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The ResD response regulator, through functional interaction with NsrR and fur, plays three distinct roles in Bacillus subtilis transcriptional control.

ABSTRACT: The ResD response regulator activates transcription of diverse genes in Bacillus subtilis in response to oxygen limitation. ResD regulon genes that are the most highly induced during nitrate respiration include the nitrite reductase operon (nasDEF) and the flavohemoglobin gene (hmp), whose products function in nitric oxide (NO) metabolism. Transcription of these genes is also under the negative control of the NO-sensitive NsrR repressor. Recent studies showed that the NsrR regulon contains genes with no apparent relevance to NO metabolism and that the ResD response regulator and NsrR coordinately regulate transcription. To determine whether these genes are direct targets of NsrR and ResD, we used chromatin affinity precipitation coupled with tiling chip (ChAP-chip) and ChAP followed by quantitative PCR (ChAP-qPCR) analyses. The study showed that ResD and NsrR directly control transcription of the ykuNOP operon in the Fur regulon. ResD functions as an activator at the nasD and hmp promoters, whereas it functions at the ykuN promoter as an antirepressor of Fur and a corepressor for NsrR. This mechanism likely participates in fine-tuning of transcript levels in response to different sources of stress, such as oxygen limitation, iron limitation, and exposure to NO.

SUBMITTER: Henares B

PROVIDER: S-EPMC3911253 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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The ResD response regulator, through functional interaction with NsrR and fur, plays three distinct roles in Bacillus subtilis transcriptional control.

Henares Bernadette B Kommineni Sushma S Chumsakul Onuma O Ogasawara Naotake N Ishikawa Shu S Nakano Michiko M MM

Journal of bacteriology 20131108 2

The ResD response regulator activates transcription of diverse genes in Bacillus subtilis in response to oxygen limitation. ResD regulon genes that are the most highly induced during nitrate respiration include the nitrite reductase operon (nasDEF) and the flavohemoglobin gene (hmp), whose products function in nitric oxide (NO) metabolism. Transcription of these genes is also under the negative control of the NO-sensitive NsrR repressor. Recent studies showed that the NsrR regulon contains genes ...[more]

PMID: 24214949

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Project description:Upon oxygen limitation, the Bacillus subtilis ResE sensor kinase and its cognate ResD response regulator play primary roles in the transcriptional activation of genes functioning in anaerobic respiration. The nitric oxide (NO)-sensitive NsrR repressor controls transcription to support nitrate respiration. In addition, the ferric uptake repressor (Fur) can modulate transcription under anaerobic conditions. However, whether these controls are direct or indirect has been investigated only in a gene-specific manner. To gain a genomic view of anaerobic gene regulation, we determined the genome-wide in vivo DNA binding of ResD, NsrR, and Fur transcription factors (TFs) using in situ DNase I footprinting combined with chromatin affinity precipitation sequencing (ChAP-seq; genome footprinting by high-throughput sequencing [GeF-seq]). A significant number of sites were targets of ResD and NsrR, and a majority of them were also bound by Fur. The binding of multiple TFs to overlapping targets affected each individual TF's binding, which led to combinatorial transcriptional control. ResD bound to both the promoters and the coding regions of genes under its positive control. Other genes showing enrichment of ResD at only the promoter regions are targets of direct ResD-dependent repression or antirepression. The results support previous findings of ResD as an RNA polymerase (RNAP)-binding protein and indicated that ResD can associate with the transcription elongation complex. The data set allowed us to reexamine consensus sequence motifs of Fur, ResD, and NsrR and uncovered evidence that multiple TGW (where W is A or T) sequences surrounded by an A- and T-rich sequence are often found at sites where all three TFs competitively bind.IMPORTANCE Bacteria encounter oxygen fluctuation in their natural environment as well as in host organisms. Hence, understanding how bacteria respond to oxygen limitation will impact environmental and human health. ResD, NsrR, and Fur control transcription under anaerobic conditions. This work using in situ DNase I footprinting uncovered the genome-wide binding profile of the three transcription factors (TFs). Binding of the TFs is often competitive or cooperative depending on the promoters and the presence of other TFs, indicating that transcriptional regulation by multiple TFs is much more complex than we originally thought. The results from this study provide a more complete picture of anaerobic gene regulation governed by ResD, NsrR, and Fur and contribute to our further understanding of anaerobic physiology.

Project description:The ResD-ResE signal transduction system plays a pivotal role in anaerobic nitrate respiration in Bacillus subtilis. The nasD operon encoding nitrite reductase is essential for nitrate respiration and is tightly controlled by the ResD response regulator. To understand the mechanism of ResD-dependent transcription activation of the nasD operon, we explored ResD-RNA polymerase (RNAP), ResD-DNA, and RNAP-DNA interactions required for nasD transcription. Full transcriptional activation requires the upstream promoter region where five molecules of ResD bind. The distal ResD-binding subsite at -87 to -84 partially overlaps a sequence similar to the consensus distal subsite of the upstream (UP) element with which the Escherichia coli C-terminal domain of the α subunit (αCTD) of RNAP interacts to stimulate transcription. We propose that interaction between αCTD and ResD at the promoter-distal site is essential for stimulating nasD transcription. Although nasD has an extended -10 promoter, it lacks a reasonable -35 element. Genetic analysis and structural simulations predicted that the absence of the -35 element might be compensated by interactions between σA and αCTD and between αCTD and ResD at the promoter-proximal ResD-binding subsite. Thus, our work suggested that ResD participates in nasD transcription activation by binding to two αCTD subunits at the proximal and distal promoter sites, representing a unique configuration for transcription activation. IMPORTANCE A significant number of ResD-controlled genes have been identified, and transcription regulatory pathways in which ResD participates have emerged. Nevertheless, the mechanism of how ResD activates transcription of different genes in a nucleotide sequence-specific manner has been less explored. This study suggested that among the five ResD-binding subsites in the promoter of the nasD operon, the promoter-proximal and -distal ResD-binding subsites play important roles in nasD activation by adapting different modes of protein-protein and protein-DNA interactions. The finding of a new type of protein-promoter architecture provides insight into the understanding of transcription activation mechanisms controlled by transcription factors, including the ubiquitous response regulators of two-component regulatory systems, particularly in Gram-positive bacteria.

Project description:The physiological relevance of bacterial iron efflux has only recently been appreciated. The Bacillus subtilis P1B4-type ATPase PfeT (peroxide-induced ferrous efflux transporter) was one of the first iron efflux pumps to be characterized, and cells lacking pfeT accumulate high levels of intracellular iron. The pfeT promoter region has binding sites for both PerR, a peroxide-sensing Fur-family metalloregulator, and the ferric uptake repressor Fur. Both Fur and PerR bind DNA with Fe(II) as a cofactor. While reaction of PerR-Fe(II) with peroxide can account for the induction of pfeT under oxidative stress, binding of Fur-Fe(II) would be expected to lead to repression, which is inconsistent with the known role of PfeT as an iron efflux protein. Here, we show that expression of pfeT is repressed by PerR, as anticipated, and induced by Fur in response to Fe(II). Activation by Fur is mediated both by antagonism of the PerR repressor and by direct transcriptional activation, as confirmed using in vitro transcription assays. A similar mechanism of regulation can explain the iron induction of the Listeria monocytogenes PfeT ortholog and virulence factor, FrvA. Mutational studies support a model in which Fur activation involves regions both upstream and downstream of the pfeT promoter, and Fur and PerR have overlapping recognition of a shared regulatory element in this complex promoter region. This work demonstrates that B. subtilis Fur can function as an iron-dependent activator of transcription.IMPORTANCE Iron homeostasis plays a key role at the host-pathogen interface during the process of infection. Bacterial growth restriction resulting from host-imposed iron starvation (nutritional immunity) highlights the importance of iron import during pathogenesis. Conversely, bacterial iron efflux pumps function as virulence factors in several systems. The requirement for iron efflux in pathogens such as Listeria monocytogenes, Streptococcus pyogenes, and Mycobacterium tuberculosis suggests that both import and efflux are needed for cells to successfully navigate rapidly changing levels of iron availability in the host. Here, we provide insight into how iron efflux genes are controlled, an aspect of bacterial iron homeostasis relevant to infectious disease processes.

Dataset Information

The ResD response regulator, through functional interaction with NsrR and fur, plays three distinct roles in Bacillus subtilis transcriptional control.

Publications

The ResD response regulator, through functional interaction with NsrR and fur, plays three distinct roles in Bacillus subtilis transcriptional control.

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