Evolution of the metabolic and regulatory networks associated with oxygen availability in two phytopathogenic enterobacteria

<p>Abstract</p> <p>Background</p> <p><it>Dickeya dadantii </it>and <it>Pectobacterium atrosepticum </it>are phytopathogenic enterobacteria capable of facultative anaerobic growth in a wide range of O₂concentrations found in plant and natural environments. The transcriptional response to O₂remains under-explored for these and other phytopathogenic enterobacteria although it has been well characterized for animal-associated genera including <it>Escherichia coli </it>and <it>Salmonella enterica</it>. Knowledge of the extent of conservation of the transcriptional response across orthologous genes in more distantly related species is useful to identify rates and patterns of regulon evolution. Evolutionary events such as loss and acquisition of genes by lateral transfer events along each evolutionary branch results in lineage-specific genes, some of which may have been subsequently incorporated into the O₂-responsive stimulon. Here we present a comparison of transcriptional profiles measured using densely tiled oligonucleotide arrays for two phytopathogens, <it>Dickeya dadantii </it>3937 and <it>Pectobacterium atrosepticum </it>SCRI1043, grown to mid-log phase in MOPS minimal medium (0.1% glucose) with and without O₂.</p> <p>Results</p> <p>More than 7% of the genes of each phytopathogen are differentially expressed with greater than 3-fold changes under anaerobic conditions. In addition to anaerobic metabolism genes, the O₂responsive stimulon includes a variety of virulence and pathogenicity-genes. Few of these genes overlap with orthologous genes in the anaerobic stimulon of <it>E. coli</it>. We define these as the conserved core, in which the transcriptional pattern as well as genetic architecture are well preserved. This conserved core includes previously described anaerobic metabolic pathways such as fermentation. Other components of the anaerobic stimulon show variation in genetic content, genome architecture and regulation. Notably formate metabolism, nitrate/nitrite metabolism, and fermentative butanediol production, differ between <it>E. coli </it>and the phytopathogens. Surprisingly, the overlap of the anaerobic stimulon between the phytopathogens is also relatively small considering that they are closely related, occupy similar niches and employ similar strategies to cause disease. There are cases of interesting divergences in the pattern of transcription of genes between <it>Dickeya </it>and <it>Pectobacterium </it>for virulence-associated subsystems including the type VI secretion system (T6SS), suggesting that fine-tuning of the stimulon impacts interaction with plants or competing microbes.</p> <p>Conclusions</p> <p>The small number of genes (an even smaller number if we consider operons) comprising the conserved core transcriptional response to O₂limitation demonstrates the extent of regulatory divergence prevalent in the Enterobacteriaceae. Our orthology-driven comparative transcriptomics approach indicates that the adaptive response in the eneterobacteria is a result of interaction of core (regulators) and lineage-specific (structural and regulatory) genes. Our subsystems based approach reveals that similar phenotypic outcomes are sometimes achieved by each organism using different genes and regulatory strategies.</p

Exploiting Thiol Modifications

Molecular oxygen may be necessary for life but with its beneficial properties comes formation of potentially toxic reactive oxygen species. One of the ways in which bacteria protect themselves is explaine

An open toolkit for tracking open science partnership implementation and impact.

Serious concerns about the way research is organized collectively are increasingly being raised. They include the escalating costs of research and lower research productivity, low public trust in researchers to report the truth, lack of diversity, poor community engagement, ethical concerns over research practices, and irreproducibility. Open science (OS) collaborations comprise of a set of practices including open access publication, open data sharing and the absence of restrictive intellectual property rights with which institutions, firms, governments and communities are experimenting in order to overcome these concerns. We gathered two groups of international representatives from a large variety of stakeholders to construct a toolkit to guide and facilitate data collection about OS and non-OS collaborations. Ultimately, the toolkit will be used to assess and study the impact of OS collaborations on research and innovation. The toolkit contains the following four elements: 1) an annual report form of quantitative data to be completed by OS partnership administrators; 2) a series of semi-structured interview guides of stakeholders; 3) a survey form of participants in OS collaborations; and 4) a set of other quantitative measures best collected by other organizations, such as research foundations and governmental or intergovernmental agencies. We opened our toolkit to community comment and input. We present the resulting toolkit for use by government and philanthropic grantors, institutions, researchers and community organizations with the aim of measuring the implementation and impact of OS partnership across these organizations. We invite these and other stakeholders to not only measure, but to share the resulting data so that social scientists and policy makers can analyse the data across projects

Reconstruction of the Core and Extended Regulons of Global Transcription Factors

The processes underlying the evolution of regulatory networks are unclear. To address this question, we used a comparative genomics approach that takes advantage of the large number of sequenced bacterial genomes to predict conserved and variable members of transcriptional regulatory networks across phylogenetically related organisms. Specifically, we developed a computational method to predict the conserved regulons of transcription factors across α-proteobacteria. We focused on the CRP/FNR super-family of transcription factors because it contains several well-characterized members, such as FNR, FixK, and DNR. While FNR, FixK, and DNR are each proposed to regulate different aspects of anaerobic metabolism, they are predicted to recognize very similar DNA target sequences, and they occur in various combinations among individual α-proteobacterial species. In this study, the composition of the respective FNR, FixK, or DNR conserved regulons across 87 α-proteobacterial species was predicted by comparing the phylogenetic profiles of the regulators with the profiles of putative target genes. The utility of our predictions was evaluated by experimentally characterizing the FnrL regulon (a FNR-type regulator) in the α-proteobacterium Rhodobacter sphaeroides. Our results show that this approach correctly predicted many regulon members, provided new insights into the biological functions of the respective regulons for these regulators, and suggested models for the evolution of the corresponding transcriptional networks. Our findings also predict that, at least for the FNR-type regulators, there is a core set of target genes conserved across many species. In addition, the members of the so-called extended regulons for the FNR-type regulators vary even among closely related species, possibly reflecting species-specific adaptation to environmental and other factors. The comparative genomics approach we developed is readily applicable to other regulatory networks

Contributions of [4Fe-4S]-FNR and Integration Host Factor to fnr Transcriptional Regulation

Maintaining appropriate levels of the global regulator FNR is critical to its function as an O(2) sensor. In this study, we examined the mechanisms that control transcription of fnr to increase our understanding of how FNR protein levels are regulated. Under anaerobic conditions, one mechanism that controls fnr expression is negative autoregulation by the active [4Fe-4S] form of FNR. Through DNase I footprinting and in vitro transcription experiments, we observed that direct binding of [4Fe-4S]-FNR to the predicted downstream FNR binding site is sufficient for repression of the fnr promoter in vitro. In addition, the downstream FNR binding site was required for repression of transcription from fnr′-lacZ fusions in vivo. No repression of fnr was observed in vivo or in vitro with the apoprotein form of FNR, indicating that repression requires the dimeric, Fe-S cluster-containing protein. Furthermore, our in vitro and in vivo data suggest that [4Fe-4S]-FNR does not bind to the predicted upstream FNR binding site within the fnr promoter. Rather, we provide evidence that integration host factor binds to this upstream region and increases in vivo expression of Pfnr under both aerobic and anaerobic conditions

Formation of Reactive Oxygen Species

<p>The four-electron reduction of molecular O₂ generates two molecules of H₂O, which is O₂ in its most reduced form. While this reduction normally occurs within the enzyme cytochrome oxidase, one-electron transfers to O₂ also occur outside of cytochrome oxidase via inadvertent reactions with other reduced electron carriers, resulting in partially reduced and reactive forms of O₂· H₂O₂ is also produced by the enzymatic or spontaneous dismutation of O₂⁻, and •OH is generated by the reaction of iron with H₂O₂ (the Fenton reaction). In addition, the reactive oxygen intermediates are produced by a variety of organisms as a defense against microbial invasion. (Illustration: Rusty Howson, sososo design)</p

Thiol Modifications of Proteins

<p>Formation of sulfenic acid from the reaction of H₂O₂ with protein thiolates leads to different protein modifications, depending on the protein. In proteins without a second sulfhydryl, the sulfenic acid (–SOH) may be stabilized (e.g., OhrR) or may react with reactive oxygen species to generate the further oxidized sulfinic (–SO₂H) (e.g., thiolperoxidase; Tpx) and sulfonic acid (–SO₃H) derivatives. Alternatively, if a second cysteinyl residue is in proximity within the same polypeptide (e.g., OxyR) or an associated protein (e.g., Yap1 and Orp1), a disulfide bond can form between the two sulfur atoms (–S–S–). Lastly, the sulfenated cysteinyl residue can react with glutathione (GSH), leading to a mixed disulfide (e.g., MetE). (Illustration: Rusty Howson, sososo design)</p

Additional Determinants within Escherichia coli FNR Activating Region 1 and RNA Polymerase α Subunit Required for Transcription Activation

The global anaerobic regulator FNR is a DNA binding protein that activates transcription of genes required for anaerobic metabolism in Escherichia coli through interactions with RNA polymerase (RNAP). Alanine-scanning mutagenesis of FNR amino acid residues 181 to 193 of FNR was utilized to determine which amino acid side chains are required for transcription of both class II and class I promoters. In vivo assays of FNR function demonstrated that a core of residues (F181, R184, S187, and R189) was required for efficient activation of class II promoters, while at a class I promoter, FF(−61.5), only S187 and R189 were critical for FNR activation. Site-directed mutagenesis of positions 184, 187, and 189 revealed that the positive charge contributes to the function of the side chain at positions 184 and 189 while the serine hydroxyl is critical for the function of position 187. Subsequent analysis of the carboxy-terminal domain of the α subunit (αCTD) of RNAP, using an alanine library in single copy, revealed that in addition to previously characterized side chains (D305, R317, and L318), E286 and E288 contributed to FNR activation of both class II and class I promoters, suggesting that αCTD region 285 to 288 also participates in activation by FNR. In conclusion, this study demonstrates that multiple side chains within region 181 to 192 are required for FNR activation and the surface of αCTD required for FNR activation is more extensive than previously observed

Fnr, NarP, and NarL Regulation of Escherichia coli K-12 napF (Periplasmic Nitrate Reductase) Operon Transcription In Vitro

The expression of several Escherichia coli operons is activated by the Fnr protein during anaerobic growth and is further controlled in response to nitrate and nitrite by the homologous response regulators, NarL and NarP. Among these operons, the napF operon, encoding a periplasmic nitrate reductase, has unique features with respect to its Fnr-, NarL-, and NarP-dependent regulation. First, the Fnr-binding site is unusually located compared to the control regions of most other Fnr-activated operons, suggesting different Fnr-RNA polymerase contacts during transcriptional activation. Second, nitrate and nitrite activation is solely dependent on NarP but is antagonized by the NarL protein. In this study, we used DNase I footprint analysis to confirm our previous assignment of the unusual location of the Fnr-binding site in the napF control region. In addition, the in vivo effects of Fnr-positive control mutations on napF operon expression indicate that the napF promoter is atypical with respect to Fnr-mediated activation. The transcriptional regulation of napF was successfully reproduced in vitro by using a supercoiled plasmid template and purified Fnr, NarL, and NarP proteins. These in vitro transcription experiments demonstrate that, in the presence of Fnr, the NarP protein causes efficient transcription activation whereas the NarL protein does not. This suggests that Fnr and NarP may act synergistically to activate napF operon expression. As observed in vivo, this activation by Fnr and NarP is antagonized by the addition of NarL in vitro