Relationships of Arc and Psp systems 17 Fig. 1. pIV-dependent induction of psp expression under different growth conditions. pIV secretin (from pGJ4) induced psp expression in E. coli [MG1655 f(pspA–lacZ); MVA44] cells grown under aerobic, microaerobic or anaerobic conditions as measured using a b-Gal assay (see Experimental procedures). WT – wild type, MVA44. Insets: Western blot analyses of pIV expression in corresponding cells using antibodies against pIV (a-pIV) (see Experimental procedures). The relative expression levels (in arbitrary units) of pIV were determined as follows: aerobic – 1.57, 2.00, 1.93, 1.84, 2.03; microaerobic – 2.13, 2.27, 2.17, 2.32, 1.77; anaerobic – 1.71, 2.35, 1.82, 2.25, 1.77, and illustrate that the overall variability in pIV expression is ~1.5-fold. aerobic respiration and upregulates genes involved in anaerobic respiration and fermentation. Notably, the ArcAB system is also known to be of particular importance under microaerobic growth conditions, where oxygen is limiting rather than being completely absent (Alexeeva et al., 2003). Recently, ArcB-independent psp induction was reported (Seo et al., 2007). We therefore explored further the rela- tionship(s) between the Psp and Arc system under differ- ent growth conditions and now report that involvement of Arc is conditional, consistent with both observations (Jovanovic et al., 2006; Seo et al., 2007). We ﬁnd that ArcAB is required to propagate pIV-dependent induction of the Psp response under microaerobic, but not under aerobic or anaerobic growth conditions. The Arc system appears to be important for Psp-signal ampliﬁcation, via the ArcB–ArcA phosphorelay. In addition, activated ArcB is by itself observed to elevate psp expression. Further, we have identiﬁed a direct binding interaction between ArcB and PspB that supports part of a Psp-speciﬁc signal transduction pathway used under microaerobic growth conditions. Notably in anaerobiosis, pIV-dependent psp expression is largely independent of PspBC (two positive regulators of the Psp response) and ArcAB, suggesting either that the repressive PspA–PspF complex is itself capable of recognizing inducing signals, or that PspBC and ArcAB are substituted by other gene products. Results PIV-dependent induction of psp expression under different growth conditions We measured pIV-dependent psp induction in strains with different complements of arc and psp genes grown under aerobic, microaerobic and anaerobic conditions (see Experimental procedures) where the ArcAB proteins are known to have differing importance (Fig. 1). We found that psp induction is dependent on ArcAB only in micro- aerobiosis and on PspBC (positive regulators) in micro- aerobiosis and aerobiosis (Fig. 1). In aerobiosis, a requirement for the ArcAB system is not apparent (Fig. 1). Under anaerobic growth, full pIV-dependent psp induction is largely independent of PspBC and the Arc system, when compared with microaerobiosis (Fig. 1). These dif- fering dependencies on Arc are consistent with the ArcAB system functioning as a microaerobic redox regulator, required under microaerobic but not aerobic or anaerobic conditions (Alexeeva et al., 2003). Notably, under microaerobic growth conditions, the requirement for ArcB is more pronounced then for ArcA. Importantly, control reactions demonstrate that the different growth conditions per se do not affect the activity from the psp promoter since psp expression in DpspA cells is similar (Fig. S1A). Further since the level of psp expression in control strains DpspA DarcB f(pspA–lacZ) and DpspA DarcA f(pspA– lacZ) are unaffected by ArcB and ArcA, we infer that the Arc proteins may contribute to relieving PspA-negative regulation in microaerobiosis (Fig. S1A). In psp induction assays we used the outer membrane (OM) secretin, pIV, to induce pspA–lacZ expression. It is believed that pIV mislocalizes to the IM thereby impairing cell membrane integrity and resulting in a psp-inducing stress signal similar to that caused by PulD (Guilvout et al., 2006). To ascertain that the strains assayed were exposed to equivalent (pIV-induced) levels of ‘stress’, we tested pIV expression and determined the amount of pIV localized within the IM. As shown in Fig. 1, pIV expression did not change greatly in the different backgrounds tested or when the cells were grown under differing aeration. Addi- tionally, the distribution of pIV between the IM and OM was similar in microaerobiosis in the wild-type (WT), arcA, arcB and pspBC deletion backgrounds (Fig. S1B). © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 74, 16–28