No abstract

The Cpx response is one of several envelope stress responses that monitor and maintain the integrity of the gram-negative bacterial envelope. While several conditions that are known or predicted to generate misfolded inner membrane proteins activate the Cpx response, the molecular nature of the Cpx inducing cue is not yet known. Studies have demonstrated that mutation of multidrug efflux pumps activates the Cpx response in many gram-negative bacteria. In Vibrio cholerae, pathway activation is due to accumulation of the catechol siderophore vibriobactin. However, the mechanism by which the Cpx response is activated by mutation of efflux pumps in Escherichia coli remains unknown. Here we show that inhibition of efflux by deletion of tolC, the outer membrane channel of several multidrug efflux pumps, activates the Cpx response in E. coli as a result of impaired efflux of the siderophore enterobactin. Enterobactin accumulation in the tolC mutant reduces activity of the nicotinamide adenine dinucleotide (NADH) oxidation arm of the aerobic respiratory chain. However, the Cpx pathway remains active in the tolC mutant when either NADH dehydrogenase I, NADH dehydrogenase II, or cytochrome bo3 is absent. Finally, we show that the Cpx response down-regulates transcription of the enterobactin biosynthesis operon. These results suggest that the Cpx response promotes adaptation to envelope stress in enteric bacteria that are exposed to iron-limited environments, which are rich in envelope-damaging compounds and conditions.

21The Cpx response is one of several envelope stress responses that monitor and 22 maintain the integrity of the gram-negative bacterial envelope. While several conditions that are 23 known or predicted to generate misfolded inner membrane proteins activate the Cpx response, 24 the molecular nature of the Cpx inducing cue is not yet known. Studies have demonstrated that 25 mutation of multidrug efflux pumps activates the Cpx response in many gram-negative bacteria. 26In Vibrio cholerae, pathway activation is due to accumulation of the catechol siderophore 27 vibriobactin. However, the mechanism by which the Cpx response is activated by mutation of 28 efflux pumps in Escherichia coli remains unknown. Here we show that inhibition of efflux by 29 deletion of tolC, the outer membrane channel of several multidrug efflux pumps, activates the 30 Cpx response in E. coli as a result of impaired efflux of the siderophore enterobactin. 31Enterobactin accumulation in the tolC mutant reduces activity of the NADH oxidation arm of the 32 aerobic respiratory chain. However, NADH dehydrogenase I, NADH dehydrogenase II, and 33 cytochrome bo 3 do not contribute to Cpx pathway activation in the E. coli tolC mutant. We show 34 that the Cpx response down-regulates transcription of the enterobactin biosynthesis operon. 35These results suggest that the Cpx response promotes adaptation to envelope stress in enteric 36 bacteria that are exposed to iron-limited environments, which are rich in envelope-damaging 37 compounds and conditions. 38 Wiriyathanawudhiwong et al., 2009). Furthermore, accumulation of several metabolites 58 increases expression of the TolC-dependent AcrAB multidrug efflux system as a compensatory 59 mechanism to increase metabolite secretion (Helling et al., 2002;Ruiz and Levy, 2014). 60Blocking metabolite secretion by mutating tolC or TolC-dependent efflux systems increases 61 sensitivity to cysteine, the siderophore enterobactin, and intermediates of heme biosynthesis, 62suggesting that metabolite accumulation is toxic (Tatsumi and Wachi, 2008; Vega and Young, 63 2014;Wiriyathanawudhiwong et al., 2009). In support of this hypothesis, numerous cellular 109 Iron limitation induces the Cpx response in the tolC mutant 110To confirm that inhibition of efflux activates the Cpx response in E. coli, we measured 111 Cpx pathway activity in a tolC mutant using a cpxP-lacZ transcriptional reporter. No change in 112 cpxP-lacZ reporter activity was observed when E. coli were grown in LB ( figure 1A). This was 113 surprising given that under similar growth conditions, expression of the periplasmic chaperone 114Spy has been shown to increase in a tolC mutant via the Cpx response (Acosta et al., 2014; 115 Rosner and Martin, 2013). Nonetheless, when E. coli were grown in M9 minimal medium we 116 observed a nearly eleven-fold increase in cpxP-lacZ activity in the tolC mutant ( figure 1A). This 117 increase was abolished in E. coli lacking cpxA ( figure 1B), suggesting that inhibition of efflux 118 generates envelope stress that ...