Bacillus subtilis CodY Operators Contain Overlapping CodY Binding Sites

Wray, Lewis V.; Fisher, Susan H.

doi:10.1128/jb.05258-11

Cited by 19 publications

(18 citation statements)

References 52 publications

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“…Interestingly, the last 9 nt (AAWWTTCWG) of the 17-nt motif correspond to the first 9 nt of a second copy of the canonical motif. This is reminiscent of a recently suggested model of a 24-nt CodY-binding site consisting of two 15-nt canonical motifs overlapping by 6 nt (26).…”

Section: Genome-scale Identification Of B Subtilis Cody-binding Regimentioning

confidence: 67%

“…The rankings of some regions increased with the increase in the CodY/DNA ratio, suggesting that their binding sites are of relatively low affinity and that binding depends on a high degree of cooperativity. Cooperative binding of CodY to DNA has been detected for many genes (13,26). The rankings of other regions decreased with the increase in the CodY/DNA ratio, suggesting that they have relatively high affinity and that binding has a lower level of cooperativity and can be competed effectively by other sequences when the CodY concentration increases.…”

Section: Genome-scale Identification Of B Subtilis Cody-binding Regimentioning

confidence: 99%

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Genome-wide identification of Bacillus subtilis CodY-binding sites at single-nucleotide resolution

Belitsky

Sonenshein

2013

Proc. Natl. Acad. Sci. U.S.A.

102

154

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The CodY protein is a global transcriptional regulator that controls, directly or indirectly, expression of more than 100 genes and operons in Bacillus subtilis. We used in vitro DNA affinity purification combined with massively parallel sequencing, to identify B. subtilis chromosomal DNA fragments that bind CodY in vitro. A nonstandard strand-specific analysis of the data allowed us to pinpoint CodY-binding sites at single-nucleotide resolution. By comparing the extent of binding at decreasing CodY concentrations, we were able to classify binding regions according to their relative strengths and construct a subset of the 323 strongest CodY-binding regions that included sites associated with nearly all genes reported to be direct CodY targets. Many of the identified sites were located within coding regions. At such sites within the ispA, rapA, and rapE genes CodY-dependent repression was demonstrated using lacZ fusions and mutational analysis.

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Section: Genome-scale Identification Of B Subtilis Cody-binding Regimentioning

confidence: 67%

Section: Genome-scale Identification Of B Subtilis Cody-binding Regimentioning

confidence: 99%

Genome-wide identification of Bacillus subtilis CodY-binding sites at single-nucleotide resolution

Belitsky

Sonenshein

2013

Proc. Natl. Acad. Sci. U.S.A.

102

154

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“…Recently, the participation of two overlapping motifs was proposed to be a general rule for CodY binding (42). The accumulated results suggest a more complex mechanism of CodY-DNA interaction than previously thought.…”

Section: Discussionmentioning

confidence: 95%

CodY-Mediated Regulation of Guanosine Uptake in Bacillus subtilis

Belitsky

Sonenshein

2011

J Bacteriol

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CodY is a global transcriptional regulator known to control expression of more than 100 genes and operons in Bacillus subtilis. Some of the most strongly repressed targets of CodY, the nupNOPQ (formerly, yufNOPQ) genes, were found to encode a guanosine transporter. Using DNase I footprinting experiments, we identified two high-affinity CodY-binding sites in the regulatory region of the nupN gene. The two sites are located 50 bp upstream and 163 bp downstream of the transcription start site. The downstream site was responsible for 6-to 8-fold nupN repression in the absence of the upstream site. When the upstream site was intact, however, only a minor contribution of the downstream site to nupN regulation could be detected under the conditions tested. Both sites contained 15-bp CodY-binding motifs with two mismatches each with respect to the consensus sequence, AATTTTCWGTTTTAA. However, the experimentally determined binding sites included additional sequences flanking the 15-bp CodY-binding motifs. An additional version of the 15-bp CodY-binding motif, with 5 mismatches with respect to the consensus but essential for efficient regulation by CodY, was found within the upstream site. The presence of multiple 15-bp motifs may be a common feature of CodY-binding sites.Bacillus subtilis cells are able to take up purine and pyrimidine nucleosides, allowing these compounds to serve as substrates for nucleotide biosynthesis via salvage pathways and as sources of carbon and nitrogen (40). Although two major transporters for pyrimidine and purine nucleosides, NupC and NupG, respectively, have been described in B. subtilis, mutants lacking NupC or NupG retain considerable ability to take up pyrimidine or purine nucleosides (19, 33). Thus, additional transporters for both pyrimidine and purine nucleosides remain to be identified (2,19,33).The B. subtilis nupNOPQ (formerly, yufNOPQ) genes appear to encode the components of an ABC transport system of unknown specificity. The first gene of this apparent operon, nupN, codes for an apparent lipoprotein that could serve as the substrate-binding, specificity-determining subunit of the transporter. The putative product of nupN is homologous (43 to 50% identity) to several proteins that are involved in purine and pyrimidine nucleoside transport in Treponema pallidum, Streptococcus mutans, and Lactococcus lactis (9,26,41). The nupO and nupPQ genes appear to encode the ATP-binding protein and integral membrane proteins typical of ABC transporters, respectively.The nupNOPQ genes were previously shown to be among the genes that are most highly repressed by CodY as detected by DNA microarray analysis and lacZ fusions (29). CodY is a global transcriptional regulator that controls expression of more than 100 genes and operons in B. subtilis.Many of the CodY-regulated genes are involved in nitrogen or carbon metabolism (14,29,(37)(38)(39). The main role of CodY in B. subtilis appears to be establishing the temporal hierarchy of utilization of various nitrogen (and carbon) compounds under cond...

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“…The DNA binding reaction mixtures contained 5 nM DNA, 25 mM 2-(Nmorpholino) ethanesulfonic acid [pH 6.0], 200 mM sodium acetate, 1 mM dithiothreitol (DTT), 1 mM Tris (2-carboxyethyl) phosphine, 1 mM EDTA, 0.05% n-octylglucoside, 5% glycerol, 100 g/ml bovine serum albumin, 2 mM GTP, and 10 mM (each) valine, leucine, and isoleucine, as described previously (17). The reaction mixtures contained various amounts of CodY and were incubated for 15 to 20 min at room temperature and separated on 5% nondenaturing polyacrylamide gels containing 50 mM bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane [pH 6.0] and 10 mM isoleucine.…”

Section: Methodsmentioning

confidence: 99%

CodY Regulates SigD Levels and Activity by Binding to Three Sites in the fla / che Operon

Ababneh

Herman

2015

J Bacteriol

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Exponentially growing cultures of Bacillus subtilis (PY79) are composed primarily of nonmotile, chained cells. The alternative sigma factor, SigD, promotes the phenotypic switch from nonmotile, chained cells to unchained, motile cells. In the present work, we investigated the role of the GTP-sensing protein CodY in the regulation of SigD. Deletion of codY resulted in a significant increase in SigD accumulation and activity and shifted the proportion of unchained cells up from ϳ15% to ϳ75%, suggesting that CodY is an important regulator of SigD. CodY was previously shown to bind to the P D3 and P fla/che promoters located upstream of the first gene in the sigD-containing fla/che operon. Using electrophoretic mobility shift assays, we found that CodY also binds to two other previously uncharacterized sites within the fla/che operon. Mutations in any one of the three binding sites resulted in SigD levels similar to those seen with the ⌬codY mutant, suggesting that each site is sufficient to tip cells toward a maximal level of CodY-dependent SigD accumulation. However, mutations in all three sites were required to phenocopy the ⌬codY mutant's reduced level of cell chaining, consistent with the idea that CodY binding in the fla/che operon is also important for posttranslational control of SigD activity. IMPORTANCEOne way that bacteria adapt quickly and efficiently to changes in environmental quality is to employ global transcriptional regulators capable of responding allosterically to key cellular metabolites. In this study, we found that the conserved GTP-sensing protein CodY directly regulates cell motility and chaining in B. subtilis by controlling expression and activity of SigD. Our results suggest that B. subtilis becomes poised for cell dispersal as intracellular GTP levels are depleted. Genetically identical populations of bacteria often exhibit phenotypic heterogeneity, possessing cells that switch between distinct physiological and/or developmental states. Phenotypic switching is most widely understood in the context of highly regulated responses to changes in environmental cues, such as nutrient status and cell density. However, phenotypic switching can also occur spontaneously, and stochastic switching between two stable phenotypes at the subpopulation level is termed phenotypic bistability (1, 2). Bistability likely confers a selective advantage to a species under unpredictable environmental conditions by increasing the chance that at least one alternative cell state will survive under any given adverse condition (2-4). For example, in a given population of antibiotic-sensitive bacteria, small subpopulations of genetically identical and yet nongrowing and antibiotictolerant bacteria known as "persisters" are detectable (5, 6). Since the persister subpopulation is nongrowing, it is able to survive a sudden onslaught of antibiotic selection.Exponentially growing populations of Bacillus subtilis are comprised of both chained, nonmotile cells and unchained, motile cells, the latter of which are presu...

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Bacillus subtilis CodY Operators Contain Overlapping CodY Binding Sites

Cited by 19 publications

References 52 publications

Genome-wide identification of Bacillus subtilis CodY-binding sites at single-nucleotide resolution

Genome-wide identification of Bacillus subtilis CodY-binding sites at single-nucleotide resolution

CodY-Mediated Regulation of Guanosine Uptake in Bacillus subtilis

CodY Regulates SigD Levels and Activity by Binding to Three Sites in the fla / che Operon

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