The Escherichia coli CRP (cAMP receptor protein), is a global regulator of transcription that modulates gene expression by activation or repression at a range of promoters in E. coli. A major function is to regulate the selection of nutrients required for growth. The anaerobic sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC27774 is capable of utilizing sulfate, nitrite and nitrate as terminal electron acceptors. In the presence of both sulfate and nitrate, sulfate is reduced preferentially despite nitrate being the thermodynamically more favourable electron acceptor. Three inverted repeat sequences upstream of the D. desulfuricans ATCC27774 nap (nitrate reduction in the periplasm) operon have high levels of similarity to the consensus sequence for the E. coli CRP DNA-binding site. In other Desulfovibrio species a putative CRP homologue, HcpR [regulator of hcp (hybrid cluster protein) transcription], has a predicted regulon comprising genes involved in sulfate reduction and nitrosative stress. The presence of CRP consensus sites within the D. desulfuricans ATCC27774 nap promoter prompted a search for CRP homologues in the genomes of sulfate-reducing bacteria. This revealed the presence of a potential CRP homologue that we predict binds to CRP consensus sites such as those of the nap operon. Furthermore, we predict that much of the core HcpR regulon predicted in other Desulfovibrio species is conserved in D. desulfuricans.

Introduction

Bacteria can adapt to changes in their environment. Environmental cues are converted into an appropriate response by transcription regulators. The CRP (cAMP receptor protein)/FNR (fumarate and nitrate reduction regulator protein) family of proteins mediate a range of responses to stimuli such as glucose starvation (CRP), lack of oxygen (FNR), carbon monoxide (CooA) and nitric oxide (DNR, NnrR). In silico analysis has provided insight into the great diversity of transcription regulators, but experimental studies are still required to establish the signals to which they respond and to confirm and revise their cognate genomic targets. Clues to what signal a transcriptional regulator responds are often inferred from the functions of the genes that they regulate.

The putative HcpR [regulator of hcp (hybrid cluster protein) transcription] regulons of the SRB (sulfate-reducing bacteria) Desulfovibrio vulgaris Hildenborough and Desulfovibrio desulfuricans G20 include genes for sulfate reduction and response to nitrosative stress. Putative HcpR regulons have been described in other delta proteo-SRB such as Geobacter sulfurreducens, Geobacter metallireducens and Desulfuromonas species [1]. The availability of the annoted genome sequence of the Gram-negative delta proteobacterium D. desulfuricans ATCC27774 has allowed us to identify an HcpR homologue in this bacterium and to compare its predicted roles with those of other Desulfovibrio species.

Global regulators CRP and FNR from Escherichia coli

The CRP is a global gene regulator in E. coli. CRP has three functional parts: the N-terminal domain, which functions as a cAMP-binding unit; a dimerization interface that is formed by an α-helix; and the C-terminal DNA-binding domain. Crystal structures for CRP in different conformations have provided insights into the mechanism behind its activity. apo (apolipoprotein)-CRP exists as a dimer and on binding cAMP an allosteric change results in the DNA recognition helix, which in apo-CRP is buried within the proteins core, becoming solvent accessible [2]. This in turn leads to an enhancement in the DNA-binding activity of CRP. Following this switch of conformation, holo-CRP binds to target DNA sites, usually within the regulatory region of a gene, and modulates transcription by repression or activation. The consensus sequence of DNA sites targeted by CRP is TGTGAN6TCACA. Since the level of cAMP synthesized by adenylate cyclase within the cell fluctuates, there is a mechanism for the control of transcription in which the signal is converted to a transcription response. In the absence of glucose, adenylate cyclase activity is activated by the enzyme II-glucose component of the phosphoenolpyruvate-dependent phosphotransferase system [3]. CRP activates transcription by a variety of direct interactions with RNAP (RNA polymerase). Activation, and indeed the promoters where activation takes place, may be classified as class I, class II or class III depending on the type of CRP–RNAP interactions. Activation at either class I or class II promoters is achieved by a single CRP dimer bound within the promoter regulatory region, whereas class III promoters require more than one CRP dimer within the promoter regulatory region or the presence of some other transcription regulator in addition to CRP [4].

Another E. coli global gene regulator, the FNR, is structurally similar to CRP. Like CRP, FNR is made up of distinct functional domains including a dimerization domain and a DNA-binding domain that binds to the specific DNA consensus sequence TTGATN4ATCAA [5]. Unlike CRP, FNR does not bind directly to an allosteric effector molecule, but instead the protein contains a [4Fe–4S]2+ iron–sulfur cluster prosthetic group that senses the effector molecule. The presence of oxygen causes the conversion of the cluster to a [2Fe–2S]2+ state that results in a decrease in the DNA-binding affinity of the protein. This in turn results in loss of transcription activation or repression. Iron-based prosthetic groups play a major role in iron sensors of a variety of bacterial sensors and regulators [6]. Thus, despite structural similarities, FNR and CRP are distinct from one another in the way that their particular signals are perceived: FNR directly senses its signal via its prosthetic group and CRP receives its signal via binding of a secondary messenger molecule.

Diauxic growth by D. desulfuricans ATCC27774

E. coli is capable of growing on a variety of carbon sources, but utilizes glucose in preference to others. Growth on medium containing both glucose and lactose would therefore result in glucose-dependent growth before lactose-dependent growth proceeds. This selection, called diauxie, allows E. coli to adapt to transient changes in its environment.

Diauxic growth is also observed when the D. desulfuricans ATCC27774 is grown in a medium containing a limited supply of sulfate and nitrate [7]. This strain is capable of using both sulfate and nitrate, in addition to other terminal electron acceptors. Nitrate reduction by D. desulfuricans ATCC27774 is mediated by the products of the napCMADGH gene cluster [8]. Although growth is more rapid and higher yields of biomass are obtained during growth on nitrate, in medium containing both sulfate and nitrate, sulfate is utilized preferentially [7]. Furthermore, the sulfate reduction pathway requires phosphorylation of sulfate to APS (adenosine-5′-phosphosulfate), this reaction being catalysed by the sat gene product with the consumption of two molecules of ATP per sulfate [9]. Thus sulfate is a thermodynamically less favourable terminal electron acceptor than nitrate not only because it does not require ATP-dependent activation, but also because its reduction yields less ATP. Therefore this diauxie does not seem to provide any obvious advantage to D. desulfuricans ATCC27774.

Since it is assumed that, when more than one terminal electron acceptor is available in the environment, bacteria will use the most thermodynamically favourable one first, the mechanism behind this seemingly degenerate sulfate–nitrate diauxie is of interest [10]. The dsrAB genes encode sulfite reductase. In contrast to napC expression, which is induced by nitrate but repressed by sulfate, dsrAB expression is constitutive [7]. Thus nitrate reduction in D. desulfuricans ATCC27774 is regulated at the transcription level. Also, nitrate reduction is subject to inhibition and/or repression by hydrogen sulfide, the end-product of sulfate reduction [7,11]. The presence of inverted repeat DNA sequences in the napCMADGH regulatory region led to the proposal that a CRP/FNR homologue might, at least in part, be responsible for this diauxie [7].

HcpR in D. desulfuricans G20 and D. vulgaris Hildenborough

A putative CRP/FNR homologue, HcpR, which is predicted to bind DNA sites similar to the consensus of CRP (TTGTGAN6TCACAA) has been identified in Desulfovibrio species by two in silico studies [1,12]. The similarity between the DNA-binding site consensus sequences for HcpR and CRP is due to the conserved arginine and glutamate residues at positions 1 and 2 of the DNA recognition helices of these proteins. The predicted regulons for HcpR in D. desulfuricans G20 and D. vulgaris Hildenborough contain genes involved in sulfate reduction and nitrosative stress [12]. Neither D. desulfuricans G20 nor D. vulgaris Hildenborough contain nap (nitrate reduction in the periplasm) operons, hence they are unable to reduce nitrate. Conserved putative regulon members for these two species include the hcp gene, which, in other bacteria, is known to be up-regulated in response to nitrosative stress, the sat gene that encodes ATP sulfurylase, and the aprsAB genes encoding APS reductase [12]. Other putative HcpR regulon members include genes encoding redox proteins, proteins of unknown function and a two component sensor regulator [12]. No signal for modulating HcpR activity has been predicted. Consistent with predictions regarding the HcpR regulon of D. vulgaris Hildenborough, He et al. [13] reported that nitrite stress resulted in up-regulation of a number of the putative D. vulgaris Hildenborough HcpR regulon members including the DVU1080–1 operon, the phsAB operon and the hcp operon. In addition, nitrate stress upon D. vulgaris Hildenborough was demonstrated to effect the transcription and also protein levels of some HcpR regulon members with nitrate resulting in up-regulation of hcp and frdX (gene encoding a ferredoxin family protein of unknown function) [14].

Recently it was reported that D. vulgaris Hildenborough responds to sulfide stress by down-regulating transcription of the HcpR-regulated genes qmoBC and cooLXUH [15]. Thus it is plausible that the sulfate–nitrate diauxie of D. desulfuricans ATCC27774 may indeed be transcriptionally repressed by the presence of sulfide. No direct evidence that HcpR binds to CRP-like elements in Desulfovibrio species has yet been presented.

Identification of an HcpR homologue in D. desulfuricans ATCC27774

The position weight matrices of E. coli CRP and putative Desulfovibrio HcpR DNA-binding sites are very similar to each other with the motif TGTGAN6TCACA being conserved for both regulators. Also, as HcpR is predicted to be a regulator of nitrosative stress response and dissimilatory sulfate reduction, these proteins were used for the prediction of a homologue in D. desulfuricans ATCC27774 capable of binding to the inverted repeats upstream of the nap gene cluster.

Four candidate CRP/HcpR homologues from D. desulfuricans ATCC27774 were identified, all of which are distantly related to E. coli CRP or other HcpR sequences (Table 1) [16]. Two D. desulfuricans ATCC27774 CRP/HcpR homologues, Ddes_0528 and Ddes_1827, are more closely related in sequence to HcpR of D. desulfuricans G20 and D. vulgaris Hildenborough. The DNA recognition helices of Ddes_0528 and Ddes_1827 were examined for arginine and glutamate residues at positions 1 and 2 as these residues are critical for CRP/HcpR DNA-binding site recognition. These residues were conserved at those positions in Ddes_0528. This suggests that D. desulfuricans ATCC27774 Ddes_0528 might bind to DNA sites with similarity to E. coli CRP consensus. We therefore propose to designate Ddes_0528 as an HcpR.

Table 1
CRP and HcpR homologues from D. desulfuricans ATCC27774

Identities between protein sequences were determined by ClustalW multiple alignments [16].

Bacterium Protein identifier Identity (%) 
  E. coli CRP D. vulgaris HcpR (DVU2547) D. desulfuricans G20 HcpR (Dde_2644) 
D. vulgaris Hildenborough DVU2547 (HcpR) 20 100 64 
D. desulfuricans G22 Dde_2644 (HcpR) 19 64 100 
D. desulfuricans ATCC27774 Ddes_0383 18 19 20 
 Ddes_0528 (HcpR) 20 25 24 
 Ddes_1827 17 25 24 
 Ddes_2092 20 15 18 
Bacterium Protein identifier Identity (%) 
  E. coli CRP D. vulgaris HcpR (DVU2547) D. desulfuricans G20 HcpR (Dde_2644) 
D. vulgaris Hildenborough DVU2547 (HcpR) 20 100 64 
D. desulfuricans G22 Dde_2644 (HcpR) 19 64 100 
D. desulfuricans ATCC27774 Ddes_0383 18 19 20 
 Ddes_0528 (HcpR) 20 25 24 
 Ddes_1827 17 25 24 
 Ddes_2092 20 15 18 

In D. vulgaris Hildenborough and D. desulfuricans G20 HcpR is co-localized with operons containing the hcp, adhE and frdX genes [1]. The frdX gene encodes an iron–sulfur protein of the ferredoxin family. From a comparison of the genomic neighbourhoods of hcpR in these species with hcpR in D. desulfuricans ATCC27774, it is apparent that there is a lack of synteny between these species with adhE and hcp in D. desulfuricans ATCC27774 being absent from the hcpR locus (Figure 1a). However, the hcpR open reading frame in D. desulfuricans ATCC27774 is part of a cluster containing genes encoding an frdX-like protein, an FMN-binding protein and an iron–sulfur cluster protein that is homologous to putative D. vulgaris Hildenborough HcpR regulon member DVU1081. The organization of the hcpR locus in D. desulfuricans ATCC27774 is different from the other Desulfovibrio species discussed. Interestingly, an inverted repeat sequence, which is a perfect match to the E. coli CRP and Desulfovibrio HcpR consensus DNA-binding sites, was found upstream of this gene cluster. Many characterized transcription regulators negatively regulate their own transcription, thus providing control by negative feedback, which is consistent with our prediction that D. desulfuricans ATCC27774 HcpR binds to DNA sites with a consensus sequence identical with E. coli CRP.

hcpR and conserved HcpR regulon members from D. desulfuricans ATCC27774

Figure 1
hcpR and conserved HcpR regulon members from D. desulfuricans ATCC27774

(a) Comparison of the hcpR genomic neighbourhoods in three Desulfovibrio species. (b) HcpR regulon members in D. desulfuricans ATCC27774 that are common to D. vulgaris Hildenborough and D. desulfuricans G20. The similarity scores for binding sites upstream of predicted operon members are given with respect to E. coli CRP position weight matrix calculated by using PRODORIC [17]. Positions are given relative to translation start codons.

Figure 1
hcpR and conserved HcpR regulon members from D. desulfuricans ATCC27774

(a) Comparison of the hcpR genomic neighbourhoods in three Desulfovibrio species. (b) HcpR regulon members in D. desulfuricans ATCC27774 that are common to D. vulgaris Hildenborough and D. desulfuricans G20. The similarity scores for binding sites upstream of predicted operon members are given with respect to E. coli CRP position weight matrix calculated by using PRODORIC [17]. Positions are given relative to translation start codons.

As a preliminary test to establish whether the HcpR regulons of D. vulgaris Hildenborough and D. desulfuricans G20 are conserved for D. desulfuricans ATCC27774 HcpR, homologues of core regulon members were identified and their putative promoter regulatory regions were scanned for CRP consensus sites by using PRODORIC [17]. The search for sites with the E. coli CRP position weight matrix was restricted to within the 350 bp of DNA upstream of the translation start codons. The sensitivity was set to exclude sites with less than 60% identity to E. coli CRP position weight matrix. Homologues for most HcpR core regulon members were identified and CRP-type sites were identified within their putative promoter regulatory regions (Figure 1b). Our findings are in partial agreement with the D. desulfuricans ATCC27774 HcpR regulon reported in an on-line database, RegPrecise, which lists 11 DNA targets with similarity to the HcpR consensus [18]. Our predictions differ from those of RegPrecise in a few ways, such as the inclusion of the nap gene cluster as an HcpR regulon member [18]. Also, searching the D. desulfuricans ATCC27774 genome for DNA sites with similarity to the CRP position weight matrix by using PRODORIC suggests the presence of many more potential HcpR/CRP-binding sites (results not shown) [17].

Contrasting roles of HcpR in D. desulfuricans and other sulfate-reducing bacteria

In D. vulgaris Hildenborough and D. desulfuricans G20- HcpR is predicted to bind to sites overlapping the predicted −10 and −35 elements of the sat promoter, whereas binding at the hcp promoters is upstream of these elements. This suggests that in these species HcpR represses sulfate reduction but activates hcp expression. There is support for this proposal [1,19]. Aligning the DNA sequences of the putative promoter regions of the sat genes of D. desulfuricans G20 and D. vulgaris Hildenborough with that of D. desulfuricans ATCC27774 reveals some conservation between these sequences. The D. desulfuricans ATCC27774 hcpR site overlaps the putative −10 consensus sequence occupying the space between this and the putative −35 sequence of sat (Figure 2a). This would suggest that the binding of HcpR to this site would repress expression of sat. There is a consensus potential −10 sequence immediately upstream of the reported transcription start site at the nap promoter, but no recognizable −35 sequence a further 17–19 bp upstream [8]. This suggests that an activator would be required to initiate transcription at the nap promoter. Three potential HcpR sites are located at −55.5, −74.5 and −117.5 with respect to the transcription start site, from which it is plausible that HcpR could activate transcription by a class I, II or III mechanism (Figure 2b).

Promoter regulatory regions of sat and nap of D. desulfuricans ATCC27774

Figure 2
Promoter regulatory regions of sat and nap of D. desulfuricans ATCC27774

Putative HcpR-binding sites are highlighted in grey, −10 and −35 promoter elements for sat are underlined and the transcription start site for nap is marked with an arrow. Shine–Dalgarno sites and start codons are in bold [7].

Figure 2
Promoter regulatory regions of sat and nap of D. desulfuricans ATCC27774

Putative HcpR-binding sites are highlighted in grey, −10 and −35 promoter elements for sat are underlined and the transcription start site for nap is marked with an arrow. Shine–Dalgarno sites and start codons are in bold [7].

In conclusion, based on identity to E. coli CRP and HcpR from D. desulfuricans G20 and D. vulgaris Hildenborough, we predict that a CRP homologue in D. desulfuricans ATCC2774, HcpR, is capable of binding to CRP-like DNA consensus sequences. However, because the HcpRs of D. desulfuricans G20 and D. vulgaris Hildenborough are highly similar with 64% identity between protein sequences, the low identity score between D. desulfuricans ATCC27774 HcpR and D. vulgaris Hildenborough HcpR (25%) suggests that these proteins are in distinct branches of the HcpR family. We also predict that D. desulfuricans ATCC27774 HcpR is a key factor in the regulation of the sulfate–nitrate diauxie, its roles possibly being to repress sat expression and activate nap expression.

Enzymology and Ecology of the Nitrogen Cycle: A Biochemical Society Focused Meeting held at University of Birmingham, U.K., 15–17 September 2010. Organized and Edited by Jeff Cole (University of Birmingham, U.K.), Rosa María Martínez-Espinosa (University of Alicante, Spain), David Richardson (University of East Anglia, Norwich, U.K.) and Nick Watmough (University of East Anglia, Norwich, U.K.).

Abbreviations

     
  • apo

    apolipoprotein

  •  
  • APS

    adenosine 5′-phosphosulfate

  •  
  • CRP

    cAMP receptor protein

  •  
  • FNR

    fumarate and nitrate reduction regulator protein

  •  
  • frdX

    gene encoding a ferredoxin family protein of unknown function

  •  
  • hcp

    hybrid cluster protein (also known as the prismane protein)

  •  
  • HcpR

    regulator of hcp transcription

  •  
  • nap

    nitrate reduction in the periplasm

  •  
  • RNAP

    RNA polymerase

  •  
  • SRB

    sulfate-reducing bacteria

Funding

We thank the University of Brimingham for funding I.T.C.

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