This suggested that rnf1 clusters might be related to N2 fixation in diazotrophs. Single clusters found in Archaea, and mainly in anaerobic Firmicutes, were clearly separated. Reporter strains of Azoarcus sp. carrying transcriptional rnfA1::gusA BH72 (BH::rnfA1-gusA) or rnfA2::gusA (BH::rnfA2-gusA) fusions were constructed to determine whether rnf expression was nitrogen-regulated. rnfA1 expression was found to be enhanced 13-fold under N2 fixation (1175 ± 89 Miller Units) as compared with the presence of ammonium (85 ± 11 Miller Units), whereas rnfA2 was found to be constitutively expressed at low levels regardless of check details the nitrogen source (ammonium, 190 ± 8 Miller Units; N2, 200 ± 15 Miller Units). Expression of nifH in Azoarcus sp. strain BH72 is found to be elevated in a similar way under growth on N2 (Egener et al., 1999) suggesting a similar regulatory circuit. Accordingly, the upstream region of the rnf1 cluster carried putative elements typical of regulation of nif gene expression: a putative −12/−24 consensus for σ54–RNA polymerase binding, a conserved palindrome sequence for integration host factor binding and putative UAS (upstream activating sequences) for NifA and NtrC binding (Fig. S2A). In contrast, no UAS or σ54 binding sequences were identified within 3 kb upstream of the rnf2 gene cluster. To investigate the role of the respective transcriptional activators putatively involved in regulating rnf1 expression, a transcriptional fusion construct rnfA1::gusA (pK18mob2rnf1-gusA) was chromosomally integrated into wild-type strain BH72, ntrBC- strain BntrBsp or nifLA- strain BHLAO respectively. Generally, rnf1 expression in all the three strains was low while growing aerobically (complex medium or ammonium) or microaerobically on ammonium (Fig. S2B). In the absence of ntrBC, expression of rnf1 was lower by a factor of two (statistically significant, P < 0.05) as compared with wild type or BHLAO under such conditions (except microaerobically on ammonium), indicating a role for NtrBC, the typical transcriptional activation system for nitrate-related genes. However, with nitrate as nitrogen source rnf1 expression was enhanced in the absence of ntrBC (Fig. S2B). For A. brasilense and R. capsulatus, it has been reported that NtrX can mediate cross-talk between the NtrBC and NtrYX two component systems (Drepper et al., 2006; Assumpcao et al., 2007). As the genome of strain BH72 harbours ntrY- and ntrX-like genes, the alternative transcription activator NtrX might complement NtrC functions. However, NifA is likely the major transcriptional activator, as high expression under conditions of nitrogen fixation was strongly reduced in the nifA mutant (P < 0.05). To understand the physiological role of Rnf proteins in Azoarcus sp. strain BH72, mutant strains were generated by deleting either the rnf1 cluster or the rnf2 cluster (Fig. S1 and Table S1). Unlike R.