# RECTA: Region Identification Based on Comparative Genomics and Transcriptomics Analysis
###### Abstract
Here, we reported an integrated computational framework of regulon identification based on comparative genomics and transcriptomics analysis (RECTA) to elucidate the GRN responses in microbes under specific conditions. To better elucidate the methodology of RECTA, we built a regulatory network responding to the acid stress in _Lactococcus lactis_ species. _Lactococcus lactis_ is one of the mesophilic Gram-positive lactic acid-producing bacteria. It has been widely applied in dairy fermentations, such as cheese and milk product [22]. Several studies have provided evidence of its essential roles in wrapping and delivering proteins or vaccinations for immune treatment, such as diabetes [23], malaria [24], tumors [25; 26], and infections [27]. Holding the advantage of higher acid tolerance to protect vectors from resolving during delivery inside of the animal body, _L. lactis_ has more potential and safety in oral drug development [28]. Moreover, it has been found that _L. lactis_, along with some _Lactobacillus_, _Bifidobacterium_, and other gut microbiota, were associated with obesity [29]. Such studies lead to the possibility and availability of _L. lactis_ in metagenomic studies to investigate the effect of microbial interaction between _L. lactis_ and other species in the human body. It is now well established that _Lactococcus_ have evolved stress-sensing systems, which enable them to tolerate harsh environmental conditions [1; 30; 31]. Among the harsh environmental conditions that microorganisms confront, acid stress is known to change the level of the alarmones (guanosine tetraphosphate and guanosine pentaphosphate), collectively referred to as (p)ppGpp [32] and leads to a stringent response to cellular regulation [33]. The reason that bacteria maintain the protection mechanism against acid stress is to withstand the deleterious effects caused by the harmful high level of protons in the exposed environment. Many mechanisms or genes related to the acid stress response (ASR) have been identified. Proton-pumping activity, the direct regulator to acid stress response, controls the intracellular pH level by pumping extra protons out of the cell [34; 35], and the increase of alkaline compound levels also counters the acidification found in _Streptococcus_[36]. Acid damage repair of cells by chaperones or proteases, such as GroES, GroEL, GrpE, HrcA, DnaK, DnaJ, Clp [37; 38], hdeA/B and Hsp3 in _E. coli_[39; 40], the arginine deaminase (ADI) system [41; 42; 43; 44] and glutamate decarboxylaseases (GAD) pathways, and so on [45; 46; 47], have been proven to be associated with the acid response. Additionally, transcriptional regulators, \(\sigma\) factors, and two-component signal transduction system (TCSTs) have also been demonstrated to be responsible for ASR by modifying gene expression [48]. These genes or pathways suggest low pH has widespread adverse effects on cell functions and inflicts response at genomic, metabolic, and macromolecular levels. To better understand the mechanism that controls the acid tolerance and response to the acid stress in _L. lactis_, we considered MG1363, a strain extensively studied for acid resistance, to carry out computational analyses [1, 49, 50, 51]. Nevertheless, to adequately describe the transcriptional state and gene regulation responsible for ASR in _L. lactis_, a GRN integrating all individual pathways is needed. The experiment was conducted by six steps and the general framework is showcased in Figure 1: (i) MG1363 co-expression gene modules (CEMs) and differentially expressed genes (DEG) were generated from microarray data by hcluster package [52] and Wilcoxon test [53] in R, respectively. Figure 1: The flowchart of constructing the global acid stress response (ASR) transcriptional network in MG1363. Step 1: microarray data was used to generate co-expressed gene clusters and differentially expressed genes (DEGs), and the MG1363 genome sequence was used to find operons.