AbrB is a global transcriptional regulator of Bacillus subtilis that represses the expression of many genes during exponential growth. Here, we demonstrate that AbrB and its homolog Abh bind to hundreds of sites throughout the entire B. subtilis genome during exponential growth. Comparison of regional binding of AbrB and Abh in wild-type, ΔabrB and Δabh backgrounds revealed that they bind as homomer and/or heteromer forms with different specificities and affinities. We found four AbrB and Abh binding patterns were major. Three of these contain pairs of TGGNA motifs connected by A/T-rich sequences, differing in arrangement and spacing. We also assessed the direct involvement of these complexes in the control of gene expression. Our data indicate that AbrB usually acts as a repressor, and that the ability of Abh to act as a transcriptional regulator was limited. We found that changes to AbrB/Abh levels affect their binding at several promoters and consequently transcriptional regulation. Surprisingly, most AbrB/Abh binding events had no impact on transcription, suggesting an interesting possibility that AbrB/Abh binding is analogous to nucleoid-associated protein binding in Escherichia coli.

Accurate identification of the DNA-binding sites of transcription factors and other DNA-binding proteins on the genome is crucial to understanding their molecular interactions with DNA. Here, we describe a new method: Genome Footprinting by high-throughput sequencing (GeF-seq), which combines in vivo DNase I digestion of genomic DNA with ChIP coupled with high-throughput sequencing. We have determined the in vivo binding sites of a Bacillus subtilis global regulator, AbrB, using GeF-seq. This method shows that exact DNA-binding sequences, which were protected from in vivo DNase I digestion, were resolved at a comparable resolution to that achieved by in vitro DNase I footprinting, and this was simply attained without the necessity of prediction by peak-calling programs. Moreover, DNase I digestion of the bacterial nucleoid resolved the closely positioned AbrB-binding sites, which had previously appeared as one peak in ChAP-chip and ChAP-seq experiments. The high-resolution determination of AbrB-binding sites using GeF-seq enabled us to identify bipartite TGGNA motifs in 96% of the AbrB-binding sites. Interestingly, in a thousand binding sites with very low-binding intensities, single TGGNA motifs were also identified. Thus, GeF-seq is a powerful method to elucidate the molecular mechanism of target protein binding to its cognate DNA sequences.

bThe 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. Bacillus subtilis undergoes either nitrate respiration or fermentation to generate ATP when oxygen becomes limited (reviewed in reference 1). Growth under oxygen-limited conditions, particularly via nitrate respiration, requires the ResD-ResE twocomponent regulatory system (2, 3). During nitrate respiration in B. subtilis, unlike the case with denitrifiers, nitrite is reduced to ammonium instead of nitric oxide (NO). However, NO is generated at low concentrations from nitrite as a by-product of nitrate respiration in B. subtilis (4), as it is in Escherichia coli (5). Since accumulation of NO is cytotoxic, B. subtilis uses flavohemoglobin (Hmp) (6) and nitrite reductase (NasDEF) (7) to reduce NO levels by conversion of NO to nitrate (or N 2 O under anaerobic conditions) (8-10) and by metabolism of nitrite to ammonium (7), respectively. NsrR, a member of the Rrf2 family, is known to control transcription of genes involved in NO detoxification in both Gram-positive and Gram-negative bacteria (reviewed in references 11 and 12). B. subtilis NsrR represses transcription of the nasD operon and hmp under anaerobic fermentative conditions (4). Transcription of these genes is dependent on the ResD response regulator and the ResE sensor kinase (6, 7). NsrR binds to the Ϫ35 region of the nasD promoter, resulting in disruption of the RNA polymerase (RNAP)-ResD-DNA complex (13). When NO is present endogenously via nitrate respiration or exogenously, NsrR-dependent repression of nasD and hmp is relieved. This derepression is attributed to the release of NsrR from the nasD promoter by direct interaction of NO with iron in the [4Fe-4S] cluster of NsrR (13,14). More genes controlled by NsrR were identified by transcriptome analysis, which was validated by transcriptional ...

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.

Bacterial bio-production during the stationary phase is expected to lead to a high target yield because the cells do not consume the substrate for growth. Bacillus subtilis is widely used for bio-production, but little is known about the metabolism during the stationary phase. In this study, we focused on the dipicolinic acid (DPA) production by B. subtilis and investigated the metabolism. We found that DPA production competes with acetoin synthesis and that acetoin synthesis genes (alsSD) deletion increases DPA productivity by 1.4-fold. The mutant showed interesting features where the glucose uptake was inhibited, whereas the cell density increased by approximately 50%, resulting in similar volumetric glucose consumption to that of the parental strain. The metabolic profiles revealed accumulation of pyruvate, acetyl-CoA, and the TCA cycle intermediates in the alsSD mutant. Our results indicate that alsSD-deleted B. subtilis has potential as an effective host for stationary-phase production of compounds synthesized from these intermediates.