# Gene Networks and Pathways Involved in _Escherichia coli_ Response to Multiple Stressors
## 4 Discussion
Among downregulated genes, four are involved in the pyrimidine biosynthetic and salvage pathway:
* _pyrC_ encodes a dihydroorotase, which catalyzes the third reaction in the de novo pyrimidine biosynthesis pathway. By looking for other genes involved in pyrimidine metabolism in our data, we found seven more genes to be downregulated: _pyrB_, _pyrD_, and _pyrI_ were downregulated under four stressors while _pyrE_, _pyrF_, _pyrG_, and _pyrL_ were downregulated under two stressors. * _carA_ is part of the _carAB_ operon. It encodes the amidotransferase component, CarA, while the CarB subunit is the synthetase component of the carbamoylphosphate synthetase, involved in L- arginine synthetic pathway, along with the de novo uridine monophosphate (UMP) biosynthetic pathway (part of pyrimidine biosynthesis). We found _carB_ to be downregulated under four stressors. * _codB_ and _codA_ form the _codBA_ operon. CodB is a cytosine permease, which brings cytosine into the cell [59], while CodA is a cytosine deaminase (CDA), which catalyzes cytosine deamination into uracil. It is one of the enzymes in the pyrimidine salvage pathway, permitting the cell to utilize cytosine for pyrimidine nucleotide synthesis [60; 61]. The downregulation of purine and pyrimidine metabolism, which we repeatedly observed here, has previously been reported as a response to acid exposure (pH 3.6) in _E. coli_ O26:H11, which induces the accumulation of certain metabolites and enzymes involved in purine metabolism [62]. Pyrimidine levels were reported to decrease to 59%, after 20 min of HOCl stress, relative to the reference levels of untreated cells [63]. The biosynthesis of nucleotides, such as cytidine and uridine, was decreased after the combined ultrasound
and mild acidic electrolyzed water treatment or electrolyzed water treatment alone, as well as the pool concentration of pyrimidines in the planktonic _E. coli_ cells. Simultaneously, ribose-5-phosphate, the precursor for nucleotide synthesis, was downregulated in planktonic _E. coli_ cells, after electrolyzed water treatment, indicating a depression in nucleotide biosynthesis [64]. In addition, levels of adenosine monophosphate (AMP) and uridine monophosphate (UMP), which are products of nucleotide degradation, were elevated in pulsed light treated _E. coli_, indicating DNA damage as well as blockage of RNA synthesis [65]. In addition to nucleotide metabolism-related genes, two genes are part of electron transport chain complexes:  
* _cyoB_ encodes subunit I of the cytochrome bo\({}_{3}\) complex. It is a part of the _cyoABCDE_ operon. Our analysis indicated that _cyoA_, _cyoC_, _cyoD_, and _cyoE_ were downregulated under two out of five stressors. * _nuoC_ is part of the _nuoABCEFGHIJKLMN_ operon, representing the 13 subunits of NADH ubiquitous oxidoreductase. Our analysis detected the downregulation of _nuoE_, _nuoF_, _nuoH_, _nuol_, and _nuo_] under four stressors, and of _nuoB_, _nuoG_, _nuoK_, _nuoL_, _nuoM_, and _nuoN_ under three stressors, while _nuoA_ was detectably downregulated under two stressors. _E. coli_ was previously reported to adapt to stressors affecting cell envelope integrity through the Cpx-mediated repression of the _nuo_ and _cyo_ operons. The _nuo_ and _cyo_ transcriptional downregulation suggested that those proteins are toxic in the presence of envelope stress [66]. The membrane-bound systems for proton and electron transport Nuo (the NADH dehydrogenases I and II) and Cyo (cytochrome o oxidase) were downregulated at high pH [67]. Our analysis detected the downregulation of these systems, or part thereof, under all five investigated stressors. Other genes that were downregulated under all studied stressors are:  
* _fadL_ encodes the component of a channel involved in the import of long-chain (C12-C18) fatty acids (LCFA) across the bacterial outer membrane [68]. It is part of the fatty acid-degrading (fad), regulon which includes eight more genes involved in fatty acids catabolism [69].