# Gene Networks and Pathways Involved in _Escherichia coli_ Response to Multiple Stressors
## 4 Discussion
_nrdH_ is part of the _nrdHIIEF_ operon, while _nrdEF_ encodes class Ib ribonucleotide reductase (RNR), catalyzing the reduction of ribonucleotides (NTP) to deoxyribonucleotides (dNTP), a key reaction for DNA synthesis, replication, and repair. _nrdE_ encodes the alpha 2 subunit, harboring the site for nucleotide reduction, and _nrfF_ encodes the beta 2 subunit, which contains the metallocafactor required for catalysis at alpha 1 subunit [22]. Our analysis indicated _nrdE_, _nrdF_ to be upregulated under at least four stressors. NrdH is a flavodoxin that mediates the generation of the tyros radical cofactor of NrdF. Oxidative stress was reported to induce _nrdHIIEF_ expression (up to 23.4-fold), suggesting that _E. coli_ overexpressed various reductases and electron donors to increase the cell's free radical scavenging capacity to cope with oxidative stress [23]. Our analysis indicated that this operon was upregulated under oxidative stress, antibiotic treatment, cold, heat, and nitrosative stress. * _yqjI (nteR)_ is part of the _yqjH-yqjI_ operon. _yqjI_ encodes a DNA-binding transcription repressor that regulates the expression of the NADPH-dependent ferric siderophore reductase yqjH [24]. YqjH is required for iron homeostasis in _E. coli_ and it is also part of the Fur regulon [25]. Our analysis indicated that _yqjH_ was upregulated under antibiotic and heat stress. In aerobic conditions, iron is present as insoluble iron hydroxides; consequently, bacteria produce siderophores, which are extracellular ferric chelators, to mobilize iron [26]. Once ferri-siderophores complexes are transferred through membranes to the cytoplasm, they are either degraded by esterase or reduced by ferric siderophore reductases to release ferrous ions [27]. * _yhcN_ encodes a putative periplasmic protein involved in response to hydrogen peroxide and acid stress. A knockout strain lacking _yhcN_ was found to be more sensitive to hydrogen peroxide and was more able to form biofilm than its parental strain [28]. YhcN was first reported to be upregulated in response to cytoplasmic acid stress by [PERSON], et al. [29]. * _ycf_ is similar to the _Proteus mirabilis umoD_ gene, involved in the flagellar synthesis and cell elongation [30]. Its expression was reported to be induced in biofilm formation, while its null mutant is deficient in biofilm formation [31]. Both _yhcN_ and _ycf_ are upregulated under all the five stressors included in our analysis, including antibiotics. As their roles remain elusive, this observation may help in exploring their molecular functions, as it suggests leads to gene function and heavy involvement in global stress response pathways. Based on the above findings, we conclude that all five stressors upregulate the transcription of various stress-responsive proteins, such as OsmB, and genes related to various reductases and electron donors, such as the _nrdHHEF_ operon. More pathways and functions have been identified by functional analysis of upregulated genes common to at least three stressors (Figure 5A). Among the sets of downregulated genes, 24 genes were downregulated under all the 5 conditions, 14 of which are involved in purine or pyrimidine biosynthesis. Interestingly, nine of the ten genes involved in the purine biosynthetic pathway (all but _purF_), were directly repressed by the regulator PurR [32; 33; 34; 35; 36; 37; 38; 39; 40]. The following are the genes involved in purine biosynthesis, specifically in IMP (inosine 5\({}^{\prime}\)-monophosphate) biosynthetic pathway from PRPP (5-phosphor biosynthesis-1-pyrophosph):  
* _prs_ encodes ribose-phosphate diphosphokinase, which transfers a pyrophosph group from ATP to ribose 5-phosphate, synthesizing PRPP that is utilized in the biosynthesis of purine and pyrimidine nucleotides [41]. * _purF_ encodes an amidophosphoribosyl transferase, which catalyzes the pathway flux-controlling step in de novo purine biosynthesis. In this reaction, 5-phospho-\(\beta\)-D-ribosyl-amine (PRA) is formed from PRPP and glutamine. It is feedback-regulated by GMP and AMP [42].