The ArcBA Two-Component System of Escherichia coli Is Regulated by the Redox State of both the Ubiquinone and the Menaquinone Pool

Abstract

ArcBA is a two-component regulatory system of Escherichia coli involved in sensing oxygen availability and the concomitant transcriptional regulation of oxidative and fermentative catabolism. Based on in vitro data, it has been postulated that the redox state of the ubiquinone pool is the determinant for ArcB kinase activity. Here we report on the in vivo regulation of ArcB activation, as determined using a lacZ reporter specifically responsive to phosphorylated ArcA. Our results indicate that upon deletion of a ubiquinone biosynthetic enzyme, regulation of ArcB in the anaerobic-aerobic transition is not affected. In contrast, interference with menaquinone biosynthesis leads to inactivation of ArcB during anaerobic growth; this phenotype is fully rescued by addition of a menaquinone precursor. This clearly demonstrates that the menaquinones play a major role in ArcB activation. ArcB shows a complex pattern of regulation when E. coli is titrated through the entire aerobiosis range; ArcB is activated under anaerobic and subaerobic conditions and is much less active under fully aerobic and microaerobic conditions. Furthermore, there is no correlation between ArcB activation and the redox state of the ubiquinone pool, but there is a restricted correlation between the total cellular ubiquinone content and ArcB activity due to the considerable increase in the size of the ubiquinone pool with increasing degrees of aerobiosis. These results lead to the working hypothesis that the in vivo activity of ArcB in E. coli is modulated by the redox state of the menaquinone pool and that the ubiquinone/ubiquinol ratio in vivo surely is not the only determinant of ArcB activity.

The relationship between redox enzyme activity and electrochemical potential—cellular and mechanistic implications from protein film electrochemistry

“…However, anaerobic growth leads to a predominance of menaquinones (MQ) that may mediate electron transfer from formate dehydrogenase to the nitrate or nitrite reductases. 40 The reduction potential of MQ/MQH 2 (E m,7 E À0.07 V) is lower than that of UQ/UQH 2 so we anticipate that lower potentials will be required to see appreciable activities from the MQ-, than UQ-, dependent enzymes. Certainly, activity from the E. coli penta-heme nitrite reductase, NrfA, a MQH 2 -dependent system is only detected below ca.…”

“…40,44,45 Testing our hypothesis requires that enzyme activities and electrochemical potentials are correlated in the cellular context. In the first instance, the activities of periplasmically facing enzymes such as NapAB will be most readily measured in chemostat cultures where the rates of substrate consumption and/or product formation can be determined by analysis of the extracellular medium.…”

Adaptation of anaerobic cultures of Escherichia coli K‐12 in response to environmental trimethylamine‐N‐oxide

“…The transient decrease in ArcA activity emphasizes the complexity of the ArcBA response. At least three signals have been proposed as modulators of ArcB activity: oxidized ubiquinone acts negatively; reduced menaquinone acts positively; and fermentation products, such as acetate, promote ArcB kinase activity and ArcA phosphorylation (Georgellis et al ., 1999; 2001; Rodriguez et al ., 2004; Bekker et al ., 2010; Rolfe et al ., 2011; 2012; Alvarez et al ., 2013). The dynamics of ArcA activity reported here suggests that introduction of TMAO to the fermenting culture causes net oxidation of the menaquinone pool, resulting in the initial dephosphorylation of ArcA followed by restoration of ArcA activity, in the final TMAO‐respiratory/fermentative steady state, perhaps mediated by enhanced production of acetate (Table 2).…”

“…Standard wet transfer to a Hybond‐C membrane (Amersham) was followed by immunodetection using rabbit ArcA antiserum and the ECL detection system (GE Healthcare). ArcA protein was purified and phosphorylated in vitro as previously described (Bekker et al ., 2010) with the exception of using 100 mM carbamoyl phosphate.…”

Genetic Proclivities of Two-Component Modulated Aerobiosis

“…In contrast, interference with menaquinone biosynthesis results in the inactivation of ArcB sensor kinase during anaerobic growth; this inactivity can fully be restored by addition of a menaquinone precursor. This vividly proves that menaquinones play a major role in ArcB activation as ArcB is activated under anaerobic and sub-aerobic conditions and is much less active under fully aerobic and micro-aerobic conditions [18].…”