# Gene Networks and Pathways Involved in _Escherichia coli_ Response to Multiple Stressors
## 4 Discussion
* _purD_ encodes a phosphoribosylamine glycine ligase, the second enzyme in the _de novo_ purine biosynthesis pathway. It catalyzes the ligation of glycine PRA to produce 5-phospho-ribosyl-glycineamide (GAR) [43]. * _purN_**and _purT_ encode two different GAR transformylases, with no significant homology; each of them can catalyze the third step in de novo purine biosynthesis, producing 5-phospho-ribosyl-N-formylglycinamide (FGAR). PurN transfers a formyl group from 10-formyl-tetrahydrofolate, while PurT utilizes formate, after the hydrolysis of 10-formyl-tetrahydrofolate by _purU_[44]. * _purL_ encodes a phosphoribosylformylglycinamide synthetase, which catalyzes the fourth step in the _E. coli_ de novo purine biosynthesis pathway. In this reaction, 5-phosphoribosyl-N-formylglycineamidine (FGAM) is formed from FGAR, glutamine, and ATP [45]. * _purM_ encodes phosphoribosylformylglycinamidine cyclo-ligase (AIR synthetase), which catalyzes the fifth step in the de novo purine biosynthesis pathway [46], in which 5-amino-1-(5-phospho-D-ribosyl)imidazole (AIR) is formed from FGAM and ATP [47]. * _purC_ encodes a phosphoribosylaminoimidazole-sucincomcarboxamide synthase, which catalyzes the formation of 4-(N-sucinylcboxamide)-5-aminoimidazole ribonucleotide (SAICAR) from CAIR [48]. This is similar to another reaction catalyzed by PurA (adenylosuccinate synthetase) that also utilizes aspartate and a ribonucleoside triphosphate [49]. Our analysis indicated that _purA_ was downregulated under antibiotic treatment and heat stress. * _purB_ encodes adenylosuccinate lyase, which catalyzes two reactions in de novo purine nucleotide biosynthesis. It converts 5-aminoimidazole-4-carboxamide SAICAR to ribonucleotide (AICAR) and catalyzes the breakdown of adenylosuccinate to AMP [38]. * _purH_ encodes the bifunctional AICAR transformylase/IMP cyclohydrolase, which catalyzes the last two steps of the de novo purine biosynthetic pathway converting AICAR to IMP [50]. Two extra genes involved in the purine biosynthetic pathway, _purK_ and _purE_, were upregulated under four stressors (heat, cold, nitrosative stress, and antibiotic treatment) but not under oxidative stress. Additionally, two genes are indirectly related to purine metabolism as they encode the enzymes that catalyze the formation of 5,10- methylenetetrahydrofolate (5,10-mTHF), which acts as a methyl group donor in the biosynthesis of various cellular components, including purines and methionine:
* _glyA_ encodes a serine hydroxymethyltransferase, which catalyzes the conversion of serine to glycine through forming 5,10-mTHF. It is activated by MetR, and repressed by MetR and PurR [51]. * _gcvT_ is part of _gcvTHP_ operon. It encodes the T-protein in the glycine cleavage system, which catalyzes glycine degradation and the formation of 5,10- methylenetetrahydrofolate 5,10-mTHF. The other two genes encode GcvH, or H-protein, and GcvP, or P protein [52; 53; 54]. Expression of the glycine cleavage enzyme system is induced by glycine, activated by GcvA, and repressed by PurR and GcvA [55; 56]. Both _gcvH_ and _gcvP_ in our analysis were downregulated under three and four stressors, respectively, indicating the many-sided roles of GcvTHP beyond amino acid metabolism. GcvA is also the activator of _gcvB_. Both were reported to be related to stress response. The _hdeAB_ operon, which encodes chaperone-like functions, was reported to be activated by GcvB and repressed by GcvA. Both HdeA _and_ HdeB protect periplasmic proteins from aggregation by acid stress [57]. _gcvB_ encodes a small regulatory RNA. _gcvB_ knockout mutant was found to be more sensitive to oxidative stress and accumulate more endogenous reactive oxygen species than wild type. The role of _gcvB_ in oxidative stress was also found to be conferred by increasing OxyR expression [58]. These findings suggest that _gcvB_ and _gcvA_ products allow _E. coli_ to survive in presence of both oxidative stress and low pH. Our findings expand their regulatory functions to cold, heat, and antibiotic treatment.