# RECTA: Region Identification Based on Comparative Genomics and Transcriptomics Analysis
## 3 Results
### Predicted Regulons Based on Motif Finding and Clustering  
Using BoBro in the DMINDA webserver, multiple motif sequences were identified from the 300 bps in the upstream of the translation start sites for each operon. Only the top five significant motifs (adjusted \(p\)-value \(<\) 0.001) were selected in each cluster, giving rise to a total of 610 (122 \(\times\) 5) identified. The motif comparison-and-clustering analysis was then performed on the 610 motifs, and 51 motif clusters were identified, with a motif similarity 0.8 as a cutoff. Intuitively, the operons sharing highly similar motifs in each motif cluster are supposed to be regulated by the same TF and tend to be in the same regulon. Hence, these 51 motif clusters correspond to 51 regulons (dataset S3). Computationally-Verified Regulon Based on Transcription Factor BLAST and Differential Gene Expression Analysis  
Among the above 51 regulons, 14 were found containing motifs significantly (E-value \(<\) 0.05) matched to known TFBSs using TOMTOM in the MEME suite, representatively. The motif logos are shown in Figure S2, and more details can be found in dataset S4. The 14 TFBS-corresponding TFs were then mapped to MG1363 using BLAST to identify the real TFs/genes regulating each regulon. As a result, eight known TFs--spo0A, lhfB, GAL80, CovR, c4494, ihfA, CovR, and RHE_PF00288--were successfully mapped to MG1363 resulting in eight TFs with multiple hits. The gene _llrA_ (llmg_0908) regulates regulons #12 and #37, _ccpA_ (llmg_0775) regulated regulons #15 and #47, _llllA_ (llmg_0496) regulates regulons #7 and #31 (Table 1). The genes _ccpA_[66, 67], _llrA_[68], _llrC_[68], and _hlLA_[69], were known to be ASR-related genes in _L. lactis_; the gene lImg_0271, without any related known TF, was found to be similar to template TF GAL80 in yeast, which has not been associated with any ASR regulation pathways yet. For all 14 significant regulons, regulons #3, #4, #20, #28, #40, and #44 are potential candidates as, currently, no related TFs in _L. lactis_ have been found (Table 1). Additionally, 86 down-regulated genes and 55 up-regulated genes (dataset S5), resulting from DGE analysis were integrated into the regulons. Regulons #10, #37, #44 and #47 were found to be lacking DEGs. Thus, gene lImg_0271, related to regulon #10, was not likely to respond to acid stress in MG1363 even though it has been successfully mapped to MG1363, and was then grouped into the potential candidate. On the contrary, _ccpA_ and _llrA_ were still retained due to their involvements in regulons #15 and #12 with DEGs, respectively. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Regulon ID** & **No. of Operons** & **DEG** & **TF Template** & **TF (Gene) BLAST in MG1363** \\ \hline
**Regulon \#2** & 82 & Y & spo0A & _llrC_ (_llmg\_0414_) \\ Regulon \#3 & 32 & Y & FoxQ1 & N/A \\ Regulon \#4 & 20 & Y & SPT2 & N/A \\
**Regulon \#7** & 49 & Y & lhfB & _hllA_ (_llmg\_0496_) \\ Regulon \#10 & 5 & N & GAL80 & lImg\_0271 \\
**Regulon \#12** & 259 & Y & CovR & _llrA_ (_llmg\_0908_) \\
**Regulon \#15** & 19 & Y & c4494 & _ccpA_ (_llmg\_0775_) \\ Regulon \#20 & 79 & Y & NHP6A & N/A \\ Regulon \#28 & 5 & Y & 1Z916 & N/A \\
**Regulon \#31** & 65 & Y & ihfA & _hllA_ (_llmg\_0496_) \\ Regulon \#37 & 10 & N & CovR & _llrA_ (_llmg\_0908_) \\ Regulon \#40 & 7 & Y & Awh & N/A \\ Regulon \#44 & 12 & N & YBR182C & N/A \\ Regulon \#47 & 5 & N & RHE\_PF00288 & _ccpA_ (_llmg\_0775_) \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Altogether, 14 significant regulons that are verified and mapped to known transcription factors (TFs). According to analyses, operon numbers and DEG determination (yes or no), matched template TFs and mapped TFs were assigned for each significant regulon, respectively, and were aligned based on regulon ID number. Five regulons containing DEGs and having the corresponding TF at the same time were bolded, being computationally verified as the regulons responsible for acid stress in MG1363.