TI - Isobutanol stress causes a loss of quinone function and ArcA activation . AB - With ArcA verified by knockout to be involved in the isobutanol response , we sought to identify the upstream isobutanol target responsible for ArcA activation . ArcA is activated through PHOSphorylation by ArcB , a membrane protein . This phosphorylaTION occurs after quinone ( primarily ubiquinone ) inhibition of ArcB autoPHOSphorylation is released . Therefore , given this two-component mechanism , the likely target for isobutanol is quinone function . Quinones are electron carriers with an isoprenoid side chain that anchors them to the membrane . They function as the primary electron carriers for respiration , and are thought to regulate the ArcA-ArcB two-component system in response to cellular redox state ( reduced Qs/QH2s cannot inhibit ArcB autoPHOSphorylation ) ( Georgellis et al , 2001 ; Malpica et al , 2004 , 2006 ) . A mechanism for the isobutanol activation of ArcA through quinone malfunction conforms to a mode of toxicity through membrane disruption . We hypothesize that isobutanol disrupts the membrane , leading to quinone malfunction ( dissociation from or disruption of interaction with the membrane ) , which results in a release of quinone inhibition on ArcB , and subsequent autoPHOSphorylation of ArcB and activation of ArcA . To support this hypothesis , we tested the effect of isobutanol on metabolic processes that require quinones . One such example is glycerol degradation , which requires quinones in the second step ( glycerol 3-phosphate dehydrogenase reaction ) . Figure 3A shows the glycolytic entry pathways for glucose and glycerol under normal aerobic growth conditions . Quinones are the only components of the respiratory chain found along the glycolytic separation of glycerol and glucose . As glycerol directly requires functional quinones to enter glycolysis and glucose does not , growth on glycerol should suffer from more significant retardation than growth on glucose if the presence of isobutanol causes quinone malfunction . Thus , growth rates were measured for E.coli in glycerol and glucose minimal media in the presence of a spectrum of isobutanol concentrations ( Materials and methods ) . Figure 3B shows the relative time to four doublings for E.coli grown in glycerol and glucose medium over a spectrum of isobutanol concentrations ( see Supplementary Figure 1 for growth curves ) . These results show that isobutanol hinders growth on glycerol more significantly than growth on glucose , and that this effect becomes more pronounced at higher concentrations of isobutanol . This result supports the hypothesis that isobutanol causes the loss of quinone function either by membrane damage or by quinone depletion , and shows possible repercussions of these findings for isobutanol production in terms of carbon-source selection , which will be discussed further in the Conclusion section . To support the hypothesis that ArcA activation is caused by the loss of quinone function , we measured ArcA -regulated genes under fermentative conditions using quantitative real-time PCR . As ubiquinone is a component of the respiratory chain , it is not needed in fermentative conditions . Thus , if the hypothesis is true , ArcA activity will not change in response to isobutanol under fermentative conditions . Fermentative expression ratios are presented alongside their aerobic equivalents in Figure 4 . The genes sdhC and oppA were selected as ArcA target genes because they were the lead genes in the significantly repressed and induced operons , respectively , shown to be regulated by ArcA in DeltaarcA experiments . The expression changes of sdhC and oppA in response to isobutanol under fermentative conditions ( red bars ) are negligible compared with their expression changes in an aerobic environment ( blue bars ) . The observed fermentative oppA repression ( average approximately two-fold ) under fermentative conditions is in contrast to the strong aerobic activation of oppA ( average approximately 14-fold ) and may have resulted from the action of other known regulators of oppABCDF expression , GcvB , Lrp or ModE . These results support a mechanism of ArcA activation through malfunction of the respiratory chain , as in the absence of respiration ( fermentation ) isobutanol fails to perturb the expression of ArcA-regulon members to the same degree or direction observed under aerobic conditions .