22 G. Jovanovic, C. Engl and M. Buck Fig. 4. Activities of the ArcBLeuZm protein. A. ArcB LeuZm has an ‘on’ kinase activity and supports induction of psp expression. Activities of overexpressed ArcBLeuZm and its corresponding phosphorelay mutants were assessed in a microaerobically grown DarcB strain (MVA59) also carrying the pﬂ–lacZ (pGJ46) transcriptional fusion construct (left panel). Activities of ArcBLeuZm and its variants were compared with ArcBWT and ArcB*. pIV-independent and -dependent induction of psp expression after complementation of the DarcB mutation (MVA63) with ArcBLeuZm and its variants is presented in the right panel. pﬂ or psp expression was measured using b-Gal assays. Vector control was pCA24N. B. ArcBLeuZm reduces cells’ Dy. E. coli DpspF DarcB cells (MVA61) expressing ArcBLeuZm and its variants in the presence and absence of pIV were treated with JC-1 dye and changes in Dy calculated. An increase in the 530/590 ratio indicates a decrease in Dy compared with DpspF DarcB strain with the vector plasmid pCA24N. psp expression. Overexpression of ArcBLeuZm in a DpspF DarcB strain (Fig. 4B) complemented the arcB mutation and reduced Dy in the presence and absence of pIV (similar to ArcB*). However as shown in Fig. 4B, overex- pression of ArcBLHe7u1Z7mA neither complemented the arcB mutation in the presence of pIV, nor reduced Dy in the absence of pIV, consistent with the inability of ArcBLHe7u1Z7mA to upregulate pﬂ transcription or stimulates psp expression (Fig. 4A). It seems that ArcBLeuZm resembles ArcB* (an active ArcB form) for the upregulation of pﬂ transcription (compare Fig. S2B and Fig. 4A) and induction of psp expression (compare Figs 2C and 4A), as well as its effect on Dy (compare Figs 3 and 4B). Introduction of the H292A or H717A mutations in ArcBLeuZm led to the same reduction in activity seen for ArcB*, demonstrating the importance of an intact phosphorelay system for ArcBLeuZm activity. PspBC propagates the signal onwards from the Arc system Since a speciﬁc binding interaction between ArcB and PspB is evident, we next explored the relationship between ArcB and PspB in the context of a PspBC-speciﬁc signal transduction pathway resulting in psp induction. In the absence of PspB (DpspB) neither pIV nor over- expression of ArcB* or ArcAWT signiﬁcantly induced psp expression (Fig. 5). Overexpression of ArcB* in the pres- ence of pIV in DpspB cells failed to induce psp expression (Fig. 5). PspBWT but not PspBLeuZm complemented DpspB for pIV-dependent induction of psp expression (Fig. 5), © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 74, 16–28