, 2010). In brief, contigs were assembled using the CAP3 sequence assembly program (Huang & Madan, 1999). In order to identify FK228 chemical structure potential protein encoding segments, three open reading frames (orfs) prediction programs were used: heuristic genemark™ (Besemer & Borodovsky, 1999), fgenesb (http:www.softberry.com) and metageneannotator (Noguchi et al., 2006). blastn and blastp queries were performed at the NCBI server (Altschul et al., 1997). Ribosome binding sites (RBS), putative promoter and terminator

sequences were predicted by metageneannotator, bprom and findterm (http:www.softberry.com), respectively. kodon software (Applied Maths N.V., Sint-Martens-Latem, Belgium) was used for the construction of the genetic map of pREN plasmid, for the prediction of the DNA secondary structures

and for the comparative mapping of pREN with its closely related plasmids. After blastp searches, protein sequences receiving top scores were retrieved from the GeneBank database. Multiple alignments of protein or nucleotide sequences were constructed using the muscle program (Edgar, 2004). jalview allowed the visualization and editing of the alignments (Waterhouse et al., 2009). For phylogenetic analysis, the alignments were further curated with gblocks (Castresana, 2000). Phylogenetic trees were constructed based on the maximum likelihood method using the phyml program (Guindon & Gascuel, 2003) and treedyn for tree rendering (Chevenet JQ1 order et al., 2006) with the WAG substitution matrix. Statistical validation for branch support (%) was conducted via a χ2-based parametric approximate likelihood-ratio test (Anisimova

& Gascuel, 2006). The MobB protein sequence was analyzed using interproscan to determine functional protein domains (Mulder & Apweiler, 2007). The full-length Reverse transcriptase nucleotide sequence of the annotated pREN plasmid was deposited in the EMBL database under Accession No.: FR714836. The plasmid content of L. rennini ACA-DC 1534 was investigated. The strain harbors more than one plasmid and plasmid assigned as pREN was further analyzed. pREN was found to be a circular molecule of 4371 bp with a 43.3% GC content. Ab initio orf calling revealed that pREN carries six putative genes located on the same DNA strand (Fig. 1). The coding sequences (3513 nucleotides in total) cover ∼80% of the plasmid. fgenesb indicated that orf1 (921 bp) and orf2 (330 bp) formed a single operon. Further analysis of this region supported this prediction. The two orfs shared a common promoter (−35 and −10 sequences) found upstream of orf1. Right after orf2, a terminator could be determined, while both orfs were preceded by typical RBS sequences. orf1 was identified as a replication initiation protein-coding gene. The deduced amino acid product (306 residues) showed the highest identity to RepA of plasmid pLJ42 from Lactobacillus plantarum (100% query coverage, 90% identity, e-value 7e−161) (Accession No.: DQ099911, direct submission).