4 mM of each primer in a final concentration of 1× master mix of the HotStarTaq Master Mix Kit (Qiagen, Basel, Switzerland). PCR targeting the 16S rRNA generrs,gyrBhousekeeping gene,pagRIAHL receptor and synthase genes, T3SS ATPasehrcN, and the insertion site of theP. agglomeransgenomic see more island carrying the pantocin genespaaABCand these genes were performed.

Primer sequences and annealing temperature (Tm) for each PCR are shown in Table1. With the exception of thegyrBamplification standard cycling conditions were used for all PCRs with an initial denaturation and activation of the HotStarTaq enzyme for 15 min at 95°C, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at the proper Tmfor 45 s, plus 30 s of elongation at 72°C for every 500 bp of expected amplicon size, ending with a final elongation for 10 min at 72°C. The protocol forgyrBamplification included, after the initial polymerase activation, 42 cycles of denaturation STA-9090 datasheet at 95°C for 30 s, 30 s annealing at 50°C where the annealing time increased by 2 s/cycle until 40 s were reached, plus 10 s elongation at 72°C where the extension time increased

by 1 s/cycle until 15 s are reached. Positive PCR amplification was verified by loading 5 μl of each reaction on a 1.2% agarose gel. Table 1 PCR primers used for gene amplification and sequencing. Gene(s) Primer name Sequence (5′-3′) Size (bp) Tm (°C) Reference gyrB gyr-320 TAARTTYGAYGAYAACTCYTAYAAAGT 970 50 [63]   rgyr-1260 CMCCYTCCACCARGTAMAGTTC     [63] hrcN hrcN-4r CGAGCAGGAYTCGATGAACG 250 50 [57]   hrcN-5rR CCGGWYTGGTATTCACCCAG     [57] 29-kbp GI mutS-rev CGCCATCGGGATCGGTTCGCC 554 60 This work   narL-rev GCCGTCTGGGCGCTGCAGAACG     Farnesyltransferase This work

paaABC paaA-fw CTCTTGCCAAAATGGATGGT 2398 55 This work   paaC-rev TTGCAAATTCTGCACTCTCG     This work pagRI pagR-fw GTGAAGGATACYTACTACAACG 1206-29 55 This work   pagI-rev CGAATGCATTGACGGCATGG     This work rrs 16S-8F AGAGTTTGATCCTGGCTCAG 1503 48 [64]   16S-533R TTACCGCGGCTGCTGGCAC     [64]   16S-609R ACTACYVGGGTATCTAAKCC     [65]   16S-1492R ACGGTTACCTTGTTACGACTT     [64] Tm = annealing temperature Sequencing of 16S rDNA, gyrB and pagRI genes PCR amplicons were purified from the PCR mix by washing twice with 50 μl of double-distilled water (ddH2O) on a MultiScreen PCR Plate (Millipore, Molsheim, France), resuspended in 30 μl of ddH2O, and quantified spectrophotometrically as described above. The cycle-sequencing reaction was performed with 20-40 ng of purified PCR product using the ABI PRISM BigDye Terminators v1.1 Cycle Sequencing Kit (S63845 supplier Applied Biosystems, Foster City, CA, U.S.A.) according to the manufacturer instructions employing the same primers used for PCR amplification. For 16S rRNA gene sequencing, additional primers 16S-609R and 16S-533R (Table1) were used to obtain complete coverage of the amplicon.