Integration of Metabolism and Virulence by
            <i>Clostridium difficile</i>
            CodY

Dineen, Sean S.; McBride, Shonna M.; Sonenshein, Abraham L.

doi:10.1128/jb.00341-10

Cited by 190 publications

(298 citation statements)

References 52 publications

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“…The method of genome-wide identification of proteinbinding regions in vitro, which we call here in vitro DNA affinity purification sequencing (IDAP-Seq), has been successfully applied previously for identification of CodY-binding regions in Staphylococcus aureus, Clostridium difficile, and Bacillus anthracis (16)(17)(18). The CodY-DNA complexes formed by incubation of fragmented, adapter-ligated chromosomal DNA with purified His-tagged CodY are isolated using immobilized metal-ion affinity purification.…”

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.

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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: 99%

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

Belitsky

Sonenshein

2013

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

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102

154

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“…First identified in Bacillus subtilis as a factor that represses stationary phase genes during exponential growth in rich medium (11), CodY is now known to provide a critical regulatory link between metabolism and pathogenesis in bacteria important for human health (5,6,(12)(13)(14)(15)(16). CodY is activated as a DNA-binding protein in vitro by at least two classes of metabolites: the branched-chain amino acids [BCAAs; i.e., isoleucine, leucine, and valine (ILV)] and GTP (17)(18)(19)(20)(21).…”

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confidence: 99%

Hierarchical expression of genes controlled by theBacillus subtilisglobal regulatory protein CodY

Brinsmade

Alexander

Livny

et al. 2014

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

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119

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Global regulators that bind strategic metabolites allow bacteria to adapt rapidly to dynamic environments by coordinating the expression of many genes. We report an approach for determining gene regulation hierarchy using the regulon of the Bacillus subtilis global regulatory protein CodY as proof of principle. In theory, this approach can be used to measure the dynamics of any bacterial transcriptional regulatory network that is affected by interaction with a ligand. In B. subtilis, CodY controls dozens of genes, but the threshold activities of CodY required to regulate each gene are unknown. We hypothesized that targets of CodY are differentially regulated based on varying affinity for the protein's many binding sites. We used RNA sequencing to determine the transcription profiles of B. subtilis strains expressing mutant CodY proteins with different levels of residual activity. In parallel, we quantified intracellular metabolites connected to central metabolism. Strains producing CodY variants F71Y, R61K, and R61H retained varying degrees of partial activity relative to the WT protein, leading to gene-specific, differential alterations in transcript abundance for the 223 identified members of the CodY regulon. Using liquid chromatography coupled to MS, we detected significant increases in branched-chain amino acids and intermediates of arginine, proline, and glutamate metabolism, as well as decreases in pyruvate and glycerate as CodY activity decreased. We conclude that a spectrum of CodY activities leads to programmed regulation of gene expression and an apparent rerouting of carbon and nitrogen metabolism, suggesting that during changes in nutrient availability, CodY prioritizes the expression of specific pathways.BCAA | ILV | RNA-seq | metabolite analysis

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“…These sequences were entered into the MEME software along with the overall CodY consensus sequence AATT TTCWGAAAATT, the C. difficile CodY consensus sequence TTYWRAA TWTTCWRAATWTTY, and 55 putative CodY-binding sequences as identified in C. difficile (15). The parameters for these analyses were as follows: any number of occurrences per sequence on the given strand of DNA with a width of 6 to 50 nt.…”

Section: Rna Extraction Reverse Transcription-pcr (Rt-pcr) and Quanmentioning

confidence: 99%

“…The PCR product was cloned into pBAD30 (15) that had been digested with EcoRI and KpnI, creating the CcpA His 6 -tagged protein vector named pBADccpA. pBADccpA was then introduced by transformation into the E. coli KS272 ara mutant strain (15). Cells containing pBADccpA were grown at 37°C in RM medium containing 100 g ml Ϫ1 of ampicillin until an OD 600 of 0.5 was reached.…”

Section: Rna Extraction Reverse Transcription-pcr (Rt-pcr) and Quanmentioning

confidence: 99%

“…For example, in log-phase cultures growing in rich media, CodY represses the tcdA and tcdB genes, which encode Clostridium difficile toxins A and B (15), as well as the Staphylococcus aureus hysA gene encoding the secreted enzyme hyaluronidase (16). Conversely, there is increasing evidence that CodY can positively regulate expression of some toxins made by low-GϩC-content Gram-positive bacteria, including production of anthrax toxin by Bacillus anthracis (17), botulinum neurotoxin by Clostridium botulinum (18), and ETX by C. perfringens (12).…”

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confidence: 99%

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NanI Sialidase, CcpA, and CodY Work Together To Regulate Epsilon Toxin Production by Clostridium perfringens Type D Strain CN3718

Freedman

McClane

2015

J Bacteriol

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Clostridium perfringens type D strains are usually associated with diseases of livestock, and their virulence requires the production of epsilon toxin (ETX). We previously showed (J. Li, S. Sayeed, S. Robertson, J. Chen, and B. A. McClane, PLoS Pathog 7:e1002429, 2011, http://dx.doi.org/10.1371/journal.ppat.1002429) that BMC202, a nanI null mutant of type D strain CN3718, produces less ETX than wild-type CN3718 does. The current study proved that the lower ETX production by strain BMC202 is due to nanI gene disruption, since both genetic and physical (NanI or sialic acid) complementation increased ETX production by BMC202. Furthermore, a sialidase inhibitor that interfered with NanI activity also reduced ETX production by wild-type CN3718. The NanI effect on ETX production was shown to involve reductions in codY and ccpA gene transcription levels in BMC202 versus wild-type CN3718. Similar to CodY, CcpA was found to positively control ETX production. A double codY ccpA null mutant produced even less ETX than a codY or ccpA single null mutant. CcpA bound directly to sequences upstream of the etx or codY start codon, and bioinformatics identified putative CcpA-binding cre sites immediately upstream of both the codY and etx start codons, suggesting possible direct CcpA regulatory effects. A ccpA mutation also decreased codY transcription, suggesting that CcpA effects on ETX production can be both direct and indirect, including effects on codY transcription. Collectively, these results suggest that NanI, CcpA, and CodY work together to regulate ETX production, with NanI-generated sialic acid from the intestines possibly signaling type D strains to upregulate their ETX production and induce disease. IMPORTANCEClostridium perfringens NanI was previously shown to increase ETX binding to, and cytotoxicity for, MDCK host cells. The current study demonstrates that NanI also regulates ETX production via increased transcription of genes encoding the CodY and CcpA global regulators. Results obtained using single ccpA or codY null mutants and a ccpA codY double null mutant showed that codY and ccpA regulate ETX production independently of one another but that ccpA also affects codY transcription. Electrophoretic mobility shift assays and bioinformatic analyses suggest that both CodY and CcpA may directly regulate etx transcription. Collectively, results of this study suggest that sialic acid generated by NanI from intestinal sources signals ETX-producing C. perfringens strains, via CcpA and CodY, to upregulate ETX production and cause disease. C lostridium perfringens is an important cause of human and livestock infections, including many diseases originating in the intestines (1, 2). The virulence of C. perfringens is largely attributable to its prolific toxin-producing ability, with different toxins involved in specific diseases (1,3,4). By definition, type D strains must produce both C. perfringens alpha toxin (CPA) and epsilon toxin (ETX). ETX is a class B NIAID priority toxin, since it is one of the most potent ba...

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Integration of Metabolism and Virulence by Clostridium difficile CodY

Cited by 190 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

Hierarchical expression of genes controlled by theBacillus subtilisglobal regulatory protein CodY

NanI Sialidase, CcpA, and CodY Work Together To Regulate Epsilon Toxin Production by Clostridium perfringens Type D Strain CN3718

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