## 4 Discussion  
In this study, we found that the mutation of the dihydrolipoamide acetyltransferase gene _aceF_ in _P. aeruginosa_ resulted in defective T3 SS and enhanced H\({}_{2}\)O\({}_{2}\) tolerance, which is due to the upregulation of RsmY/Z. By using _PrsmY-lacZ_ and _PrsmZ-lacZ_ transcriptional fusions, we found that the activities of the _rsmY/Z_ promoters were elevated in the \(\Delta\)aceF mutant. The transcription of _rsmY/Z_ is under the direct regulation of the two-component regulatory system GacS-GacA. However, we did not observe an upregulation of _gacS_ or _gacA_ in the \(\Delta\)aceF mutant (data not shown). This warrants further studies to examine whether the activity of GacS-GacA is enhanced (e.g., phosphorylation of GacA) or whether other regulatory genes are involved in the regulation of _rsmY/Z_. _P. aeruginosa_ produces catalases and alkyl hydroperoxide reductases to detoxify ROS [43]. The expression of these oxidative response genes is activated by the transcriptional regulator OxyR upon oxidative stresses [44]. OxyR is an LysR-type transcriptional regulator that consists of an N-terminal DNA-binding domain and a C-terminal regulatory domain. The presence of H\({}_{2}\)O\({}_{2}\) leads to the formation of an intramolecular disulfide bond in the C-terminal regulatory domain, which results in the activation of OxyR [45; 46]. In the \(\Delta\)aceF mutant, _alhpB_ and _katB_ were upregulated, and _alhpC_ and _katA_ were slightly downregulated. However, the mRNA levels of _oxyR_ were similar to wild-type PA14. These results suggest that AceF is unlikely to influence the protein level or function of OxyR. Since the deletion of _rsmY_ and _rsmZ_ reduced the expression levels of _alhpB_ and _katB_, it might be possible that an unknown regulatory gene is involved in the regulation of _rsmY_ and _rsmZ_ and subsequent _alhpB_ and _katB_ in the \(\Delta\)aceF_ mutant. In addition to OxyR, the stress sigma factor RpoS positively regulates the catalase genes _katA_ and _katB_[47; 48]. In _Pseudomonas fluorescens_, the expression of _rpoS_ is activated by GacA and repressed by RsmA [49]. Further studies are warranted to examine whether the mutation of _aceF_ affects the level of RpoS. The stringent response has been shown to control the expression of the catalase genes in _P. aeruginosa_ and promotes the bacterial survival under oxidative stresses [47; 50]. The stringent response is controlled by the molecule guanosine tetra- and penta-phosphate (p)ppGpp [51]. The homeostasis of (p)ppGpp is controlled by two enzymes, namely RelA and SpoT. RelA is a (p)ppGpp synthetase that is activated by amino acid starvation. SpoT functions as both (p)ppGpp synthetase and hydrolase [52]. It has been demonstrated that in _E. coli_, acyl carrier protein (Acyl-ACP) activates the (p)ppGpp hydrolysis activity of SpoT, whereas uncharged ACP activates the (p)ppGpp synthetase activity [53], indicating a connection between carbon metabolism and the stringent response. The pyruvate dehydrogenase connects the glycolysis and the TCA cycle by converting pyruvate to acetyl-CoA that influx into the TCA cycle. Thus, the mutation of _aceF_ might impede the TCA cycle, which might affect the activity of SpoT. Further studies are needed to examine whether the stringent response is activated in the \(\Delta\)aceF mutant. Previously, we found that the mutation of another carbon metabolism gene _eno_ results in the downregulation of _alupB_ and _alupC_, which increases bacterial susceptibility to oxidative stresses [32]. The _eno_ encoded enolase is a glycolytic enzyme that reversibly catalyzes the dehydration of 2-phosphoglycerate to phosphoenolpyruvate [54]. In addition, enolase interacts with ribonuclease (RNaseE), polynucleotide phosphorylase (PNPase) and the RNA helicase RhIB to form the RNA degradosome, which plays important roles in RNA processing and degradation [55; 56]. Further studies are warranted to elucidate whether the downregulation of _alupB_ and _alupC_ is due to defective assembly of the RNA degradosome or carbon metabolism.