Positive transcriptional feedback controls hydrogenase expression in Alcaligenes eutrophus H16

The protein HoxA is the central regulator of the Alcaligenes eutrophus H16 hox regulon, which encodes two hydrogenases, a nickel permease and several accessory proteins required for hydrogenase biosynthesis. Expression of the regulatory gene hoxA was analyzed. Screening of an 8-kb region upstream of hoxA with a promoter probe vector localized four promoter activities. One of these was found in the region immediately 5' of hoxA; the others were correlated with the nickel metabolism genes hypA1, hypB1, and hypX. All four activities were independent of HoxA and of the minor transcription factor sigma(54). Translational fusions revealed that hoxA is expressed constitutively at low levels. In contrast to these findings, immunoblotting studies revealed a clear fluctuation in the HoxA pool in response to conditions which induce the hox regulon. Quantitative transcript assays indicated elevated levels of hyp mRNA under hydrogenase-derepressing conditions. Using interposon mutagenesis, we showed that the activity of a remote promoter is required for hydrogenase expression and autotrophic growth. Site-directed mutagenesis revealed that P(MBH), which directs transcription of the structural genes of the membrane-bound hydrogenase, contributes to the expression of hoxA under hydrogenase-derepressing conditions. Thus, expression of the hox regulon is governed by a positive feedback loop mediating amplification of the regulator HoxA. These results imply the existence of an unusually large (ca. 17,000-nucleotide) transcript.