Transcriptional control of several aerobically induced cytochrome structural genes in Rhodobacter sphaeroides

Graduate Program in Cell and Molecular Biology, University of Wisconsin-Madison, Madison, WI 53706, USA Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA 1 Author for correspondence: Timothy J. Donohue. Tel: +1 608 262 4663. Fax: +1 608 262 9865. e-mail: tdonohue@bact.wisc.edu ABSTRACT To decipher how the synthesis of energy-transducing enzymes responds to environmental cues, the response of three Rhodobacter sphaeroides aerobic cytochrome gene promoters was analysed under different conditions. Two of these promoters are upstream of structural genes ( ctaD and coxII ) for individual subunits of the cytochrome aa 3 respiratory complex. The third promoter is that for the cycFG operon, which encodes two c -type cytochromes of unknown function, cytochrome c 554 and CycG. Primer extension analysis identified a single oxygen-responsive transcription start site for each gene. Utilizing operon fusions to Escherichia coli lacZ as a measure of promoter activity, transcription from the ctaD, coxII and cycFG promoters was approximately twofold higher when cells were grown at high (30%) oxygen tensions than under low (2%) oxygen or anaerobic (photosynthetic) conditions. Analysis of promoter function using specific host mutations indicated that loss of the R. sphaeroides FNR homologue, FnrL, causes a small, but reproducible, increase in cycFG and coxII transcription when cells are grown at 2% oxygen. However, neither the FnrL mutation nor alterations in sequences related to a consensus target site for the E. coli FNR protein increased function of any of these three promoters to that seen under aerobic conditions in wild-type cells. From this we conclude that FnrL is not solely responsible for reduced transcription of these three aerobic cytochrome genes under low oxygen or anaerobic conditions. When activity of these three promoters was monitored after cells were shifted from anaerobic (photosynthetic) conditions to a 30% oxygen atmosphere, it took several cell doublings for LacZ levels to increase to those found in steady-state 30% oxygen cultures. From these results, it appears that activity of these promoters is also regulated by a stable molecule whose synthesis or function responds slowly to the presence of high oxygen tensions. Keywords: cytochromes, gene expression, transcriptional control, anaerobic regulation, oxygen regulation