Sequence analysis A PCR-based strategy, employing the primer pair llsAFor-llsARev, was employed to screen for the presence of the LLS structural gene, llsA. These and other primers corresponding GW2580 purchase to regions both within (1113for, 1114rev, 1115 rev, 1118rev, 1120rev) and surrounding (araCrev) the LIPI-3 of L. monocytogenes F2365 were employed to amplify flanking DNA sequences which were subsequently sequenced (MWG Biotech) (Table 4). Primer Lin1080_F1, which
was designed to amplify from the conserved gene, corresponding to lin1080 in strain CLIP11262, was used to determine the position of LIPI-3 in L. innocua strains relative to this locus. Overlapping sequences were assembled and a consensus sequence was determined using the Seqmanager programme (Lasergene 6) and deposited in Genbank (accession numbers KJ394487, KJ394488, KJ394489 and KJ394490). Putative open reading frames (ORFs)
were identified and pair-wise alignment of protein sequences was carried out using Needlemann-Wunsch global alignment algorithms accessed via the European Nec-1s mouse Bioinformatics Institute (EBI) web server.
Shading of multiple-aligned sequences was carried out using the Boxshade programme (version 3.2) accessed via the European Molecular Biology web server (EMBnet). Table 4 Primers Endonuclease used in this study Primer name Sequence (5′ to 3′)* PllsAchgA(LI) GGCTGCAGAATCCGCGTTCTTG PllsAchgB(LI) GAGGTTTTAGGGCTTTGCTC PhelpFsoe(LI) GAGCAAAGCCCTAAAACCTCGAGATCTGCTGG PhelpRsoe GATGATTGTGATTTAATATTCATGGGTTTCACTCTC PllsAchgC ATGAATATTAAATCACAATCATC PllsAchgD TGGAATTCCCAGCTCCATTGTCTC pORI280For CTCGTTCATTATAACCCTC pORI280Rev CGCTTCCTTTCCCCCCAT Lin1080_F1 CGGTACGGATTGTGAATGAAGTGG llsAFor CGATTTCACAATGTGATAGGATG llsARev GCACATGCACCTCATAAC 1113for GTTATGAGGTGCATGTGC P005091 1114rev GTCTGGGATATGTAGTCC 1115 rev CACTAGCATGATGTTTATAGGGG 1118rev CATGACAAGCAGTGCCTGTTGATACAGC 1120rev CGTTCCCCCTCCTTTTTAGAGCAG araCrev CTCTCCTTTTCATTAGCCTGC actA1-f AATAACAACAGTGAACAAAGC actA1-r TATCACGTACCCATTTACC plcB2-f TTGTGATGAATACTTACAAAC plcB2-r TTTGCTACCATGTCTTCC actA3-f CGGCGAACCATACAACAT plcB3-r TGTGGTAATTTGCTGTCG *Restriction site in bold and SOE overhang italicised. Constitutive expression of the LIPI-3 cluster of L. innocua strain FH2051 The L.