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

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 aa3 respiratory complex. The third promoter is that for the cycFG operon, which encodes two c-type cytochromes of unknown function, cytochrome c554 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 delta 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.

Respiratory pathways of Rhodobacter sphaeroides 2.4.1T: identification and characterization of genes encoding quinol oxidases

“…The aa 3 -type cytochrome c oxidase, encoded by the cta genes, is a classic member of the heme-copper superfamily of bacterial oxidases containing the binuclear heme a 3 -copper (Cu B ) center as the active site [2]. cta Genes are repressed when the O 2 tension is lowered, in accordance with the proposed role of the aa 3 oxidase as a low-a¤nity oxidase functional under high O 2 conditions [3].…”

Overexpression and Purification of CytochromecOxidase fromRhodobacter sphaeroides

“…A 207-bp 5Ј-region in front of the coxI was found to be sufficient for the expression of subunit I; this region appears to contain an oxygen-regulated promoter for coxI (9). Similarly, an oxygen-regulated promoter for coxII/III has been located 150 bp upstream of the coxII coding region (20). Constructs made with either orientation of the coxII/III operon, with respect to that of coxI, showed equal overexpression, consistent with the presence of separate promoters for the two operons.…”

Oxygen intervention in the regulation of gene expression: the photosynthetic bacterial paradigm

“…This terminal oxidase has a low affinity for oxygen, and is used for aerobic respiration when oxygen concentrations are high. The predicted regulation of these genes by FnrL at first appears to be unsubstantiated by transcription analyses [64]. However, there are two known cases in which genes can be both directly and indirectly regulated by FnrL [55,65], and the aa 3 -type cytochrome c oxidase genes might represent additional examples.…”