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, 9 (1), e86507
eCollection

IHF Is Required for the Transcriptional Regulation of the Desulfovibrio Vulgaris Hildenborough Orp Operons

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IHF Is Required for the Transcriptional Regulation of the Desulfovibrio Vulgaris Hildenborough Orp Operons

Anouchka Fiévet et al. PLoS One.

Abstract

Transcriptional activation of σ(54)-dependent promoters is usually tightly regulated in response to environmental cues. The high abundance of potential σ(54)-dependent promoters in the anaerobe bacteria, Desulfovibrio vulgaris Hildenborough, reflects the high versatility of this bacteria suggesting that σ(54) factor is the nexus of a large regulatory network. Understanding the key players of σ(54)-regulation in this organism is therefore essential to gain insights into the adaptation to anaerobiosis. Recently, the D. vulgaris orp genes, specifically found in anaerobe bacteria, have been shown to be transcribed by the RNA polymerase coupled to the σ(54) alternative sigma factor. In this study, using in vitro binding experiments and in vivo reporter fusion assays in the Escherichia coli heterologous host, we showed that the expression of the divergent orp promoters is strongly dependent on the integration host factor IHF. Bioinformatic and mutational analysis coupled to reporter fusion activities and mobility shift assays identified two functional IHF binding site sequences located between the orp1 and orp2 promoters. We further determined that the D. vulgaris DVU0396 (IHFα) and DVU1864 (IHFβ) subunits are required to control the expression of the orp operons suggesting that they form a functionally active IHF heterodimer. Interestingly results obtained from the in vivo inactivation of DVU0396, which is required for orp operons transcription, suggest that several functionally IHF active homodimer or heterodimer are present in D. vulgaris.

Conflict of interest statement

Competing Interests: Co-author Eric Cascales is a PLOS ONE Editorial Board member. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. IHF is involved in the transcriptional activation of both operons orp1 and orp2.
(A) Schematic representation of the transcriptional elements of promoter regions of orp1, DVU2106 and orp2. The positions of the σ54 and σ70 promoters are indicated by bent arrows and the DVU2106-binding sites are indicated by solid line boxes. The lacZ reporter fusions with the promoter regions of orp1 (B), DVU2106 (C) and orp2 (D) are represented on the left. The transcript start sites are indicated by bent arrows. The positions of the −10 and −35 sequences of the σ70 promoter and the −12 and −24 sequences of the σ54 promoters are indicated by black rectangles. The activities measured in various backgrounds are shown on the right: E.coli wild-type strain (dark-grey) and E.coli ΔihfA strain (light-grey). The activity is the average of three independent measurements (the error bars show the standard deviations).
Figure 2
Figure 2. The integrator Host Factor (IHF) interacts with the promoter regions of orp1 and orp2.
Shown is the gel shift assay of the promoter regions of orp1 (A) and orp2 (B) using purified E.coli IHF heterodimer (lane 1, no protein; lane 2, 4 nM; lane 3, 8 nM; lane 4, 12 nM; lane 5, 16 nM).
Figure 3
Figure 3. Sequence analyses of the orp1 and orp2 promoter regions.
(A) The σ54 and σ70 promoters of the ORP system are indicated by bent arrows. The solid-line boxes indicate the palindromic DVU2106-binding sites. The dashed boxes represent the three putative IHF-binding sequences identified by comparison to the consensus sequence of the E.coli IHF-binding site (5′-WATCARxxxxTTR-3′). (B) DNA sequence alignment between the consensus sequence of E.coli IHF-binding site and each putative IHF-binding sequence of orp1 and orp2 promoter regions. The sequence identity between the consensus sequence of the E.coli IHF-binding site and the different orp putative IHF-binding site are indicated in bold.
Figure 4
Figure 4. orp1 IHF-2 and orp2 IHF sites are involved in transcriptional activation of orp1 and orp2, respectively.
The lacZ reporter fusions with the promoter regions of (A) orp1 IHF1-mut, mutated for the putative IHF-binding site 1, (B) orp1 IHF2-mut, mutated for the putative IHF-binding site 2, (C) orp1 IHF-mut, mutated for both IHF-binding sites, (D) DVU2106 IHF-mut, mutated for putative IHF-binding site of orp2 promoter and (E) orp2 IHF-mut, mutated for the putative IHF-binding site are represented on the left. The nature of these mutations is described in materials and methods and in figure S4. Promoters σ54 and σ70 are indicated by bent arrows. The putative IHF-binding sites are indicated by boxes: solid-line boxes for wild-type sites and dashed boxes for mutated sites. The activities measured in various backgrounds are shown on the right: E.coli wild-type strain (dark-gray) and E.coli ΔihfA strain (light-gray). The measures shown in black and indicated by (+) correspond to the β-galactosidase activity observed in a wild-type E.coli strain carrying the fusion between wild-type promoters and lacZ. The activity is the average of three independent measurements (the error bars show the standard deviations).
Figure 5
Figure 5. The integrator Host Factor (IHF) interacts with the site 2 of promoter region of orp1 and the putative IHF binding site detected in orp2.
Shown is the gel shift assays of the promoter regions of (A) orp1 IHF1-mut, (B) orp1 IHF2-mut, (C) orp1 IHF-mut and (D) orp2 IHF-mut, using purified E.coli IHF (lane 1, no protein; lane 2, 4 nM; lane 3, 8 nM; lane 4, 12 nM; lane 5, 16 nM). Lane 6 is from Figure 2 and corresponds to the gel shift assays of wild-type promoter fragments using 16 nM of purified E.coli IHF.
Figure 6
Figure 6. Protein sequence alignment of IHFβ and IHFα subunits of E.coli and DvH.
(A) The figure shows the sequence of IHFβ from E. coli aligned with the sequences of the two putative IHFβ from DvH, DVU1864 and DVU2973. (B) The sequence of IHFα from E.coli was aligned with the sequences of the two putative IHFα, DVU0396 and DVUA004 from DvH using ClustalW (http://www.ebi.ac.uk). Highlighted residues correspond to crucial amino-acids for IHF function in E. coli.
Figure 7
Figure 7. The heterodimer DVU0396-DVU1864 from DvH is involved in the transcriptional activation of orp operons.
The lacZ reporter fusions with the promoter regions of orp1 (A) and orp2 (B) are represented on the left. The positions of the −12 and −24 sequences of the σ54 promoters are indicated by black rectangles. The transcript start sites are indicated by bent arrows. The activities measured in various backgrounds are shown on the right: E.coli wild-type strain (black), E.coli ΔihfA strain (dark-grey), E.coli ΔihfA strain producing DVU0396 (middle-grey) and E.coli ΔihfA strain producing DVU0396-DVU1864 (light-grey). The activity is the average of three independent measurements (the error bars show the standard deviations).
Figure 8
Figure 8. Protein sequence alignment of putative IHFβ and IHFα subunits from DvH.
The figure shows the sequence of DVU1864 aligned with the sequences of the two putative IHFβ from DvH, DVU1864 and DVU2973 and the sequence of DVU0396 (IHFα) aligned with the sequences of three putative IHFα from DvH using ClustalW (http://www.ebi.ac.uk). Highlighted residues correspond to crucial amino-acids for IHF function in E. coli.
Figure 9
Figure 9. Schematic representation of the regulatory mechanisms of the orp gene cluster in DvH.
The positions of the σ54 and σ70 are indicated by bent arrows, the DVU2106-binding sequences are indicated by hatched rectangles and IHF-binding sequences are indicated by blotted rectangles. The DVU2106 transcriptional regulator and IHF play a positive role in the expression of the σ54-dependent orp1 and orp2 operons. DVU2106 exerts a negative retrocontrol on its own σ70-dependent expression.

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The Agence nationale de la recherche ANR (ANR-12-ISV8-0003-01) funds this research project. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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