Transcriptional regulation of puc operon expression in Rhodobacter sphaeroides: Involvement of an integration host factor-binding sequence

The putative overlapping consensus sequences (-129 to -105) for binding of fumarate nitrate reductase regulator- and integration host factor (IHF)-like proteins to puc operon upstream DNA of Rhodobacter sphaeroides was protected from DNase I digestion by purified Escherichia coli IHF. The binding of E. coli IHF to the purported IHF-binding site in the puc upstream DNA is highly sequence-specific. The recorded binding affinity was significantly lower than that of E. coli IHF to the λ attP site. Employing site-directed changes in the DNA sequence within the -129 to -105 region, a loss in IHF binding, as monitored through gel retardation analysis, was correlated with alterations in puc operon expression monitored through the use of puc::lacZ transcriptional fusions. These results suggest that the IHF-binding site is involved in repression of puc operon transcription by oxygen as well as modulation of puc operon transcription levels by incident light intensity. Mutations specific to the upstream half of the putative fumarate nitrate reductase regulator-binding site of the puc upstream DNA did not show any physiological effects under the experimental conditions employed. Taken together, these studies reveal that the DNA sequence between -129 to -105 may involve facilitation of the interaction between upstream and downstream cisacting regulatory sequences involved in puc operon expression.