Interdependent expression of the ccoNOQP-rdxBHIS loci in Rhodobacter sphaeroides 2.4.1

Abstract

The rdxBHIS gene cluster of Rhodobacter sphaeroides 2.4.1, located downstream of the ccoNOQP operon encoding the cbb(3) cytochrome c oxidase, is required for the posttranscriptional modification of the cbb(3) cytochrome c oxidase. The cbb(3) cytochrome c oxidase is the main terminal oxidase under microaerobic conditions, as well as a component of the signal transduction pathway controlling photosynthesis gene expression. Because of the intimate functional and positional relationships of the ccoNOQP operon and the rdxBHIS gene cluster, we have examined the transcriptional activities of this DNA region in order to understand their expression and regulation. Northern blot analysis and reverse transcription-PCR, together with earlier complementation analysis, suggested that the ccoNOQP-rdxBHIS cluster is transcribed as ccoNOQP-, ccoNOQP-rdxBH-, rdxBH-, and rdxIS-specific transcripts. Multiple transcriptional start sites have been identified by primer extension analyses: five for ccoN, four for rdxB, and one for rdxI. Transcription from P1(N) of ccoN and P1(B) of rdxB is dependent on the presence of FnrL. LacZ fusion analysis support the above-described studies, especially the importance of FnrL. Expression of the cco-rdx cluster is closely related to photosynthesis gene expression, suggesting that transcript stoichiometry and presumably the stoichiometry of the gene products are critical factors in controlling photosynthesis gene expression.

FIG. 1.

Northern blot analysis of the ccoNOQP-rdxBHIS gene cluster. Total RNA was isolated from R. sphaeroides 2.4.1 and the ccoN::Km (CCON) and ccoP::Tp (CCOP1) mutants grown under phototrophic conditions. Insertion of the Km^r and Tp^r cassettes within the ccoN and ccoP genes, respectively, is indicated. Labeled lines with asterisks above the open bar designate the 5′ and 3′ ends of the riboprobes used. The arrows under the open bar indicate the deduced transcripts. The 3,434-bp ccoNOQP operon (1,608, 725, 204, and 873 bp, respectively) and the 4,268-bp rdxBHIS gene cluster (1,434, 459, 2,214, and 159 bp, respectively) are separated by 235 bp. Approximately 10 μg of total RNA was loaded onto each lane. The ³²P-labeled riboprobes corresponding to the rdxB, rdxH, rdxI, rdxS, ccoN, and 23S rRNA genes were used for hybridization as described in Materials and Methods. Molecular size ladders are shown to the left. The arrow in the ccoN hybridization panel indicates the transcript corresponding to the position of the longest transcript which was observed with the rdxB and rdxH riboprobes.

FIG. 2.

RT-PCR of R. sphaeroides 2.4.1 total RNA. The locations of the oligonucleotides used for RT-PCR are shown by the arrows. The PCR products amplified by using the primers a, b, c, d, and e were subjected to electrophoresis on a 1% agarose gel (PCR product with primers a plus d and a plus e) or a 2% agarose gel (PCR products with primers a plus b and a plus c). The positions and sizes of the 1-kb plus DNA ladder from Invitrogen are indicated to the left (lanes M). The RT-PCR lacking a template failed to detect any contamination (lane 1), and total RNA without reverse transcriptase also failed to detect DNA contamination in the template (lanes 2), with the cDNA sample (lanes 3), and with positive control DNA, i.e., pJR235 containing the ccoNOQP-rdxBHIS genes in pRK415 (lanes 4).

FIG. 3.

Primer extension experiments and nucleotide sequence upstream of the ccoN gene. (A) Total RNAs isolated from the wild type grown under aerobic conditions (+O₂) and under photosynthetic conditions (−O₂), the wild type containing ccoNOQP in pRK415 [p(ccoNOQP)], the wild type containing ccoNOQP-rdxBH in pRK415 [p(ccoNOQP-rdxBH)], the fnrL mutant, and the wild type containing the fnrL gene in pRK415 [p(fnrL)] were used for primer extension experiments with primer 126. The 5′ ends (P1_N to P5_N) are indicated on the left. Sizes of the primer extension products were determined by comparison to the sequencing ladder obtained from the same primer. P3_N, P4_N, and P5_N were confirmed by extended gel electrophoresis (data not shown). (B) Quantification of signal levels of P1_N and P5_N. Since the level of P2_N was very low and the bands for P3_N and P4_N are indistinguishable, we did not quantitate these signals. The levels of P1_N and P5_N of wild-type cells grown aerobically were considered to be 100%, and other values are given relative to that value. ND, not detectable. (C) Nucleotide numbering is relative to the putative translational start site (designated +1) of the ccoN gene, which is shown in italics. The binding sites of the primers used in the primer extension experiments are underlined with arrows, and the number under the arrow is the primer number used for the experiment. The 5′ ends and directions of transcription are marked by arrows above the determined 5′ ends and are indicated as P1_N, P2_N, P3_N, P4_N, and P5_N. The BamHI site was used in order to subclone the ccoNOQP operon into pRK415. Two putative FnrL binding sites are double underlined. A putative σ⁵⁴ motif is indicated by an underline. The nucleotides mentioned above are in boldface, except the sequence for primer 167.

FIG. 4.

Primer extension experiments and nucleotide sequence of the upstream regions of rdxB (A and C) and rdxI (B and D). (A) Total RNAs isolated from the wild type (lanes 1 and 4), the wild type containing the rdxBH genes in pRK415 (lanes 2 and 6), and the fnrL mutant (lanes 3 and 5) were used for primer extension experiments. The strains were grown under aerobic conditions. The levels of P2_B and P1_I of the wild type were considered to be 100%, and other levels are compared to these values. The P1_B transcript from another primer extension experiment is shown by a separate gel (small box inside the gel). DNA sequence and transcripts (P1_B to P4_B) are indicated on the left and right, respectively. (B) Total RNAs isolated from the wild type (lane 1), the wild type containing the rdxBHIS genes in pRK415 (lane 2), and the wild type containing the rdxIS genes in pRK415 (lane 3) were used for primer extension experiments with primer 101. The strains were grown aerobically. DNA sequence and transcript (P1_I) are indicated on the left and right, respectively. (C) The translational start site (designated +1) for rdxB is indicated with an arrow above the sequence which is shown in italics. A putative FnrL binding sequence is doubly underlined. (D) The translational start site (designated +1) for rdxI is indicated with an arrow above the sequence, which is shown in italics. The 5′ ends and directions are marked by arrows above the determined 5′ ends. The nucleotides mentioned above are in boldface. The binding sites of the primers used in the primer extension experiments are underlined with an arrow, and the numbers under the arrows indicate the primers used for the experiments.

FIG. 5.

β-Galactosidase activities of the various rdxBHIS::lacZ fusions in the wild-type and fnrL mutant strains. The positions of relevant RNA 5′ ends and 5′ endpoints of lacZ fusions are indicated. Strains were grown aerobically (sparged with 69% N₂-30% O₂-1% CO₂) and photosynthetically (sparged with 95% N₂-5% CO₂) to an OD₆₀₀ of 0.4 ± 0.1. Activities are expressed as nanomoles of o-nitrophenyl-β-d-galactopyranoside hydrolyzed per minute per milligram of protein extract. Values with the ranges indicated are the average of two independent determinations. pCF1010 is the control plasmid vector containing the promoterless lacZ used to construct the rdxBHIS::lacZ fusions as described in Materials and Methods. Error bars indicate standard deviations.

FIG. 6.

Northern blot analysis of R. sphaeroides 2.4.1 grown under aerobic conditions. Total RNA was isolated from the wild type containing pRK415, the wild type containing ccoNOQP in pRK415 [p(ccoNOQP)], the wild type containing ccoNOQP-rdxBH in pRK415 [p(ccoNOQP-rdxBH)], the wild type containing rdxIS in pRK415 [p(rdxIS)], the wild type containing rdxBH in pRK415 [p(rdxBH)], and the wild type containing rdxBHIS in pRK415 [p(rdxBHIS)]. The strains were grown in Sistrom's minimal medium A containing tetracycline, by sparging with 69% N₂-30% O₂-1% CO₂, to an OD₆₀₀ of 0.3 ± 0.05. Approximately 10 μg of total RNA was loaded onto each lane. Riboprobes specific for puf, puc, and the 23S rRNA gene were used for hybridization as described in Materials and Methods. The 2.7- and 0.5-kb transcripts for puf are present (6, 11). The levels of puc and puf were normalized to the level of rRNA. The mRNA level in the wild type containing pRK415 was considered 100%, and other values are given relative to that value.

FIG. 7.

Diagrams illustrating the genetic organization of the cco(fix)NOQP-rdxBHIS (fix/ccoGHIS) cluster and their flanking regions in different organisms. Homology searching was done with rdxH and rdxS because rdxB and rdxI have high homology to bacterial ferredoxin and CPx-type metal transporter which are normally found in many other bacteria. The DDBJ/EMBL/GenBank accession numbers of the sequences are as follows: R. sphaeroides, U58092 and AF202779; Caulobacter crescentus, AE005673; Neisseria meningitidis, AE002098 and AL162759; Vibrio cholerae, AE004222; Campylobacter jejuni, AL111168; Helicobacter pylori, AE001439 and AE000511; Agrobacterium tumefaciens, AE007869 and AE008688; and Pseudomonas aeruginosa, AE004091. C. crescentus OmpW is a putative outer membrane protein. The fnrL and fnr genes encode anaerobic transcriptional regulators. NMB1454 and NMB1453 are rdxBH homologues. VC1435 and Atu1531 are hypothetical proteins, and hemZ corresponds to the genes encoding isoenzymic forms of coproporphyrinogen III oxidase. NMB1515, which is composed of 436 amino acids, shows similarity to the central and carboxyl regions of the R. sphaeroides RdxI protein (737 amino acids) and is a putative transporter. cj1492c-cj1491c is a two-component regulatory system. cj1155c is an rdxI homologue. Atu1532 and Atu1533 are proteins related to glyoxylate and bleomycin resistance. The rdxB homologue of V. cholerae and the rdxB and rdxI homologues of H. pylori are not indicated. The rdxH homologue was not found for H. pylori.