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, 193 (13), 3207-19

The Anaerobe-Specific Orange Protein Complex of Desulfovibrio Vulgaris Hildenborough Is Encoded by Two Divergent Operons Coregulated by σ54 and a Cognate Transcriptional Regulator

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The Anaerobe-Specific Orange Protein Complex of Desulfovibrio Vulgaris Hildenborough Is Encoded by Two Divergent Operons Coregulated by σ54 and a Cognate Transcriptional Regulator

Anouchka Fiévet et al. J Bacteriol.

Abstract

Analysis of sequenced bacterial genomes revealed that the genomes encode more than 30% hypothetical and conserved hypothetical proteins of unknown function. Among proteins of unknown function that are conserved in anaerobes, some might be determinants of the anaerobic way of life. This study focuses on two divergent clusters specifically found in anaerobic microorganisms and mainly composed of genes encoding conserved hypothetical proteins. We show that the two gene clusters DVU2103-DVU2104-DVU2105 (orp2) and DVU2107-DVU2108-DVU2109 (orp1) form two divergent operons transcribed by the σ(54)-RNA polymerase. We further demonstrate that the σ(54)-dependent transcriptional regulator DVU2106, located between orp1 and orp2, collaborates with σ(54)-RNA polymerase to orchestrate the simultaneous expression of the divergent orp operons. DVU2106, whose structural gene is transcribed by the σ(70)-RNA polymerase, negatively retrocontrols its own expression. By using an endogenous pulldown strategy, we identify a physiological complex composed of DVU2103, DVU2104, DVU2105, DVU2108, and DVU2109. Interestingly, inactivation of DVU2106, which is required for orp operon transcription, induces morphological defects that are likely linked to the absence of the ORP complex. A putative role of the ORP proteins in positioning the septum during cell division is discussed.

Figures

Fig. 1.
Fig. 1.
orp gene cluster organization in D. vulgaris Hildenborough. (A) PCR products of intergenic regions between DVU2103-DVU2104 (a), DVU2104-DVU2105 (b), DVU2105-DVU2106 (c), DVU2106-DVU2107 (d), DVU2107-DVU2108 (e), DVU2108-DVU2109 (f), and DVU2109-DVU2110 (g) from RNA (lanes 1), cDNA (lanes 2), and genomic DNA (lanes 3). Molecular size markers are indicated on the left and right (in bp). (B) Structural organization of the orp gene cluster. The arrows indicate the direction of transcription. (C) Relative quantification of orp gene expression by real-time PCR of DVU2104, DVU2106, and DVU2108 transcripts. The averages of three independent biological samples are shown, with standard deviations calculated from replicates. The amount of DVU2108 transcripts was taken as a reference (arbitrary units).
Fig. 2.
Fig. 2.
Identification of signal transcription of orp1, orp2, and DVU2106. (A) Primer extension analysis using RNA extracted from D. vulgaris Hildenborough. The arrowheads indicate the primer extension products generated using primers DVU2105_rev(+1) (annealing within the DVU2105 gene) (left) and DVU2107_rev (annealing within the DVU2107 gene) (right). Lanes C, T, A, and G correspond to the sequence reads generated using the same primers. Sequences around the transcriptional +1 position are indicated, and the sequence patterns are shown on the right. (B) Sequences of the DVU2105-DVU2106 and DVU2106-DVU2107 intergenic regions with the positions of the transcriptional start sites. The nucleotide numbers correspond to the locations in the genome sequence (27). The predicted σ54 promoters of orp2 and orp1 are boxed. The predicted Shine-Dalgarno (SD) consensuses are shown in boldface, and the initiation codons of DVU2105 and DVU2107 are underlined.
Fig. 3.
Fig. 3.
Sequence analyses of the orp1 and orp2 promoters. The −12/−24 elements of the orp1 and orp2 σ54 promoters are indicated by solid-line boxes, and the putative −10/−35 elements of the DVU2106 σ70 promoter are indicated by dashed boxes. The drawing emphasizes the positions of the two putative palindromic DVU2106-binding sequences. The positions and lengths of the probes used for EMSA are indicated by arrows.
Fig. 4.
Fig. 4.
The σ54-RNA polymerase interacts with the promoter regions of orp1 and orp2. Shown is EMSA of the promoter regions of orp1 (A), orp2 (B), and the 5′ (C) and 3′ (D) fragments of the orp2 promoter with a reconstituted σ54-RNA polymerase complex (Eσ54) (lane 1, no protein; lane 2, 25 nM; lane 3, 100 nM; lane 4, 250 nM). Competition experiments using a double-stranded consensus σ54-binding box (lane 5, molecular ratio, probe-competitor, 1:5; lane 6, molecular ratio, 1:25) or consensus Fur-binding box (lane 7, molecular ratio, probe-competitor, 1:5; lane 8, molecular ratio, 1:25). Retarded probe-Eσ54 complexes are indicated by asterisks. As a control, a 260-bp fragment of the σ70-dependent E. coli sci-1 promoter (indicated by ∼) was incubated in the presence of Eσ54 (250 nM; lane 9). The arrow in panel D indicates an unrelated amplicon obtained by PCR when the 5′ fragment of the orp2 promoter was amplified.
Fig. 5.
Fig. 5.
The σ54-dependent transcriptional regulator DVU2106 interacts with the intergenic region of orp1 and orp2. Shown is EMSA of the promoter regions of orp1 (A), orp2 (B), and the 5′ (C) and 3′ (D) fragments of the orp2 promoter using purified DVU2106c (lane 1, no protein; lane 2, 100 nM; lane 3, 200 nM; lane 4, 500 nM). Competition experiments using a double-stranded putative palindromic DVU2106-binding sequence (lane 5, molecular ratio, probe-competitor, 1:5; lane 6, molecular ratio, 1:25) or a consensus σ54-binding box (lane 7, molecular ratio, probe-competitor, 1:5; lane 8, molecular ratio, 1:25). Retarded probe-DVU2106c complexes are indicated by asterisks. As a control, a 260-bp fragment of the σ70-dependent E. coli sci-1 promoter (indicated by ∼) was incubated in the presence of DVU2106c (500 nM; lane 9). The arrow in panel D indicates an unrelated amplicon obtained by PCR when the 5′ fragment of the orp2 promoter was amplified.
Fig. 6.
Fig. 6.
Activities of the reporter fusions in the heterologous host E. coli. The lacZ reporter fusions with the promoter regions of orp1 (A), DVU2106 (B), and orp2 (C) are represented on the left. The transcript start sites are indicated by bent arrows. The positions of the −10 and −35 consensus sequences of the σ70 promoter and of the −12 and −24 consensus sequences of the σ54 promoters are indicated by black rectangles. The β-galactosidase activities of these reporter fusions in various backgrounds are shown on the right: the E. coli wild-type strain and its isogenic rpoN derivative in the absence (dark-gray and white bars, respectively) or presence (light-gray and black bars, respectively) of DVU2106 production. The activity is the average of three independent measurements (the error bars show the standard deviations).
Fig. 7.
Fig. 7.
DVU2106 represses its own transcription. (A) The promoter of DVU2106. The putative −10 and −35 elements of the σ70 DVU2106 promoter are underlined and in boldface. The proposed Shine-Dalgarno consensus is shown in boldface, and the initiation codon is underlined. The DVU2106-binding site is indicated by a box showing its overlap with the putative −10 element. (B) EMSA of the 5′ fragment of the orp2 promoter using the σ70-RNA polymerase complex (Eσ70) (lane 1, no protein; lane 2, 25 nM; lane 3, 100 nM; lane 4, 250 nM), the core enzyme (E) (lane 8, RNAP devoid of the sigma factor, 250 nM), or the purified 2106 protein (lane 9, 200 nM; lane 10, 500 nM). Competition experiments between Eσ70 and 2106c (lane 5, 250 nM Eσ70 plus 200 nM 2106c; lane 6, 250 nM Eσ70 plus 500 nM 2106c; lane 7, 250 nM Eσ70 plus 500 nM unrelated E. coli NtrC EBP).
Fig. 8.
Fig. 8.
Endogenous pulldown experiments using C-terminally strep-tagged 2103, 2108, and 2109 as bait. (A) SDS-PAGE of the affinity purification fractions. The strep-tagged (St) protein used as bait is indicated at the top of each lane, and the orp gene clusters identified by mass spectrometry are indicated by arrows. The degradation products of DVU2109 are indicated by plus signs. The molecular mass markers are indicated in kDa. (B) Summary of the ORP interaction network deduced from panel A (DVU2108 partners, solid line; DVU2109 partners, dashed line, DVU2103 partners, dotted line).
Fig. 9.
Fig. 9.
Effect of DVU2106 inactivation on cell morphology. (A and B) Phase-contrast micrographs of cells of wild-type D. vulgaris Hildenborough (A) and DvH(p2106::2106PAS) (B). (C) Histograms plotting cell length frequency distributions of wild-type D. vulgaris Hildenborough and DvH(p2106::2106PAS).
Fig. 10.
Fig. 10.
Schematic representation of the regulatory mechanisms of the orp gene cluster in D. vulgaris Hildenborough. The positions of the σ54 and σ70 promoters are indicated by bent arrows, and the DVU2106-binding sequences are indicated by hatched rectangles. The DVU2106 transcriptional regulator plays a positive role in the expression of the σ54-dependent orp1 and orp2 operons and exerts a negative retrocontrol on its own σ70-dependent expression.

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