Nevertheless, the MGEs also include regions unique to the Pf-5 genome that could contribute to the bacterium’s fitness in the soil or rhizosphere. Methods Strains and plasmids Wild type variants of P. fluorescens Pf-5 [5], P. fluorescens SBW25 [73], and P. fluorescens

Q8r1-96 [74] were used in the study. Pseudomonas strains were grown at 28°C in King’s B medium [75], while E. coli strains were grown in LB [76] or 2xYT [76] at 25°C or 37°C. When appropriate, antibiotic NU7026 manufacturer supplements were used at the following concentrations: tetracycline, 12.5 μg/ml; chloramphenicol, 35 μg/ml; and ampicillin, 100 μg/ml. DNA manipulations and sequence analyses Plasmid DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, JQ-EZ-05 ligation, and transformation were carried out using standard protocols [76]. All primers were developed with Oligo 6.65 Software (Molecular Biology Insights, West Cascade, Colo.), and routine PCR amplifications were performed with Taq DNA polymerase (Promega, Madison, Wisc.) according to the manufacturer’s recommendations. Sequencing of prophage 01 from P. fluorescens Q8r1-96 was carried out essentially as described by Mavrodi et al. [77]. Briefly, the Q8r1-96 gene library was screened by PCR with oligonucleotide primers col1 (5′ GCT GCT GGG CAA TGG TAA CAC 3′) and col2 (5′ CTG CCG ACT GCT CAC

CTA TC 3′) and a positive cosmid clone was shotgun sequenced by using the EZ::TN™ transposition

system (Epicentre Technologies, Madison, Wisc.). DNA sequencing was carried out by using an ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, Calif.), and sequence data were compiled and analyzed with Vector NTI 9.1.0 (Invitrogen Corp., Carlsbad, Calif.) and OMIGA 2.0 (Accelrys, San Diego, Calif.) software packages. Database searches for similar see more protein sequences were performed using the NCBI’s BLAST network service, and searches against Unoprostone PROSITE, Profile, HAMAP, and Pfam collections of protein motifs and domains were carried out by using the MyHits Internet engine [78]. Signal peptides were predicted with SignalP v. 3.0. [79]. The nucleotide sequence of prophage 01 from P. fluorescens Q8r1-96 has been deposited in GenBank under accession number EU982300. DNA hybridization The 3.12-kb prophage 01 probe was amplified by PCR from P. fluorescens Q8r1-96 genomic DNA with the oligonucleotide primers orf11-1 (5′ CAT TCG TGT GCC GCT GTT CTA 3′) and orf14-2 (5′ TGA CCA GGC GAA CAG CGT CTG 3′). The 1.79-kb P. fluorescens SBW25-specific prophage 01 probe was amplified from genomic DNA of SBW25 with oligonucleotides SBW3 (5′ GAA CTC ACC AGC GTC CTT AAC 3′) and SBW4 (5′ GGG CAG CTC CTT GGT GAA GTA 3′). Amplification was carried out with Expand Long DNA polymerase (Roche Applied Science, Indianapolis, IN) according to manufacturer’s recommendations.