# RECTA: Region Identification Based on Comparative Genomics and Transcriptomics Analysis
## 4 Discussion and Conclusions  
Implementation of the novel computational pipeline RECTA resulted in the construction of an eight-regulons enrolled ASR regulatory network. The framework provides a useful tool and will be a starting point toward a more systems-level understanding of the question [80]. The identified motifs and regulons suggest acid resistance is a coordinated response regarding regulons, although most of these have not been identified or experimentally verified. From the three well-identified regulons--_llrA_, _llrC_, and _hlla_--it appears the gene regulation is also complex, as these regulons also interact with other proteins and TFs. The F0/F1 ATPase is directly involved in the concentration regulations of the intracellular proton. Other pathways are responsible for repairing the damage caused by acid stress, such as DNA repair, protein repair, and cell envelops alterations. However, there were also several reported ASR-related genes or transporters such as _htrA_ in _Clostridium_ spp. [81], CovS/CovR acid response regulator in _Streptococcus_[82], cyclopropane fatty acid (cfa) synthase for cell-membrane modification [83], and oxidative damage protectant genes like _sodA_, _nox-1_ and _nox-2_[84] that failed to map to MG1363. Using more gene expression datasets for CEM and DGE analyses could be a way to strengthen the result of our computational pipeline, which might cover more significant regulons to construct a more solid and complete regulatory network. Homology mapping at the genomic level showed very a long evolutionary distance between MG1363 and currently well-annotated model species. Hence, the functional analysis for MG1363 is limited, and it is hard to apply gene functional enrichment to verify our prediction results. With more expression datasets and experiments about protein-protein interactions, the ASR mechanism can be largely improved in _L. lactic_ MG1363. In summary, through the implementation of RECTA, we found that the ASR at the transcriptome level in MG1363 is an orchestrated complex network. Functional annotation shows these regulons are involved in many levels of biological processes, including but not limited to DNA expression, transcription, and metabolism. Our method builds a TF-regulons-GRN relationship so that the new ASR-related genes can be predicted. Besides, the low false positive and true negative rate indicate the RECTA pipeline as sensitive and reasonable. In fact, considering the high accuracy, we regarded _ccpA_ as the putative regulon, though not connected to any related functional modules, while more robust methods are required. Such results expand current pathways to those that can corroborate cell structures--cell wall, cell membranes, and so on--and related functions. Our findings suggest that acid has profound adverse effects and inflicts a systems-level response. Such predicted response pathways can inform better resistance design. Looking forward to the acid tolerance advantage of _L. lactis_, which makes its prospective application in drug and vaccine delivery, the effects on anti-obesity research, and metagenomic studies, the ASR-related GRN in _L. lactis_ shows an excellent research value. Fully understanding its theory may contribute to the development of _Lactococcus_ therapy and can even expand to other close species by genetic modification. Furthermore, our computational pipeline provides an effective method to construct a reliable GRN based on regulon prediction, integrating CEMs, DGE analysis, motif finding, and comparative genomics study. It has a durable application power and can be effectively applied to other bacterial genomes, where the elucidation of the transcriptional regulation network is needed. In this study, we designed a computational framework, RECTA, for acid-response regulon elucidation. This tool integrates differential gene expression, co-expression analysis, cis-regulatory motif identification, and comparative genomics to predict and validate regulons associated with acid response.