AerR, a second aerobic repressor of photosynthesis gene expression in Rhodobacter capsulatus

Abstract

Open reading frame orf192, which is located immediately upstream of the aerobic repressor gene crtJ, was genetically and biochemically demonstrated to code for a second aerobic repressor (AerR) of photosynthesis gene expression in Rhodobacter capsulatus. Promoter-mapping studies indicate that crtJ has its own promoter but that a significant proportion of crtJ expression is promoted by read-through transcription of orf192 (aerR) transcripts through crtJ. Disruption of aerR resulted in increased photopigment biosynthesis during aerobic growth to a level similar to that of disruption of crtJ. Like that reported for CrtJ, beta-galactosidase assays of reporter gene expression indicated that disruption of aerR resulted in a two- to threefold increase in aerobic expression of the crtI and pucB operons. However, unlike CrtJ, AerR aerobically represses puf operon expression and does not aerobically repress bchC expression. Gel mobility shift analysis with purified AerR indicates that AerR does not bind to a bchC promoter probe but does bind to the crtI, puc, and puf promoter probes. These results indicate that AerR is a DNA-binding protein that targets genes partially overlapping a subset of genes that are also controlled by CrtJ. We also provide evidence for cooperative binding of AerR and CrtJ to the puc promoter region.

FIG. 1.

Expression patterns of aerR and crtJ. (A) β-Galactosidase activities of various aerR and crtJ reporter plasmids in the wild-type strain SB1003 cells grown under aerobic (black bars) and anaerobic (open bars) conditions. ONP, o-nitrophenyl-β-d-galactopyranoside. (B) Anaerobic and aerobic β-galactosidase activities present in various regulatory mutant strains containing plasmid pES15. Wild type is strain SB1003, crtJ is strain CD2-4, aerR is strain ES8, and regA is strain MS01. β-Galactosidase values represent the average of at least three independent assays.

FIG. 2.

Assays for polarity of the aerR::Kan^r insertion mutations. (A) Strains ES7 and ES8 contain the same Kan^r gene insertion in two different orientations. (B) Spectral analysis of membrane fractions from the wild-type strain SB1003, the two aerR-disrupted mutants ES7 and ES8, and the crtJ-disrupted strain CD2-4, grown under anaerobic conditions. (C) Immunoblot analysis of the amounts of FLAG CrtJ synthesized in strains SB1003-FLAG, ES8-FLAG, and ES7-FLAG.

FIG. 3.

(A) β-Galactosidase analysis of bchC::lacZ expression from aerobically grown SB1003, ES7, ES7B, ES7C, and CD2-4 cells containing plasmid pDAY23Ω. The β-galactosidase activities are the means of three independent analyses, with the values representing micromoles of o-nitrophenyl-β-d-galactopyranoside hydrolyzed per minute per milligram of protein. Standard deviations are indicated by the error bars. (B) Alignment of the helix-turn-helix DNA-binding region of CrtJ and PpsR. The lengths (in amino acid numbers) of the two proteins are indicated in brackets. The mutated codons that are found in the photosynthetically competent suppressors of strain ES7 are indicated with an arrow.

FIG. 4.

Measurement of photosynthesis gene expression in the wild-type strain SB1003, in the crtJ-disrupted strain CD2-4, in the aerR-disrupted strain ES8, and in the strain CD3 with a deletion of aerR-crtJ. The four graphs represent aerobic expression levels of the pucB::lacZ fusion plasmid pLHIIZ (26), the crtI::lacZ fusion plasmid pCrtI:ZΩ, the bchC::lacZ fusion plasmid pDAY23Ω, and the pufQ::lacZ fusion plasmid pCB532Ω (4). β-Galactosidase activities are as described for Fig. 3.

FIG. 5.

Gel mobility shift assays with purified AerR. The first lane of each gel had only ³²P-labeled probe and heparin (1,000-fold excess), while the second lane had the same probe and heparin preincubated with 2 μM AerR. puc, crtAI, puf, and bchC represent the various promoter probes that were used with each probe containing two CrtJ recognition sequences. P- represents the mobility of the unshifted probe, while -S represents the mobility of the AerR-shifted probe.

FIG. 6.

Cooperation between AerR and CrtJ in binding to the puc promoter region. (A) Gel mobility shift of CrtJ binding to the downstream puc palindrome in the absence or presence of AerR. Lane 1 is of probe only, while lanes 2 to 4 contain probe plus CrtJ at 141, 281, and 656 nM, respectively. Lanes 5 to 7 contain AerR at 24, 48, and 72 nM, respectively. Lanes 8 to 10 contain CrtJ at 141 nM in all lanes, as well as AerR at 24, 48, and 72 nM, respectively. Lanes 11 to 13 contain 281 nM CrtJ in each lane as well as AerR at 24, 48, and 72 nM, respectively. Lanes 14 to 16 contain 656 nM CrtJ in each lane as well as AerR at 24, 48, and 72 nM, respectively. (B) The graphs show a plot of the percentage of shifted ³²P-labeled pucB probe versus the amount of CrtJ in the assay. Filled circles represent the DNA-binding isotherm of CrtJ with no AerR present, empty circles represent the CrtJ DNA-binding isotherm obtained in the presence of 24 nM AerR, and filled inverted triangles represent the CrtJ DNA-binding isotherm obtained in the presence of 48 nM AerR. The left, middle, and right graphs represent the binding curves obtained with a probe containing only the upstream CrtJ recognition palindrome, with a probe containing only the downstream recognition palindrome, and with a probe containing both recognition palindromes, respectively.

FIG. 7.

A diagram depicting CrtJ and AerR circuits that repress individual transcripts. Both proteins affect transcription initiation (lines with a single arrowhead) at promoters that contain distantly removed CrtJ recognition palindromes (lines with double arrows). Only CrtJ represses the bchC promoter that contains two closely spaced palindromes (8 bp apart), and only AerR represses the puf promoter, which does not contain CrtJ recognition palindromes.

FIG. 8.

A model depicting possible interactions between CrtJ and AerR at promoters that contain distantly spaced palindromes. The number of AerR symbols drawn relative to that of CrtJ symbols is arbitrary.