# RECTA: Region Identification Based on Comparative Genomics and Transcriptomics Analysis
## 3 Results
### A Model of Regulatory Network in Response to pH Change  
According to the results outlined above, we are presenting a working model of the transcriptional regulatory network for acid stress response in MG1363 (Figure 3). The network consists of two transmembrane proteins (dataset S7), eight regulons, nine functional modules, and 33 orthologous genes known for ASR in other bacteria that are also contributing in MG1363. The network is subjected to respond to the change of intracellular proton level. The signal is captured by H\({}^{+}\) sensor and regulons are initiated to be regulated. Although significance was not shown for _rcfB_ in our computational results, it has been reported to recognize and regulate promoter P170 [72], P1, and P3 [73; 74], which are activated by boxes A, C and D (ACiD-box) and essential to acid response [75]. With the ACiD box, operons like _groESL_, _lacticin 481_ and _lacZ_ have been proved to be  
Figure 3: A working model of the transcriptional gene regulatory network in response to pH change in _L. lactis_. The mechanism is activated by the change of proton signal in a cell. Regulon _RcfB_ is assumed to be the overall activator for the rest seven regulons and controls the ASR functional module solely. Three kinds of literature were verified; significant ASR-related regulons, _llrA_, _llrC_, and _hllaA_, and two insufficiently significant regulons, _llrD_ (regulon #39) and regulon #8 (llmg_1803) were predicted via our workflow but with results under a 0.8 motif similarity cutoff or a hit could not be found; one putative significant regulon NHP6A controls the seven functional modules which are experimentally verified in the close species MG1363. The other significant regulon _ccpA_ failed to be confirmed by any literature-proved genes or transporters. Two extra functional modules, GAD, and urea degradation show no direct connection to all seven of the regulons. One or more homology genes are found in MG1363 for all the nine modules using BLAST. The solid arrows indicate regulation between regulons/TFs and functional modules/genes, and the dashed arrows indicate uncertain control processes. Additionally, two ovals indicate two trans-membrane proteins; one is confirmed as F0/F1 ATPase and the other one, with the dashed line, whose related information we cannot find in the public-domain literature. regulated by _rcfB_, while _als_, _aldB_, etc., have not [75]. The homologous comparative study also predicted the existence of the ACiD box in _llrA_[68; 76]. With such evidence, we separated _rcfB_ from regulon #39 and predicted that _rcfB_ is first triggered by H\({}^{+}\) sensor and acts as the global initiator that controls the other seven regulons. It is reasonable that _rcfB_-related regulon #39 failed to show significant TF matching results after CEM treatment in the operon clustering step. The _rcfB_ regulator worked as a trustworthy global factor; its differential expression should be less significant than regulons directly responding to acid stress, thus leading to the failure of being predicted by the RECTA pipeline. Nevertheless, the low number of microarray data sets (8) also limited the real performance to the ASR. However, the mechanism of how H\({}^{+}\) sensor is activating and regulating the GRN and _rcfB_ remains unclear. In the seven regulons, three--_llrA_, _llrC_ and _hlla_--were verified through literature to be related to ASR; regulons #8 and #39 showed less significant in regulon prediction; NHP6A was considered as putative regulon due to its failure to map in MG1363; and _ccpA_ was another putative regulon without literature support. The six downstream regulons (_llrA_, _llrC_, _hlla_, NHP6A, regulon #39, and regulon #8) other than _ccpA_, interact with each other to regulate six ASR-related functional modules, including the ADI system, DNA repair, LDH, protein repair, envelope alterations, and F0/F1 adenosine triphosphate (F0/F1 ATPase).