Transcription Factor Family-Based Reconstruction of Singleton Regulons and Study of the Crp/Fnr, ArsR, and GntR Families in Desulfovibrionales Genomes

Lawrence Berkeley National Laboratory, Berkeley, CA 94710, USA.
Journal of bacteriology (Impact Factor: 2.81). 10/2012; 195(1). DOI: 10.1128/JB.01977-12
Source: PubMed


Accurate detection of transcriptional regulatory elements is essential for high-quality genome annotation, metabolic reconstruction,
and modeling of regulatory networks. We developed a computational approach for reconstruction of regulons operated by transcription
factors (TFs) from large protein families and applied this novel approach to three TF families in 10 Desulfovibrionales genomes. Phylogenetic analyses of 125 regulators from the ArsR, Crp/Fnr, and GntR families revealed that 65% of these regulators
(termed reference TFs) are well conserved in Desulfovibrionales, while the remaining 35% of regulators (termed singleton TFs) are species specific and show a mosaic distribution. For regulon
reconstruction in the group of singleton TFs, the standard orthology-based approach was inefficient, and thus, we developed
a novel approach based on the simultaneous study of all homologous TFs from the same family in a group of genomes. As a result,
we identified binding for 21 singleton TFs and for all reference TFs in all three analyzed families. Within each TF family
we observed structural similarities between DNA-binding motifs of different reference and singleton TFs. The collection of
reconstructed regulons is available at the RegPrecise database (

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Available from: Alexey Kazakov
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    • "Data set 1 includes HcpR-like sequences of Desulfovibrio and closely related d-proteobacteria (plus an outgroup: Porphyromonas). Data set 2 is a larger matrix that includes HcpR, Dnr, Nnr and other CRP/FNR-like proteins from proteobacteria , bacteroidetes and cyanobacteria, as these sequences have been found to be closely related to the HcpR [16] [23] [24]. The cyanobacteria sequences were used to root the trees of data set 2, after being determined that these formed a separated group that was distantly related to the other sequences in our sampling (Supplementary data, Fig. S1). "
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    ABSTRACT: 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.
    Full-text · Article · Aug 2015 · FEBS Open Bio
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    • "Frontiers in Microbiology | Terrestrial Microbiology July 2014 | Volume 5 | Article 382 | 2 2004; Kazakov et al., 2013b "
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    ABSTRACT: We surveyed the eight putative cyclic-di-GMP-modulating response regulators (RRs) in Desulfovibrio vulgaris Hildenborough that are predicted to function via two-component signaling. Using purified proteins, we examined cyclic-di-GMP (c-di-GMP) production or turnover in vitro of all eight proteins. The two RRs containing only GGDEF domains (DVU2067, DVU0636) demonstrated c-di-GMP production activity in vitro. Of the remaining proteins, three RRs with HD-GYP domains (DVU0722, DVUA0086, and DVU2933) were confirmed to be Mn(2+)-dependent phosphodiesterases (PDEs) in vitro and converted c-di-GMP to its linear form, pGpG. DVU0408, containing both c-di-GMP production (GGDEF) and degradation domains (EAL), showed c-di-GMP turnover activity in vitro also with production of pGpG. No c-di-GMP related activity could be assigned to the RR DVU0330, containing a metal-dependent phosphohydrolase HD-OD domain, or to the HD-GYP domain RR, DVU1181. Studies included examining the impact of overexpressed cyclic-di-GMP-modulating RRs in the heterologous host E. coli and led to the identification of one RR, DVU0636, with increased cellulose production. Evaluation of a transposon mutant in DVU0636 indicated that the strain was impaired in biofilm formation and demonstrated an altered carbohydrate:protein ratio relative to the D. vulgaris wild type biofilms. However, grown in liquid lactate/sulfate medium, the DVU0636 transposon mutant showed no growth impairment relative to the wild-type strain. Among the eight candidates, only the transposon disruption mutant in the DVU2067 RR presented a growth defect in liquid culture. Our results indicate that, of the two diguanylate cyclases (DGCs) that function as part of two-component signaling, DVU0636 plays an important role in biofilm formation while the function of DVU2067 has pertinence in planktonic growth.
    Full-text · Article · Jul 2014 · Frontiers in Microbiology
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    • "An important issue in such studies is to connect TFs to the cognate TF binding sites (TFBSs) identified by phylogenetic footprinting and other computational techniques (Conlan et al., 2005; Wels et al., 2006; Liu et al., 2008). This problem is either solved experimentally or addressed computationally, for instance for regulons controlled by local TF from specific protein families (Rigali et al., 2004; Francke et al., 2008; Sahota and Stormo, 2010; Ahn et al., 2012; Kazakov et al., 2013). Phylogenetic profiling of TF genes and motifs upstream of candidate regulon members is an alternative bioinformatics approach for assigning TFs to putative regulons (Rodionov and Gelfand, 2005). "
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    ABSTRACT: DNA-binding transcription factors (TFs) are essential components of transcriptional regulatory networks in Bacteria. LacI-family TFs (LacI-TFs) are broadly distributed among certain lineages of bacteria. The majority of characterized LacI-TFs sense sugar effectors and regulate carbohydrate utilization genes. The comparative genomics approaches enable in silico identification of TF-binding sites and regulon reconstruction. To study function and evolution of LacI-TFs, we performed genomics-based reconstruction and comparative analysis of their regulons. For over 1,300 LacI-TFs from over 270 bacterial genomes, we predicted their cognate DNA-binding motifs and identified target genes. Using the genome context and metabolic subsystem analyses of reconstructed regulons we tentatively assigned functional roles and predicted candidate effectors for 78% and 67% of the analyzed LacI-TFs, respectively. Nearly 90% of the studied LacI-TFs are local regulators of sugar utilization pathways, whereas the remaining 125 global regulators control large and diverse sets of metabolic genes. The global LacI-TFs include the previously known regulators CcpA in Firmicutes, FruR in Enterobacteria, and PurR in Gammaproteobacteria, and the three novel regulators, GluR, GapR, and PckR, that are predicted to control the central carbohydrate metabolism in three lineages of Alphaproteobacteria. Phylogenetic analysis of regulators combined with the reconstructed regulons provides a model of evolutionary diversification of LacI-TFs. The obtained genomic collection of in silico reconstructed regulons in Bacteria is available in the RegPrecise database ( It provides a framework for future structural and functional classification of the LacI protein family and identification of molecular determinants of the DNA and ligand specificity. The inferred regulons can be also used for functional gene annotation and reconstruction of sugar catabolic networks in diverse bacteria.
    Full-text · Article · Jun 2014 · Frontiers in Microbiology
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