located in close proximity of, or overlapping with, the actual promoter sequence [10]. Previously, many studies aimed at detection of intergenic regulatory elements on a genome-wide scale [11,12] have employed consensus sequences that were deduced from experi- mentally determined binding sites in order to identify similar regulatory elements elsewhere in the genome [3]. When experi- mental evidence is limited or absent, comparative genomics or other de novo prediction methods can be employed to detect conserved intergenic cis-acting elements in a genome [13–16]. The species Lactobacillus plantarum is frequently encountered in vegetable, meat and dairy fermentations and is also present among the natural inhabitant of mammalian gastrointestinal tracts [17]. The genome of L. plantarum WCFS1 [18] was predicted to encode two sigma factors besides s70, namely the rpoN-encoded s54 factor that has been shown to exclusively control the expression of the mannose specific PTS system in this strain [9], and the sigH encoded sigma factor of which the target genes in L. plantarum remain unknown to date, but that resembles the Bacillus subtilis 168 transcription factor sigH that is involved in sporulation. In this study, we performed a model-based genome-wide prediction of conserved motifs in the intergenic sequence upstream of protein-encoding genes within the genome of L. plantarum WCFS1. In silico models of recognizable s70-promoter sequence elements were generated using position weight matrices (PWM; [19,20]), which were subsequently used for genome-wide predic- tion of promoter sites. Using this approach we predicted 3874 candidate s70 promoters in the L. plantarum WCFS1 genome at p- value cut-offs of 1024. Subsequently, transcriptome datasets obtained using strand-specific genome-wide tiling DNA micro- arrays were used to validate the transcriptionally active proportion of the in silico predicted s70-promoter sequence elements [12,21,22], enabling the validation of 568 of the predicted promoters by their proximity (#40 nucleotides) to 1167 transcrip- tion start sites inferred from experimentally estimated transcript boundaries. Results Prediction of conserved motifs in the intergenic regions of L. plantarum With 3.3 million nucleotides, the genome of L. plantarum WCFS1 is among the largest Lactobacillus genomes [18] and comprises 3137 predicted genes, of which 3051 are protein- encoding (Refseq NC_004567, [23]). The intergenic regions encompass approximately 16 percent of the entire genome nucleotide sequence (530640 out of 3308275 nucleotides). Using the genome annotation of L. plantarum WCFS1, 2209 intergenic sequences (see Methods) were used to search for conserved motifs using MEME [24], resulting in the detection of eight distinct sequence motifs with E-values below 1023 (see Figure 1, Results S1 and Figure S1). The element with the lowest E-value (motif 1; E-value 2.76102239; Figure 1) displayed clear resemblance to the consensus Shine and Dalgarno sequence (SD; 59-AGAAAG- GAGGTGATC-39) of bacteria [25]. The SD sequence of L. plantarum WCFS1 is found because the intergenic sequences used to search for conserved motifs generally include the untranslated regions of the protein-encoding genes that encompass the SD sequence. In addition to the SD sequence, two motifs (motif 2, motif 3; Figure 1) were identified that resemble cis-acting elements described for sigma factor s70 (rpoD) [6] dependent promoters. These motifs encompassed one or both of the two canonical consensus s70 elements that were previously designated the 235 Prediction and Validation of Sigma70 Promoters and 210 box (consensus sequence 59-TTGACA-39and 59- TATAAT-39, respectively) [4]. Both motif 2 (E-value 1.16102149) and motif 3 (E-value 3.8610282) encompass sequences that resemble the 210 box, while motif 3 also includes the 235 box. Genome-wide prediction of s70 promoters in L. plantarum The detection of L. plantarum related s70-promoter elements enabled the genome-wide prediction of s70-promoters that share similarity with the identified motifs. The two s70-promoter associated position-specific weight matrices (PWMs) generated by MEME (motif 2 and motifs 3) were employed to predict the positions of both sequence elements in the L. plantarum WCFS1 chromosome, using MAST [26]. These predictions generated 939 and 1141 occurrences of the extended 210 (based on motif 2) and the 235 210 promoter motifs (based on motif 3) at a p-value cut- off of 1024, respectively. Occurrences were filtered for partially overlapping promoters, resulting in the identification of 1879 putative s70 promoters. Previous studies have revealed that the two s70-promoter elements (235 and 210 boxes) may flank a spacer sequence of slightly variable length in different promoters (16–20 nt) [27]. To accommodate such spacer-length variation in our predictions, the PWM of the 235 210 promoter (based on motif 3) was manually modified to allow for length variations ranging from 16 to 22 nt. MAST-based searches using these modified PWMs yielded 1995 additional non-overlapping pre- dicted promoter sequences. Overall, these predictions generated a total of 3874 putative promoter sequences that displayed high-level similarity to the consensus motif (Bonferroni corrected p- values,0.05). Detection of putative transcription start sites As described in previous studies, the high sensitivity of in silico prediction of s70-promoters using PWMs usually generates many false-positive identifications [3,28–30]. To overcome this limita- tion, putative transcription start sites (TSSs) inferred from 59-ends of transcripts are commonly employed to corroborate predicted s70-promoter locations [12,21,31]. In this study, we determined genome-wide putative TSSs from tiling-array derived transcription data obtained for L. plantarum WCFS1 grown in rich laboratory medium (MRS) at 37uC. Transcriptional activity was assigned to genomic regions by employing a global threshold approach [32], leading to the identification of 1167 distinct transcriptionally active regions (TARs) larger than 120 nucleotides [33]. The 1167 TARs cover 1995 of the 3137 predicted genes in the L. plantarum WCFS1 genome, likely reflecting the transcriptionally active fraction of the entire genomic coding capacity under the growth conditions employed in this study. The 59-ends of TARs may be the result of transcription initiation but can also arise from other events, such as degradation, processing or cross-hybridisation. Although in the majority of cases 59-end points of TARs are probably the TSSs [12] false-positive identifications are possible (see Results S2, Figure S2 and S3). Inspection of signal strength patterns at the 59-end of different TARs, revealed sharper increases in signal intensity for highly expressed TARs compared to weakly expressed TARs. Conse- quently, the assignment of putative TSSs to weakly expressed TARs leads to more ambiguous TSS positioning as compared to those assigned to highly expressed TARs. Therefore, TARs were grouped according to their mean signal intensity (see Methods), which allowed the discrimination between TSSs assigned to highly and weakly expressed TARs. To consistently assign TSSs to TARs an algorithm was developed that identifies the maximum increase PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e45097