Fine-Tuning Modulation of Oxidation-Mediated Posttranslational Control of Bradyrhizobium diazoefficiens FixK2 Transcription Factor.

FixK2 is a CRP/FNR-type transcription factor that plays a central role in a sophisticated regulatory network for the anoxic, microoxic and symbiotic lifestyles of the soybean endosymbiont Bradyrhizobium diazoefficiens. Aside from the balanced expression of the fixK2 gene under microoxic conditions (induced by the two-component regulatory system FixLJ and negatively auto-repressed), FixK2 activity is posttranslationally controlled by proteolysis, and by the oxidation of a singular cysteine residue (C183) near its DNA-binding domain. To simulate the permanent oxidation of FixK2, we replaced C183 for aspartic acid. Purified C183D FixK2 protein showed both low DNA binding and in vitro transcriptional activation from the promoter of the fixNOQP operon, required for respiration under symbiosis. However, in a B. diazoefficiens strain coding for C183D FixK2, expression of a fixNOQP'-'lacZ fusion was similar to that in the wild type, when both strains were grown microoxically. The C183D FixK2 encoding strain also showed a wild-type phenotype in symbiosis with soybeans, and increased fixK2 gene expression levels and FixK2 protein abundance in cells. These two latter observations, together with the global transcriptional profile of the microoxically cultured C183D FixK2 encoding strain, suggest the existence of a finely tuned regulatory strategy to counterbalance the oxidation-mediated inactivation of FixK2 in vivo.

Proteome dynamics during post-desiccation recovery reveal convergence of desiccation and gamma radiation stress response pathways in Deinococcus radiodurans.

Deinococcus radiodurans is inherently resistant to both ionizing radiation and desiccation. Fifteen months of desiccation was found to be the LD50 dose for D. radiodurans. Desiccated cells of D. radiodurans entered 6h of growth arrest during post-desiccation recovery (PDR). Proteome dynamics during PDR were mapped by resolving cellular proteins by 2-dimensional gel electrophoresis coupled with mass spectrometry. At least 41 proteins, represented by 51 spots on proteome profiles, were differentially expressed throughout PDR. High upregulation in expression was observed for DNA repair proteins involved in single strand annealing (DdrA and DdrB), nucleotide excision repair (UvrA and UvrB), homologous recombination (RecA) and other vital proteins that contribute to DNA replication, recombination and repair (Ssb, GyrA and GyrB). Expression of CRP/FNR family transcriptional regulator (Crp) remained high throughout PDR. Other pathways such as cellular detoxification, protein homeostasis and metabolism displayed both, moderately induced and repressed proteins. Functional relevance of proteomic modulations to surviving desiccation stress is discussed in detail. Comparison of our data with the published literature revealed convergence of radiation and desiccation stress responses of D. radiodurans. This is the first report that substantiates the hypothesis that the radiation stress resistance of D. radiodurans is incidental to its desiccation stress resistance.

Disparate response to microoxia and nitrogen oxides of the Bradyrhizobium japonicum napEDABC, nirK and norCBQD denitrification genes.

Expression of the Bradyrhizobium japonicum napEDABC, nirK and norCBQD denitrification genes requires low oxygen (O2) tension and nitrate (NO3-), through a regulatory network comprised of two coordinated cascades, FixLJ-FixK2-NnrR and RegSR-NifA. To precisely understand how these signals are integrated in the FixLJ-FixK2-NnrR circuit, we analyzed ß-Galactosidase activities from napE-lacZ, nirK-lacZ and norC-lacZ fusions, and performed analyses of NapC and NorC levels as well as periplasmic nitrate reductase (Nap) activity, in B. japonicum wildtype and fixK2 and nnrR mutant backgrounds. While microoxic conditions (2% O2 at headspace) were sufficient to induce expression of napEDABC and nirK genes and this control depends on FixK2, norCBQD expression requires, in addition to microoxia, nitric oxide gas (NO) and both FixK2 and NnrR transcription factors. Purified FixK2 protein directly interacted and activated transcription in collaboration with B. japonicum RNA polymerase (RNAP) from the napEDABC and nirK promoters, but not from the norCBQD promoter. Further, recombinant NnrR protein bound exclusively to the norCBQD promoter in an O2-sensitive manner. Our work suggest a disparate regulation of B. japonicum denitrifying genes expression with regard to their dependency to microoxia, nitrogen oxides (NOx), and the regulatory proteins FixK2 and NnrR. In this control, expression of napEDABC and nirK genes requires microoxic conditions and directly depends on FixK2, while expression of norCBQD genes relies on NO, being NnrR the candidate which directly interacts with the norCBQD promoter.

Characterization of LnmO as a pathway-specific Crp/Fnr-type positive regulator for leinamycin biosynthesis in Streptomyces atroolivaceus and its application for titer improvement.

The cyclic adenosine monophosphate (cAMP) receptor protein/fumarate and nitrate reductase regulatory protein (Crp/Fnr) family of transcriptional regulators are pleiotropic transcriptional regulators that control a broad range of cellular functions. Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140. We previously cloned and characterized the lnm biosynthetic gene cluster from S. atroolivaceus S-140. We here report inactivation of lnmO in S. atroolivaceus S-140 and overexpression of lnmO in the S. atroolivaceus S-140 wild-type and ∆lnmE mutant SB3033 to investigate its role in LNM biosynthesis. Bioinformatics analysis revealed LnmO as the only regulator within the lnm gene cluster, exhibiting high sequence similarity to known Crp/Fnr family regulators. The inactivation of lnmO in S. atroolivaceus S-140 completely abolished LNM production but caused no apparent morphological changes, supporting that LnmO is indispensable and specific to LNM biosynthesis. Overexpression of lnmO in S. atroolivaceus S-140 and SB3033 resulted in three- and fourfold increase in LNM and LNM E1 production, respectively, supporting that LnmO acts as a positive regulator. While all of the Crp/Fnr family regulators studied to date appeared to be pleiotropic, our results support LnmO as the first Crp/Fnr family regulator that is pathway-specific. LnmO joins the growing list of regulators that could be exploited to improve secondary metabolite production in Streptomyces. Engineered strains overproducing LNM and LNM E1 will facilitate further mechanistic studies and clinical evaluation of LNM and LNM E1 as novel anticancer drugs.

The structure of Bradyrhizobium japonicum transcription factor FixK2 unveils sites of DNA binding and oxidation.

FixK2 is a regulatory protein that activates a large number of genes for the anoxic and microoxic, endosymbiotic, and nitrogen-fixing life styles of the α-proteobacterium Bradyrhizobium japonicum. FixK2 belongs to the cAMP receptor protein (CRP) superfamily. Although most CRP family members are coregulated by effector molecules, the activity of FixK2 is negatively controlled by oxidation of its single cysteine (Cys-183) located next to the DNA-binding domain and possibly also by proteolysis. Here, we report the three-dimensional x-ray structure of FixK2, a representative of the FixK subgroup of the CRP superfamily. Crystallization succeeded only when (i) an oxidation- and protease-insensitive protein variant (FixK2(C183S)-His6) was used in which Cys-183 was replaced with serine and the C terminus was fused with a hexahistidine tag and (ii) this protein was allowed to form a complex with a 30-mer double-stranded target DNA. The structure of the FixK2-DNA complex was solved at a resolution of 1.77 Å, at which the protein formed a homodimer. The precise protein-DNA contacts were identified, which led to an affirmation of the canonical target sequence, the so-called FixK2 box. The C terminus is surface-exposed, which might explain its sensitivity to specific cleavage and degradation. The oxidation-sensitive Cys-183 is also surface-exposed and in close proximity to DNA. Therefore, we propose a mechanism whereby the oxo acids generated after oxidation of the cysteine thiol cause an electrostatic repulsion, thus preventing specific DNA binding.

Surface Plasmon Resonance as a Tool to Elucidate the Molecular Determinants of Key Transcriptional Regulators Controlling Rhizobial Lifestyles.

Bacteria must be provided with a battery of tools integrated into regulatory networks, in order to respond and, consequently, adapt their physiology to changing environments. Within these networks, transcription factors finely orchestrate the expression of genes in response to a variety of signals, by recognizing specific DNA sequences at their promoter regions. Rhizobia are host-interacting soil bacteria that face severe changes to adapt their physiology from free-living conditions to the nitrogen-fixing endosymbiotic state inside root nodules associated with leguminous plants. One of these cues is the low partial pressure of oxygen within root nodules.Surface plasmon resonance (SPR) constitutes a technique that allows to measure molecular interactions dynamics at real time by detecting changes in the refractive index of a surface. Here, we implemented the SPR methodology to analyze the discriminatory determinants of transcription factors for specific interaction with their target genes. We focused on FixK2, a CRP/FNR-type protein with a central role in the complex oxygen-responsive regulatory network in the soybean endosymbiont Bradyrhizobium diazoefficiens. Our study unveiled relevant residues for protein-DNA interaction as well as allowed us to monitor kinetics and stability protein-DNA complex. We believe that this approach can be employed for the characterization of other relevant transcription factors which can assist to the better understanding of the adaptation of bacteria with agronomic or human interest to their different modes of life.

Roles of the Crp/Fnr Family Regulator ArcR in the Hemolysis and Biofilm of Staphylococcus aureus.

Staphylococcus aureus is an opportunistic human pathogen that is often involved in severe infections such as pneumonia and sepsis in which bacterial virulence factors play a key role. Infections caused by S. aureus are often difficult to eradicate, particularly when they are associated with biofilm. The physiological roles of the Crp/Fnr family regulator ArcR are elusive in S. aureus. In this study, it was found that the deletion of arcR increased the hemolytic ability and biofilm formation in S. aureus. Differential gene expression analysis by RNA-seq and real-time quantitative reverse transcription PCR showed that genes associated with hemolytic ability (hla and hlb) and biofilm formation (icaA, icaB, icaC and icaD) were significantly upregulated compared with those in the wild-type strain. The results revealed that ArcR regulated the expression of the hla and ica operon by binding to their promoter regions, respectively. This study provided new insights into the functional importance of ArcR in regulating the virulence and biofilm of S. aureus.

RedB, a Member of the CRP/FNR Family, Functions as a Transcriptional Redox Brake.

Phylogenetic and sequence similarity network analyses of the CRP (cyclic AMP receptor protein)/FNR (fumarate and nitrate reductase regulatory protein) family of transcription factors indicate the presence of numerous subgroups, many of which have not been analyzed. Five homologs of the CRP/FNR family are present in the Rhodobacter capsulatus genome. One is a member of a broadly disseminated, previously uncharacterized CRP/FNR family subgroup encoded by the gene rcc01561. In this study, we utilize mutational disruption, transcriptome sequencing (RNA-seq), and chromatin immunoprecipitation sequencing (ChIP-seq) to determine the role of RCC01561 in regulating R. capsulatus physiology. This analysis shows that a mutant strain disrupted for rcc01561 exhibits altered expression of 451 genes anaerobically. A detailed analysis of the affected loci shows that RCC01561 represses photosynthesis and favors catabolism over anabolism and the use of the Entner-Doudoroff shunt and glycolysis over that of the tricarboxylic acid (TCA) cycle to limit NADH and ATP formation. This newly characterized CRP/FNR family member with a predominant role in reducing the production of reducing potential and ATP is given the nomenclature RedB as it functions as an energy and redox brake. Beyond limiting energy production, RedB also represses the expression of numerous genes involved in protein synthesis, including those involved in translation initiation, tRNA synthesis and charging, and amino acid biosynthesis. IMPORTANCE CRP and FNR are well-characterized members of the CRP/FNR family of regulatory proteins that function to maximize cellular energy production. In this study, we identify several new subgroups of the CRP/FNR family, many of which have not yet been characterized. Using Rhodobacter capsulatus as a model, we have mutationally disrupted the gene rcc01561, which codes for a transcription factor that is a member of a unique subgroup of the CRP/FNR family. Transcriptomic analysis shows that the disruption of rcc01561 leads to the altered expression of 451 genes anaerobically. Analysis of these regulated genes indicates that RCC01561 has a novel role in limiting cellular energy production. To our knowledge, this is first example of a member of the CRP/FNR family that functions as a brake on cellular energy production.

Hybrid Transcriptional Regulators for the Screening of Target DNA Motifs in Organohalide-Respiring Bacteria.

The bioremediation of persistent organohalide molecules under anoxic conditions mostly relies on the bacterial process called organohalide respiration (OHR). Organohalide-respiring bacteria (OHRB) are phylogenetically diverse anaerobic bacteria that share the capacity to use organohalides as terminal electron acceptors in an energy-conserving process. The reductive dehalogenase (rdh) gene clusters encode for proteins specialized in the respiration of one or a limited number of organohalides. One particular OHRB may harbor up to several dozens of rdh gene clusters suggesting a wide potential for bioremediation. To avoid wasting energy in producing unnecessary proteins, rdh gene clusters often include a transcriptional regulator. In organohalide-respiring Firmicutes, RdhK is a dedicated transcriptional regulator of OHR and represents a subfamily of proteins among the CRP/FNR superfamily of regulators. RdhK proteins are composed of an effector-binding domain (EBD) which recognizes a given organohalide and subsequently controls the interaction of its C-terminal DNA-binding domain (DBD) with a DNA motif (referred to as dehalobox, or DB) located in the promoter region of the target rdh genes. The two binding partners (i.e. an organohalide molecule and a DB sequence) of RdhK proteins are interdependent which impairs the exploration of OHR regulatory networks. Here, we propose a strategy relying on hybrid proteins to efficiently screen the DNA target of a single RdhK protein without prior knowledge on its effector. To demonstrate the potential of the method, two hybrids with alternative fusion points were designed based on RdhK6 EBD and RdhK1 DBD from Desulfitobacterium hafniense. Electrophoretic mobility shift assay was performed with purified hybrids along with the parental proteins and their binding properties were further tested in vivo through a ß-galactosidase reporter assay. Along with revealing new RdhK6 features, we show that both hybrids resulted in active regulatory proteins with distinct binding patterns. While Hybrid A was less specific for the DNA motif, Hybrid B successfully mimicked the binding behavior of the parental proteins and thus represents a promising template for the design of new RdhK hybrids to screen yet uncharacterized RdhK proteins and also possibly other members of the CRP/FNR superfamily.

Interactions among Redox Regulators and the CtrA Phosphorelay in Dinoroseobacter shibae and Rhodobacter capsulatus.

Bacteria employ regulatory networks to detect environmental signals and respond appropriately, often by adjusting gene expression. Some regulatory networks influence many genes, and many genes are affected by multiple regulatory networks. Here, we investigate the extent to which regulatory systems controlling aerobic-anaerobic energetics overlap with the CtrA phosphorelay, an important system that controls a variety of behavioral processes, in two metabolically versatile alphaproteobacteria, Dinoroseobacter shibae and Rhodobacter capsulatus. We analyzed ten available transcriptomic datasets from relevant regulator deletion strains and environmental changes. We found that in D. shibae, the CtrA phosphorelay represses three of the four aerobic-anaerobic Crp/Fnr superfamily regulator-encoding genes (fnrL, dnrD, and especially dnrF). At the same time, all four Crp/Fnr regulators repress all three phosphorelay genes. Loss of dnrD or dnrF resulted in activation of the entire examined CtrA regulon, regardless of oxygen tension. In R. capsulatus FnrL, in silico and ChIP-seq data also suggested regulation of the CtrA regulon, but it was only with loss of the redox regulator RegA where an actual transcriptional effect on the CtrA regulon was observed. For the first time, we show that there are complex interactions between redox regulators and the CtrA phosphorelays in these bacteria and we present several models for how these interactions might occur.

PgRsp Is a Novel Redox-Sensing Transcription Regulator Essential for Porphyromonas gingivalis Virulence.

Porphyromonas gingivalis is one of the etiological agents of chronic periodontitis. Both heme and oxidative stress impact expression of genes responsible for its survival and virulence. Previously we showed that P. gingivalis ferric uptake regulator homolog affects expression of a gene encoding a putative Crp/Fnr superfamily member, termed P. gingivalis redox-sensing protein (PgRsp). Although PgRsp binds heme and shows the highest similarity to proteins assigned to the CooA family, it could be a member of a novel, separate family of proteins with unknown function. Expression of the pgrsp gene is autoregulated and iron/heme dependent. Genes encoding proteins engaged in the oxidative stress response were upregulated in the pgrsp mutant (TO11) strain compared with the wild-type strain. The TO11 strain showed higher biomass production, biofilm formation, and coaggregation ability with Tannerella forsythia and Prevotella intermedia. We suggest that PgRsp may regulate production of virulence factors, proteases, Hmu heme acquisition system, and FimA protein. Moreover, we observed growth retardation of the TO11 strain under oxidative conditions and decreased survival ability of the mutant cells inside macrophages. We conclude that PgRsp protein may play a role in the oxidative stress response using heme as a ligand for sensing changes in redox status, thus regulating the alternative pathway of the oxidative stress response alongside OxyR.

Expanding the Regulon of the Bradyrhizobium diazoefficiens NnrR Transcription Factor: New Insights Into the Denitrification Pathway.

Denitrification in the soybean endosymbiont Bradyrhizobium diazoefficiens is controlled by a complex regulatory network composed of two hierarchical cascades, FixLJ-FixK2-NnrR and RegSR-NifA. In the former cascade, the CRP/FNR-type transcription factors FixK2 and NnrR exert disparate control on expression of core denitrifying systems encoded by napEDABC, nirK, norCBQD, and nosRZDFYLX genes in response to microoxia and nitrogen oxides, respectively. To identify additional genes controlled by NnrR and involved in the denitrification process in B. diazoefficiens, we compared the transcriptional profile of an nnrR mutant with that of the wild type, both grown under anoxic denitrifying conditions. This approach revealed more than 170 genes were simultaneously induced in the wild type and under the positive control of NnrR. Among them, we found the cycA gene which codes for the c 550 soluble cytochrome (CycA), previously identified as an intermediate electron donor between the bc 1 complex and the denitrifying nitrite reductase NirK. Here, we demonstrated that CycA is also required for nitrous oxide reductase activity. However, mutation in cycA neither affected nosZ gene expression nor NosZ protein steady-state levels. Furthermore, cycA, nnrR and its proximal divergently oriented nnrS gene, are direct targets for FixK2 as determined by in vitro transcription activation assays. The dependence of cycA expression on FixK2 and NnrR in anoxic denitrifying conditions was validated at transcriptional level, determined by quantitative reverse transcription PCR, and at the level of protein by performing heme c-staining of soluble cytochromes. Thus, this study expands the regulon of NnrR and demonstrates the role of CycA in the activity of the nitrous oxide reductase, the key enzyme for nitrous oxide mitigation.

Regulation of organohalide respiration.

Organohalide respiration (OHR) is an anaerobic metabolism by which bacteria conserve energy with the use of halogenated compounds as terminal electron acceptors. Genes involved in OHR are organized in reductive dehalogenase (rdh) gene clusters and can be found in relatively high copy numbers in the genomes of organohalide-respiring bacteria (OHRB). The minimal rdh gene set is composed by rdhA and rdhB, encoding the catalytic enzyme involved in reductive dehalogenation and its putative membrane anchor, respectively. In this chapter, we present the major findings concerning the regulatory strategies developed by OHRB to control the expression of the rdh gene clusters. The first section focuses on the description of regulation patterns obtained from targeted transcriptional analyses, and from transcriptomic and proteomic studies, while the second section offers a detailed overview of the biochemically characterized OHR regulatory proteins identified so far. Depending on OHRB, transcriptional regulators belonging to three different protein families are found in the direct vicinity of rdh gene clusters, suggesting that they activate the transcription of their cognate gene cluster. In this chapter, strong emphasis was laid on the family of CRP/FNR-type RdhK regulators which belong to members of the genera Dehalobacter and Desulfitobacterium. Whereas only chlorophenols have been identified as effectors for RdhK regulators, the protein sequence diversity suggests a broader organohalide spectrum. Thus, effector identification of new regulators offers a promising alternative to elucidate the substrates of yet uncharacterized reductive dehalogenases. Future work investigating the possible cross-talk between OHR regulators and their possible use as biosensors is discussed.

FnrL and Three Dnr Regulators Are Used for the Metabolic Adaptation to Low Oxygen Tension in Dinoroseobacter shibae.

The heterotrophic marine bacterium Dinoroseobacter shibae utilizes aerobic respiration and anaerobic denitrification supplemented with aerobic anoxygenic photosynthesis for energy generation. The aerobic to anaerobic transition is controlled by four Fnr/Crp family regulators in a unique cascade-type regulatory network. FnrL is utilizing an oxygen-sensitive Fe-S cluster for oxygen sensing. Active FnrL is inducing most operons encoding the denitrification machinery and the corresponding heme biosynthesis. Activation of gene expression of the high oxygen affinity cbb3-type and repression of the low affinity aa3-type cytochrome c oxidase is mediated by FnrL. Five regulator genes including dnrE and dnrF are directly controlled by FnrL. Multiple genes of the universal stress protein (USP) and cold shock response are further FnrL targets. DnrD, most likely sensing NO via a heme cofactor, co-induces genes of denitrification, heme biosynthesis, and the regulator genes dnrE and dnrF. DnrE is controlling genes for a putative Na+/H+ antiporter, indicating a potential role of a Na+ gradient under anaerobic conditions. The formation of the electron donating primary dehydrogenases is coordinated by FnrL and DnrE. Many plasmid encoded genes were DnrE regulated. DnrF is controlling directly two regulator genes including the Fe-S cluster biosynthesis regulator iscR, genes of the electron transport chain and the glutathione metabolism. The genes for nitrate reductase and CO dehydrogenase are repressed by DnrD and DnrF. Both regulators in concert with FnrL are inducing the photosynthesis genes. One of the major denitrification operon control regions, the intergenic region between nirS and nosR2, contains one Fnr/Dnr binding site. Using regulator gene mutant strains, lacZ-reporter gene fusions in combination with promoter mutagenesis, the function of the single Fnr/Dnr binding site for FnrL-, DnrD-, and partly DnrF-dependent nirS and nosR2 transcriptional activation was shown. Overall, the unique regulatory network of the marine bacterium D. shibae for the transition from aerobic to anaerobic growth composed of four Crp/Fnr family regulators was elucidated.

The Phytohormone Ethylene Enhances Cellulose Production, Regulates CRP/FNRKx Transcription and Causes Differential Gene Expression within the Bacterial Cellulose Synthesis Operon of Komagataeibacter (Gluconacetobacter) xylinus ATCC 53582.

Komagataeibacter (formerly Gluconacetobacter) xylinus ATCC 53582 is a plant-associated model organism for bacterial cellulose (BC) biosynthesis. This bacterium inhabits the carposphere where it interacts with fruit through the bi-directional transfer of phytohormones. The majority of research regarding K. xylinus has been focused on identifying and characterizing structural and regulatory factors that control BC biosynthesis, but its ecophysiology has been generally overlooked. Ethylene is a phytohormone that regulates plant development in a variety of ways, but is most commonly known for its positive role on fruit ripening. In this study, we utilized ethephon (2-chloroethylphosphonic acid) to produce in situ ethylene to investigate the effects of this phytohormone on BC production and the expression of genes known to be involved in K. xylinus BC biosynthesis (bcsA, bcsB, bcsC, bcsD, cmcAx, ccpAx and bglAx). Using pellicle assays and reverse transcription quantitative polymerase chain reaction (RT-qPCR), we demonstrate that ethephon-derived ethylene enhances BC directly in K. xylinus by up-regulating the expression of bcsA and bcsB, and indirectly though the up-regulation of cmcAx, ccpAx, and bglAx. We confirm that IAA directly decreases BC biosynthesis by showing that IAA down-regulates bcsA expression. Similarly, we confirm that ABA indirectly influences BC biosynthesis by showing it does not affect the expression of bcs operon genes. In addition, we are the first to report the ethylene and indole-3-acetic acid (IAA) induced differential expression of genes within the bacterial cellulose synthesis (bcs) operon. Using bioinformatics we have identified a novel phytohormone-regulated CRP/FNRKx transcription factor and provide evidence that it influences BC biosynthesis in K. xylinus. Lastly, utilizing current and previous data, we propose a model for the phytohormone-mediated fruit-bacteria interactions that K. xylinus experiences in nature.

An HcpR paralog of Desulfovibrio gigas provides protection against nitrosative stress.

Desulfovibrio gigas belongs to the group of sulfate reducing bacteria (SRB). These ubiquitous and metabolically versatile microorganisms are often exposed to reactive nitrogen species (RNS). Nonetheless, the mechanisms and regulatory elements involved in nitrosative stress protection are still poorly understood. The transcription factor HcpR has emerged as a putative regulator of nitrosative stress response among anaerobic bacteria. HcpR is known to orchestrate the expression of the hybrid cluster protein gene, hcp, proposed to be involved in cellular defense against RNS. According to phylogenetic analyses, the occurrence of hcpR paralog genes is a common feature among several Desulfovibrio species. Within the D. gigas genome we have identified two HcpR-related sequences. One of these sequences, hcpR1, was found in the close vicinity of the hcp gene and this finding prompted us to proceed with its functional characterization. We observed that the growth of a D. gigas strain lacking hcpR1 is severely impaired under nitrosative stress. An in silico search revealed several putative targets of HcpR1 that were experimentally validated. The fact that HcpR1 regulates several genes encoding proteins involved in nitrite and nitrate metabolism, together with the sensitive growth phenotype to NO displayed by an hcpR1 mutant strain, strongly supports a relevant role of this factor under nitrosative stress. Moreover, the finding that several Desulfovibrio species possess HcpR paralogs, which have been transmitted vertically in the evolution and diversification of the genus, suggests that these sequences may confer adaptive or survival advantage to these organisms, possibly by increasing their tolerance to nitrosative stress.