S phenolica were grown in K YTSS broth (2.5 g?L21 tryptone, 4 g?L21 yeast extract, 20 g?L21 sea salts (Sigma)) at 30uC. Antibiotic concentrations used to maintain the plasmids were 100 mg?mL21 ampicillin or 50 mg?mL21 kanamycin. D. discoideum AX3 cells were SR-3029 custom synthesis obtained from the Dicty Stock Center and maintained in liquid culture (HL5) with shaking (150 rpm) 25033180 at 22uC [22]. Environmental bacteria were collected by submerging a 166518-60-1 web Turtox tow net (Envco, New Zealand) with a 20 mm pore-size Nitex mesh spanning a 30.48 cm diameter mouth in estuary water for one minute. Water samples (200 mL) collected from estuaries of the Rio Grande delta were blended with a handheld homogenizer (PRO Scientific; Oxford, CT), and vacuum filtered through Whatman filter paper number 3 (GE Healthcare, Little Chalfont, UK). A second vacuum filtration was performed on the filtrate through 0.45 mM pore-size membranes (Millipore, Bedford, MA). Filters were incubated separately in a small volume of 0.15 M sterile NaCl for one hour shaking at RT. The suspensions were plated on thiosulfate-citrate-bile saltssucrose (TCBS) agar (BD, Franklin Lakes, NJ) and/or marine agar 2216 (BD, Franklin Lakes, NJ). Following incubation for 16 hours at 30uC, colony forming units (CFUs) were isolated and cultured in LB broth. A polymorphic 22-kb region was sequenced for both isolates, DL2111 and DL2112, for strain identification. Sequences were submitted to GenBank (accession number JX669612 and JX669613).rized in Table 2. DNA sequencing was performed at the University of Alberta Applied Genomics Centre and species were identified using BLASTn.Protein Secretion ProfilesOvernight cultures of bacterial strains were diluted to 1:100 in 3 mL of fresh LB containing appropriate antibiotics and incubated until they reached late mid-logarithmic growth phase (OD600 ,0.6). L-arabinose (0.1 ) was added to induce expression of the PBAD promoter in pBAD24 and pBAD18. Bacteria were pelleted at high speed in a tabletop microcentrifuge for 5 minutes. Supernatants were filtered through 0.22 mm low protein-binding polyvinylidine fluoride (PVDF) syringe filters (Millipore). Proteins were precipitated with 20 trichloroacetic acid (TCA) for 15 minutes on ice, pelleted by centrifugation at 14,0006 g for 5 minutes at 4uC, and washed twice with ice-cold acetone to remove residual TCA. Protein pellets were resuspended in 40 mL SDS-PAGE lysis buffer (40 glycerol; 0.24 M Tris-HCl, pH 6.8; 8 SDS; 0.04 bromophenol blue; 5 b-mercaptoethanol) and boiled for 10 minutes. 300 mL of bacterial culture was centrifuged at 14,0006 g for 5 minutes. Bacterial pellets were resuspended inDNA Sequence Analysis and Protein Structure Prediction AnalysisNucleotide sequence analyses and alignments were performed with MacVector software (version 11.0.2).16S Ribosomal SequencingPrimers binding to conserved 16S ribosomal gene sequences were used to PCR-amplify the 16S 
ribosomal sequences from environmental bacterial isolates. Primer sequences are 
summaTable 3. RGVC isolates.DL Number 2111 2112 4211 4215 NSerogroup None (rough) None (rough) O123 O113 OVasH sequence compared to V52 frameshift, H116D, Q278L, T449A, T456I frameshift, H116D, Q278L, T449A, T456I H116D, T449A H116D, T441S, P447S, T449V H116D, T449Adoi:10.1371/journal.pone.0048320.tFigure 1. Ability of RGVC isolates to kill E. coli. Rough RGVC isolates DL2111 and DL2112, and smooth RGVC isolates DL4211 and DL4215 were tested for their ability to confer T6SS-mediated prokaryotic.S phenolica were grown in K YTSS broth (2.5 g?L21 tryptone, 4 g?L21 yeast extract, 20 g?L21 sea salts (Sigma)) at 30uC. Antibiotic concentrations used to maintain the plasmids were 100 mg?mL21 ampicillin or 50 mg?mL21 kanamycin. D. discoideum AX3 cells were obtained from the Dicty Stock Center and maintained in liquid culture (HL5) with shaking (150 rpm) 25033180 at 22uC [22]. Environmental bacteria were collected by submerging a Turtox tow net (Envco, New Zealand) with a 20 mm pore-size Nitex mesh spanning a 30.48 cm diameter mouth in estuary water for one minute. Water samples (200 mL) collected from estuaries of the Rio Grande delta were blended with a handheld homogenizer (PRO Scientific; Oxford, CT), and vacuum filtered through Whatman filter paper number 3 (GE Healthcare, Little Chalfont, UK). A second vacuum filtration was performed on the filtrate through 0.45 mM pore-size membranes (Millipore, Bedford, MA). Filters were incubated separately in a small volume of 0.15 M sterile NaCl for one hour shaking at RT. The suspensions were plated on thiosulfate-citrate-bile saltssucrose (TCBS) agar (BD, Franklin Lakes, NJ) and/or marine agar 2216 (BD, Franklin Lakes, NJ). Following incubation for 16 hours at 30uC, colony forming units (CFUs) were isolated and cultured in LB broth. A polymorphic 22-kb region was sequenced for both isolates, DL2111 and DL2112, for strain identification. Sequences were submitted to GenBank (accession number JX669612 and JX669613).rized in Table 2. DNA sequencing was performed at the University of Alberta Applied Genomics Centre and species were identified using BLASTn.Protein Secretion ProfilesOvernight cultures of bacterial strains were diluted to 1:100 in 3 mL of fresh LB containing appropriate antibiotics and incubated until they reached late mid-logarithmic growth phase (OD600 ,0.6). L-arabinose (0.1 ) was added to induce expression of the PBAD promoter in pBAD24 and pBAD18. Bacteria were pelleted at high speed in a tabletop microcentrifuge for 5 minutes. Supernatants were filtered through 0.22 mm low protein-binding polyvinylidine fluoride (PVDF) syringe filters (Millipore). Proteins were precipitated with 20 trichloroacetic acid (TCA) for 15 minutes on ice, pelleted by centrifugation at 14,0006 g for 5 minutes at 4uC, and washed twice with ice-cold acetone to remove residual TCA. Protein pellets were resuspended in 40 mL SDS-PAGE lysis buffer (40 glycerol; 0.24 M Tris-HCl, pH 6.8; 8 SDS; 0.04 bromophenol blue; 5 b-mercaptoethanol) and boiled for 10 minutes. 300 mL of bacterial culture was centrifuged at 14,0006 g for 5 minutes. Bacterial pellets were resuspended inDNA Sequence Analysis and Protein Structure Prediction AnalysisNucleotide sequence analyses and alignments were performed with MacVector software (version 11.0.2).16S Ribosomal SequencingPrimers binding to conserved 16S ribosomal gene sequences were used to PCR-amplify the 16S ribosomal sequences from environmental bacterial isolates. Primer sequences are summaTable 3. RGVC isolates.DL Number 2111 2112 4211 4215 NSerogroup None (rough) None (rough) O123 O113 OVasH sequence compared to V52 frameshift, H116D, Q278L, T449A, T456I frameshift, H116D, Q278L, T449A, T456I H116D, T449A H116D, T441S, P447S, T449V H116D, T449Adoi:10.1371/journal.pone.0048320.tFigure 1. Ability of RGVC isolates to kill E. coli. Rough RGVC isolates DL2111 and DL2112, and smooth RGVC isolates DL4211 and DL4215 were tested for their ability to confer T6SS-mediated prokaryotic.
