg ml–1 for plates or 50 µg ml–1 for liquid growth), gentamicin (50 µg ml–1), carbenicillin (500 µg ml–1). Table data removed from full text. Table identifier and caption: 10.1371/journal.pone.0076030.t001 Strains, plasmids and oligonucleotides used in this study. lacZ Reporter Fusion and β-galactosidase Assay: The H3-T6SS left-lacZ and H3-T6SS right-lacZ transcriptional fusions were constructed by PCR amplification of respectively 486 and 494 bp upstream DNA region from the lip3 or hsiB3 gene by using TSO15/TSO16 and TSO17/TSO18 primers (Table 1). PCR amplification products were directly cloned into the pMini-CTX::lacZ vector [18], yielding pTS12 and pTS13, in pCR2.1, and pTS19 and pTS20 in MiniCTX-‘lacZ. Nucleotide sequences were verified by sequencing (GATC). The promoter fragment was integrated at the CTX phage attachment site in PAO1 and isogenic mutants using established protocols [18]. Overnight culture, grown in TSB, was diluted in TSB to OD600 = 0.1. Growth and ß-galactosidase activity were monitored by harvesting samples at different time intervals. ß-galactosidase activity was measured according Miller [19], based on o-nitrophenyl-β-D-galactopyranoside hydrolysis. ß-galactosidase activities were expressed in Miller units. Construction of the ΔclpV2ΔcplV3 Mutant: To generate the ΔclpV2cplV3 mutant, the pTS27 mutator plasmid [14] was mobilized in the P. aeruginosa strain PAO1ΔclpV3 [20]. Mutants which had undergone a double recombination event, resulting in the non-polar deletion of the clpV2 gene, were verified by PCR with the primers OA14 and OA17 that flank clpV2. Construction of sfa2 and sfa3 Mutants: To generate sfa2 and sfa3 mutants, internal fragments of 490 and 511 bp were respectively amplified with TSO118-TSO119 and TSO120-TSO121 and cloned in the pCR2.1, resulting in pSBC56 and pSBC57. The mutator plasmids were electroporated into P. aeruginosa PAO1 and the mutant bacteria selected on PIA medium containing Carbenicillin. The insertions were verified by PCR with the primer pairs TSO39-TSO40-TSO45 and TSO41-TSO42-TSO46 that hybridize outside and inside of the sfa2 and sfa3 genes. The slow killing assay was performed as described previously [14]. Each independent assay consisted of three replicates. E. coli OP50 was used as a control. L4 to adult stage C. elegans were removed from food and placed on unseeded NGM plates for 24 hours at 25°C. 50 worms were then picked onto plates containing overnight grown bacteria. Worms were evaluated for viability on a daily basis. Animal survival was plotted using the PRISM 5.0 program. Survival curves are considered significantly different from the control when P-values are <0.05. Prism calculates survival fractions using the product limit (Kaplan-Meier) method. Prism compares survival curves by two methods: the log-rank test (also called the Mantel-Cox test) and the Gehan-Breslow-Wilcoxon test. Paired Student’s t tests were performed using Excel software. In the figures, * means P-values ≤0.05, ** ≤0.01, and *** ≤0.001.
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