integration of metabolism and virulence by clostridium difficile cody â€

JOURNAL OF BACTERIOLOGY, Oct. 2010, p. 5350–5362 Vol. 192, No. 200021-9193/10/$12.00 doi:10.1128/JB.00341-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Integration of Metabolism and Virulence byClostridium difficile CodY�†

Sean S. Dineen,‡§ Shonna M. McBride,‡ and Abraham L. Sonenshein*Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111

Received 26 March 2010/Accepted 2 August 2010

CodY, a global regulatory protein that monitors the nutrient sufficiency of the environment by respondingto the intracellular levels of GTP and the branched-chain amino acids, was previously shown to be a potentrepressor of toxin gene expression in Clostridium difficile during growth in rich medium. In the intestinal tract,such derepression of toxin synthesis would lead to destruction of epithelial cells and the liberation of potentialnutrients for the bacterium. CodY is likely to play an important role in regulating overall cellular physiologyas well. In this study, DNA microarray analysis and affinity purification of CodY-DNA complexes were used toidentify and distinguish the direct and indirect effects of CodY on global gene transcription. A codY nullmutation resulted in >4-fold overexpression of 146 genes (organized in 82 apparent transcription units) andunderexpression of 19 genes. In addition to the toxin genes, genes for amino acid biosynthesis, nutrienttransport, fermentation pathways, membrane components, and surface proteins were overexpressed in the codYmutant. Genome-wide analysis identified more than 350 CodY binding regions, many of which are likely tocorrespond to sites of direct CodY-mediated regulation. About 60% of the CodY-repressed transcription unitswere associated with binding regions. Several of these genes were confirmed to be direct targets of CodY by gelmobility shift and DNase I footprinting assays.

The ability to sense and adapt to excess or limiting nutrientsis a universal trait in the bacterial world. For Clostridium dif-ficile, a Gram-positive, anaerobic, spore-forming, intestinalbacterium, this ability may be essential for its proliferation inthe gastrointestinal tract (18). In the hospital environment, C.difficile is now the most common cause of antibiotic-associatedcolitis, a disease that has increased in incidence and severity inrecent years due to the emergence of highly virulent “epi-demic” C. difficile strains (19). Antibiotic treatment is thoughtto induce C. difficile infection (CDI) by disrupting the colonicmicroflora, thereby allowing C. difficile to colonize (19). Mosthighly virulent C. difficile strains produce two large proteintoxins, A and B, that act by glycosylating members of the hostcell’s Rho family of small GTPases (16, 17, 52). Recent work ofLyras and colleagues (25) has shown that toxin B, but not toxinA, is essential for virulence in a hamster model of CDI. SomeC. difficile strains, including the current epidemic strain typeNAP-1/027, also produce a binary ADP-ribosylating toxin,CDT, that is reported to increase colonization (42) but is notknown to be essential for pathogenicity (19, 32).

The genes encoding toxins A and B (tcdA and tcdB) liewithin a 19.6-kb pathogenicity locus (PaLoc) that also includesthe tcdR, tcdE, and tcdC genes (4, 11). In broth cultures of C.difficile, expression of tcdA and tcdB is induced when nutrients

become limiting and cells enter stationary phase (8, 14). Aclear link between nutrient limitation and toxin gene expres-sion came from the discovery that expression of all the PaLocgenes is repressed by the global transcriptional regulator CodY(7). CodY represses toxin gene expression by binding to theputative promoter region for the tcdR gene (7), whose productis a sigma factor that directs transcription from the tcdA andtcdB gene promoters as well as from its own promoter (30, 31).

CodY proteins, first discovered in Bacillus subtilis and foundin many other low-G�C-content Gram-positive bacteria, ap-pear to have in common the ability to repress during rapidgrowth genes whose products are not needed when nutrientsare in excess and to release this repression under nutrient-poorconditions (46). One interpretation of this effect is that CodYproteins help to regulate the synthesis and distribution of pyru-vate and 2-oxoglutarate, two key intermediates in central me-tabolism (47). Functions commonly regulated by CodY in var-ious bacteria include carbon overflow metabolism; the Krebscycle; synthesis of certain amino acids; uptake and catabolismof amino acids, peptides, and sugars; genetic competence; mo-tility; and sporulation (3, 6, 10, 12, 21, 33, 37). For most ofthese genes, CodY acts as a transcriptional repressor. Only inthe case of the B. subtilis ackA gene has CodY been shown toact as a direct positive regulator (44).

CodY proteins also have species-specific roles, particularlywith respect to virulence gene expression, in C. difficile (7),Staphylococcus aureus (27, 28, 37), Streptococcus pneumoniae(12), Streptococcus pyogenes (29), Streptococcus mutans (21),Listeria monocytogenes (3), Bacillus cereus (13) and Bacillusanthracis (51). Although the number of genes whose expres-sion is affected by a codY mutation in any given bacterium islarge, in only a few cases has the direct binding of CodY to theregulatory region of a regulated gene been demonstrated. As aresult, our knowledge of the extent to which the CodY regulon

* Corresponding author. Mailing address: Department of MolecularBiology and Microbiology, Tufts University School of Medicine, 136Harrison Avenue, Boston, MA 02111. Phone: (617) 636-6761. Fax:(617) 636-0337. E-mail: [email protected].

‡ These authors contributed equally to this work.§ Present address: Beckman Coulter Genomics, Danvers, MA

01923.† Supplemental material for this article may be found at http://jb

.asm.org/.� Published ahead of print on 13 August 2010.

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is defined by the direct action of CodY, as opposed to an effectof CodY on the synthesis or activity of other regulatory pro-teins, is rather limited.

Structural studies of B. subtilis CodY suggest that the di-meric protein binds to DNA through winged helix-turn-helixmotifs located in the C-terminal domain (15, 22). Interactionof B. subtilis CodY with DNA is enhanced in the presence oftwo different kinds of effectors whose activities account forCodY’s response to nutritional conditions. GTP and thebranched-chain amino acids (BCAAs) act synergistically toincrease the affinity of CodY for DNA (38, 45). C. difficileCodY also responds to GTP and BCAAs in vitro; that is, CodYbinding to the tcdR promoter region is enhanced synergisticallyin response to these effectors (7). A CodY consensus bindingmotif, AATTTTCWGAAAATT, has been identified upstreamof many of the genes regulated by CodY in Lactococcus lactisand B. subtilis (2, 6, 10).

In the work described here, we have identified many genesregulated by C. difficile CodY. Using microarray analysis, wefound more than 140 genes that appeared to be directly orindirectly under CodY control. The products of these genesare active in many aspects of metabolism and cell function.Affinity purification was used to pinpoint direct targets ofCodY, allowing us to distinguish between transcription unitsdirectly and indirectly influenced by CodY. For selected genes,CodY was confirmed to interact directly with their putativepromoter regions by electrophoretic mobility shift and DNaseI footprinting experiments. Many apparent direct targets ofCodY-mediated regulation contain sequences with similarityto the consensus CodY binding motif.

MATERIALS AND METHODS

Strains and growth conditions. Bacterial strains and plasmids used in thisstudy are described in Table 1. C. difficile strains were grown in TY medium (8),in tryptone-yeast extract (TY) medium supplemented with 1% glucose (TYG) orin brain heart infusion medium (Difco) supplemented with yeast extract (5g/liter) and 0.1% cysteine (BHIS). Media for growth of C. difficile were supple-mented with 10 �g of thiamphenicol ml�1 as needed. C. difficile strains weregrown in an atmosphere of 10% H2, 5% CO2, and 85% N2 in an anaerobicchamber (Coy Laboratory Products) at 37°C. Escherichia coli strains were grown

at 37°C in L medium (24) supplemented with 50 �g of ampicillin ml�1, asneeded.

DNA preparation. Genomic DNA (gDNA) was extracted from C. difficilestrain 630 that had been grown overnight in 10 ml of BHIS broth. Cells werepelleted and washed in 1 ml of TE buffer (10 mM Tris-HCl [pH 8.0] and 1 mMEDTA) and then resuspended in 400 �l of TE-glucose buffer (50 mM glucose, 25mM Tris-HCl [pH 8.0], and 10 mM EDTA) containing 25 mg of lysozyme per ml.The suspension was incubated for 2 h at 37°C before addition of N-lauroylsarcosine to a final concentration of 7%. After addition of 15 �l of RNase A (10mg/ml), the suspension was incubated for 15 min at 37°C. To this mixture, 10 �lof proteinase K (8 units; Sigma-Aldrich) was added, and the solution was incu-bated for 30 min at 37°C. TE buffer was used to bring the volume up to 1 ml. TheDNA was extracted three times with phenol-chloroform-isoamyl alcohol (25:24:1) and once with chloroform. DNA was precipitated with 3 volumes of iso-propanol and 1/10 volume of 3 M sodium acetate (pH 5.2), washed with 70%ethanol, air dried, and resuspended in 100 �l of TE buffer.

RNA preparation. Overnight cultures of C. difficile grown in TYG mediumwere diluted to an optical density at 600 nm (OD600) of 0.05 in TY medium andincubated anaerobically at 37°C. Samples were removed for RNA isolation at anOD600 of 0.4 (exponential phase), diluted with an equal volume of ice-coldacetone-ethanol (1:1), and stored at �80°C. The cell suspensions were thawedand centrifuged at 4°C; the pellets were air dried, washed twice with 500 �l of TEbuffer (pH 7.6), and resuspended in 1 ml of buffer RLT from the Qiagen RNeasykit. Silica glass beads (0.1 mm) were added, and cells were disrupted using aMini-BeadBeater (BioSpecs Products). Silica beads were removed by centrifu-gation, and total RNA was isolated from the supernatant fluid using a QiagenRNeasy kit. DNA contaminating the RNA samples was removed using an Am-bion TURBO DNA-free kit for reverse transcription-PCR (RT-PCR) and 5�rapid amplification of cDNA ends (RACE) analysis. Independent samples ofeach culture were used to provide DNA templates to verify by PCR the main-tenance of the integrated plasmid at the codY locus (oligonucleotides used arelisted in Table S1 in the supplemental material). In addition, the characteristicsmooth, partly translucent colony morphology of the codY mutant strain wasused as an indicator of the retention of the integrated plasmid in the mutantstrain culture.

Microarray analysis. The C. difficile microarray based on the genome se-quence of strain 630 was obtained from the Bacterial Microarray Group at St.George’s Hospital, University of London (B�G@S), and its design has beendescribed previously (34, 48). The array layout, representing 3,688 of the 3,776identifiable open reading frames, can be found through the B�G@S website(http://bugs.sgul.ac.uk/A-BUGS-20) and ArrayExpress (accession numberA-BUGS-20). A Genisphere 3DNA Array 900 MPX microarray kit was used forcDNA synthesis, labeling, and hybridization with specific protocol modificationsas described by O’Connor et al. (34). Ten micrograms of starting RNA was usedfor the cDNA synthesis reactions. The microarray slides were scanned using aScanArray 4000 (Packard Instrument Co.), and fluorescence intensities werequantified using ImaGene (BioDiscovery) software. Raw expression data from

TABLE 1. Bacterial strains and plasmids

Strain or plasmid Characteristics or description Referenceor source

StrainsE. coli

TOP10 Invitrogen

C. difficile630 Wild-type 53JIR8094 Ems derivative of strain 630 34JIR8094(pSD21) JIR8094 containing pSD21 7JIR8094::pSD21 codY mutant 7

PlasmidspJIR1456 E. coli-C. perfringens shuttle vector; Tmr 26pSD21 Internal region of C. difficile codY gene cloned in pJIR1456; Tmr 7pCR2.1 E. coli cloning vector; Apr InvitrogenpSD30 494-bp DNA fragment containing the CD2344 gene promoter region cloned in pCR2.1; Apr This studypSD31 780-bp DNA fragment containing the glgC gene promoter region cloned in pCR2.1; Apr This studypSD32 861-bp DNA fragment containing the hisZ gene promoter region cloned in pCR2.1; Apr This study

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three arrays, representing three biological replicates, were normalized and av-eraged using GeneSpring (Agilent Technologies) software. A dye swap wasperformed with one of the three replicates. A gene was defined as differentiallyexpressed if it exhibited at least a 4-fold change in transcript level in the mutantversus the wild-type strain and if the change was statistically significant by aStudent’s t test (P � 0.005). All such genes are listed in Table S2 in the supple-mental material.

Quantitative reverse transcriptase PCR (qRT-PCR) analysis. SuperScript IIreverse transcriptase (Invitrogen) was used to generate randomly primed cDNApools from 1 �g of RNA, as instructed by the manufacturer, from each of threestrains, JIR8094, JIR8094 (pSD21), and JIR8094::pSD21. To control for chro-mosomal DNA contamination, mock cDNA synthesis reaction mixtures contain-ing no reverse transcriptase were used as negative controls in subsequent am-plifications. cDNA samples were diluted 4-fold and used as templates forquantitative PCR of transcripts from rpoC (primers oMC44/oMC45), ilvC(oMC152/oMC153), CD2344 (oMC154/oMC155), glgC (oMC156/oMC157),hisZ (oMC158/oMC159), speA (oMC160/oMC161), CD1276 (oMC162/oMC163), spoIIE (oMC164/oMC165), CD1476 (oMC166/oMC167), andCD1803 (oMC168/oMC169), using Qiagen SYBR green PCR mix and anMXP3005 thermocycler (Stratagene/Agilent Technologies). The sequences of allprimers are listed in Table S1 in the supplemental material. Reactions wereperformed in a final volume of 25 �l using 4 �l of diluted cDNA, and a 1 �Mconcentration of each primer. Reactions were performed in triplicate usingcDNA extracted from a minimum of two biological replicates. Amplificationincluded 40 cycles of the following steps: 15 s at 95°C, 60 s at 50°C, and 30 s at72°C. Results were calculated using the comparative cycle threshold method(41), where the amount of target mRNA is normalized relative to an internalcontrol transcript (rpoC), and then that ratio is compared to the ratio in theparental strain (JIR8094).

Affinity purification of CodY-DNA complexes and analysis using the IlluminaGenome Analyzer II. We adapted a method developed by C. D. Majerczyk et al.(27) to screen the C. difficile genome for CodY binding sites. Four samples of 7.5�g of gDNA were each dissolved in 500 �l of sterile deionized water and shearedby sonication (Branson 250 microtip sonicator) in five 30-s cycles, on ice. Fol-lowing shearing, gDNA was purified using a Qiagen PCR Purification kit ac-cording to the manufacturer’s instructions, and resuspended in 30 �l of Qiagenbuffer EB. For each sample to be bar-coded, 5 �g of sheared, purified gDNA wasblunted using a Quick Blunting kit from New England Biolabs, cleaned with aQiagen PCR Purification kit, and eluted in 30 �l of buffer EB. A dAMP residuewas added (A-tailing) to the 3� ends of the DNA fragments using dATP andDNA polymerase Klenow fragment (exo-) (New England Biolabs); the frag-ments were cleaned again with the Qiagen PCR Purification kit and eluted in 30�l of buffer EB. To generate double-stranded adapters, equal amounts of com-plementary oligonucleotides (e.g., BC1a and BC1b primers for bar code 1) (seeTable S1 in the supplemental material) were mixed, heated to 95°C, and slowlycooled to �40°C. (The b series of oligonucleotides was phosphorylated at the 5�ends after synthesis). An adapter (0.125 nmol) (see Table S1), specific to eachbar code, was then ligated to the sheared, blunted, A-tailed gDNA using a QuickLigation Kit (New England Biolabs). Each sample was then run on a 2% agarosegel, and DNA corresponding to the 400- to 500-bp size range was recovered fromthe gel using a Qiagen Gel Extraction kit. Each purified, bar-coded sample wassplit into 10 aliquots, each of which was subjected to 15 cycles of PCR amplifi-cation using primers Olj139 and Olj140 (see Table S1). The PCRs were thencleaned using a Qiagen PCR Purification kit and pooled for each bar code, andthe DNA products were quantified.

In a total volume of 250 �l, 5 �g of bar-coded C. difficile strain 630 gDNA wasused in a binding reaction with 200 nM purified His6-tagged C. difficile CodY (7)or His10-tagged B. subtilis aconitase protein (43) (as a negative control) in abinding buffer containing 10 mM (each) isoleucine, leucine, and valine (BCAAs)and 2 mM GTP, 20 mM Tris-Cl, pH 8.0, 50 mM sodium glutamate, 10 mMMgCl2, 0.05% Nonidet P-40, 5% glycerol, and E. coli tRNA (25 �g/ml). After 25min at room temperature, the binding reaction mixture was combined with a0.1-ml packed volume of Talon Co2� Metal Affinity Resin (Clontech) that hadbeen preequilibrated with binding buffer. Following incubation at room temper-ature for 20 min, the resin was washed six times with 0.25 ml of binding buffer.The resin was then resuspended in 100 �l of 10 mM Tris, pH 8.0, boiled for 5min, allowed to come to room temperature, incubated with proteinase K (100�g/ml) at 65°C for 2 h, and then boiled for 5 min. After centrifugation for 1 minat 3,000 � g, 100 �l of the supernatant fluid was transferred to a fresh tube andpurified using a PCR purification kit (Qiagen). The sample was then subjected to10 cycles of PCR amplification using primers Olj139 and Olj40, purified with thePCR purification kit, and quantified. To verify that the pulldown was successful,semiquantitative PCR was performed with primers for the rpoC gene (oMC44/

oMC45) and a known CodY target gene, tcdR (oLB51/oLB52). Samples werethen subjected to 40-nucleotide (nt), single-ended sequence analysis using theIllumina Genome Analyzer II by the Tufts University Nucleic Acid and ProteinCore Facility. Each pulldown experiment was performed in duplicate. CodY-bound and gDNA (control) samples were labeled independently using separatebar codes (1 to 4).

Reference alignment and assembly were performed using MAQ (23) againstthe C. difficile 630 complete genome reference sequence (NCBI accessionNC_009089.1). Using a series of BioPerl scripts, 5� tag positions were identifiedand then shifted over by one-half the average DNA fragment length, as deter-mined by an Agilent Bioanalyzer DNA 1000 chip. Tag counts for each positionwere averaged using a sliding window, creating a coverage map for the entiregenome. From this analysis, the CodY/total DNA enrichment ratio at everyposition was found. In control samples (aconitase-enriched DNA or unselectedgDNA), no region was enriched more than 5-fold above background. Regions ofenrichment in the CodY sample were considered when the CodY samplesshowed an average enrichment ratio greater than 5-fold for more than 100consecutive base pairs. Using a method described by Pepke et al. (35), the falsediscovery rate at 5-fold enrichment was determined to be 0.01. To focus on thesites most likely to be bona fide targets of CodY, we selected the 350 most highlyenriched regions (see Table S3 in the supplemental material), all of which wereenriched at least 17-fold in at least one experiment. The calculated z scores forthese genes ranged from 13 to 100, indicating that the chance occurrence of thislevel of enrichment is infinitely small.

The sequences of 293 enriched regions were searched for conserved motifsusing the MEME suite, version 4.1.0 (1). The parameters were as follows: 0 to 1occurrences per fragment on either strand of DNA and a width of 6 to 50 nt. Themotif that resulted was found in 280 sequences with an overall P value of5.2E�68. The sequence with least adherence to the motif had a P value of3.6E�0.03.

RT-PCR analysis. Reverse transcription was performed using SuperScript IIreverse transcriptase (Invitrogen) with 50 ng of total RNA and 2 pmol of gene-specific primer (see Table S1 in the supplemental material), according to themanufacturer’s protocol. Two-microliter samples of the reverse transcriptionreactions were used as templates for PCR with gene-specific primers (Table S1).PCRs were performed using 27 cycles of denaturation (94°C for 30 s), annealing(50°C for 30 s), and extension (72°C for 1 min). For each gene tested, the amountof RNA used and the number of cycles chosen for the experiment were belowsaturation for both the reverse transcription and the PCR. As a control forchromosomal DNA contamination, RNA was used directly for PCR amplifica-tion without reverse transcription.

Gel mobility shift assays. DNA fragments containing promoter regions of theilvC (423 bp) and CD2344 (494 bp) genes were amplified from C. difficile strain630 chromosomal DNA by PCR using specific primers (OSD107/OSD108 forilvC; OSD124/OSD125 for CD2344) (see Table S1), agarose gel-purified, andlabeled with [�-32P]ATP using T4 polynucleotide kinase (Invitrogen) as de-scribed by the manufacturer. For amplification of DNA fragments containing theglgC (780 bp) and hisZ (861 bp) promoters, primers OSD127 and OSD128,respectively, were radioactively labeled with [�-32P]ATP using T4 polynucleotidekinase (Invitrogen) as described previously (20) and used in conjunction withOSD126 and OSD129, respectively, to amplify the promoter regions. LabeledDNA was mixed with increasing amounts of His6-tagged CodY protein (7) in10-�l reaction mixtures that contained 20 mM Tris-Cl (pH 8.0), 50 mM sodiumglutamate, 10 mM MgCl2, 5 mM EDTA, 0.05% (vol/vol) Nonidet P-40 (Igepal;Sigma-Aldrich), 5% (vol/vol) glycerol, and 250 ng of calf thymus DNA. Whereindicated, 10 mM (each) isoleucine, leucine, and valine (BCAAs) and 2 mMGTP was included. After incubation for 30 min at room temperature, bindingreaction mixtures were loaded on a 12% nondenaturing polyacrylamide gelprepared in Tris-glycine buffer, and electrophoresis was carried out in 35 mMHEPES–43 mM imidazole buffer (pH 7.4), as described previously (45). When 10mM BCAAs were present in the binding reaction mixture, BCAAs at the sameconcentration were also added to the electrophoresis buffer. Gels were driedunder vacuum and exposed to a phosphorimager screen before detection with aMolecular Dynamics Storm 860 Imager. Gel images were analyzed using Image-Quant, version 1.2, Macintosh software.

DNase I footprinting assays. DNA fragments containing promoter regions forCD2344 (494 bp), glgC (780 bp), and hisZ (861 bp) were amplified by PCR fromC. difficile strain 630 chromosomal DNA using specific primers (OSD124/OSD125, CD2344; OSD126/OSD127, glgC; OSD128/OSD129, hisZ) (see TableS1 in the supplemental material) and then cloned in plasmid pCR2.1 (Invitro-gen), yielding pSD30 (CD2344), pSD31 (glgC), and pSD32 (hisZ). PrimersOSD124, OSD126, OSD174, and OSD128 were radioactively labeled with[�-32P]ATP using T4 polynucleotide kinase (Invitrogen), as described previously

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(20), and used in conjunction with OSD125, OSD170, OSD126, and OSD169,respectively, to amplify the CD2344, glgC (top strand labeled), glgC (bottomstrand labeled), and hisZ promoter regions, respectively, by PCR using C. difficilestrain 630 chromosomal DNA as a template. Binding reaction mixtures wereprepared as described for the gel mobility shift assays except the reaction volumewas 20 �l. All reaction mixtures contained 10 mM (each) BCAAs and 2 mMGTP. After incubation for 30 min at room temperature, 6 mM MgCl2, 6 mMCaCl2, and 0.15 or 0.25 U of RQ1 DNase I (Promega) were added to eachreaction mixture, and incubation was continued for 1 min at room temperature.The reactions were stopped by the addition of 1 �l of 0.5 M EDTA and trans-ferred to an ice bath. Samples were extracted with phenol-chloroform, and theDNA was ethanol-precipitated and resuspended in 4 �l of sequencing gel loadingbuffer (40). The samples were heated to 80°C for 5 min and then subjected toelectrophoresis in an 8 M urea–6% polyacrylamide gel. Dideoxy sequencingreactions were performed with primers OSD124, OSD126, OSD174, andOSD128 and pSD30, pSD31, or pSD32 as template DNA using a Sequenase kit(US Biochemical) and [�-35S]dATP.

Primer extension. Total RNA (5 or 10 �g) was subjected to reverse transcrip-tion using SuperScript II reverse transcriptase (Invitrogen) and 2 pmol of gene-specific primer (OSD125 for CD2344; OSD170 for glgC) according to the man-ufacturer’s protocol in a 20-�l reaction mixture. Ten microliters of sequencinggel loading buffer was added to the sample, and the samples were heated to 80°Cfor 5 min and then subjected to electrophoresis in an 8 M urea–6% polyacryl-amide gel. Dideoxy sequencing reactions were performed with primers OSD125and OSD170 using a Sequenase kit (US Biochemical) and [�-35S]dATP.

5� RACE analysis. Mapping of the 5� end of RNA transcripts was performedusing 115 ng of total RNA and a 5� Rapid Amplification of cDNA Ends (RACE)kit (version 2.0; Invitrogen) according to the manufacturer’s protocol. First-strand cDNA synthesis was performed with gene-specific primers (OSD179 forhisZ or OSD88 for tcdR). Nested PCR amplification of the first-strand cDNAproducts was conducted using gene-specific primers (OSD178 for hisZ; OSD181for tcdR) and the abridged anchor primer supplied with the kit. The nested PCRproducts were used as templates in sequencing reactions with gene-specificprimers (OSD178 for hisZ; OSD181 for tcdR).

Microarray data accession number. The data discussed in this publicationhave been deposited in NCBI’s Gene Expression Omnibus (9) and are accessiblethrough GEO Series accession number GSE23192 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?accGSE23192).

RESULTS AND DISCUSSION

Identification of CodY-regulated genes by microarray anal-ysis. We used DNA microarray analysis to compare transcriptlevels in a codY null mutant strain, JIR8094::pSD21, in whichthe codY gene has been interrupted by integration of an un-stable plasmid (7), and a strain that expresses wild-type levelsof CodY. The control strain was JIR8094 (pSD21), in whichplasmid pSD21 remains primarily extrachromosomal (7). Do-ing so allowed us to grow both strains in the presence ofthiamphenicol. RNA for microarray analysis was isolated fromcells grown in TY medium to mid-exponential phase. Thecultures were plated before RNA isolation to verify the uni-form appearance of colonies with the characteristic smooth,partly translucent appearance of the codY mutant. The cultureswere also tested by PCR to verify that the plasmid remainedintegrated in strain JIR8094::pSD21. Note that we have previ-ously shown that in strain JIR8094::pSD21 the phenotype isdue to disruption of the codY gene and not to any polar effecton downstream genes or to any mutation located elsewhere onthe chromosome (7).

We identified 146 genes putatively organized in 82 transcrip-tion units whose transcripts were at least 4-fold more abundantin the codY mutant than in the strain producing wild-type levelsof CodY and for which P values of �0.005 were obtained.These genes are listed in full in Table S2 and summarized inTable 2. In addition, 19 genes in 15 apparent transcriptionunits were underexpressed by more than 4-fold in the codY

mutant, with P values of �0.005 (Table 2; see also Table S2 inthe supplemental material). In agreement with previous results(7), expression of four genes of the PaLoc (tcdR, tcdB, tcdE,and tcdA) was greatly derepressed (79- to 167-fold) in the codYmutant. Most of the other genes of identifiable function thatwere overexpressed in the codY mutant are involved in metab-olism, including biosynthesis of amino acids (isoleucine, valine,leucine, histidine, and arginine); glycogen synthesis; transportof amino acids, oligopeptides, and sugars; proteolysis; synthesisof butyrate from �-aminobutyrate, succinate, and acetyl-coen-zyme A (CoA); ATP synthesis; and sporulation. Most of thesefunctions have been seen to be under CodY control in otherbacterial species as well (3, 6, 10, 12, 28, 33). In addition, genesfor apparent cell surface (CD0440 and CD1803) and cyclicdi-GMP signaling (CD0757 and CD1476) proteins were over-expressed in the codY mutant. In contrast, the genes of thepathway responsible for the conversion of arginine to the poly-amines putrescine and spermidine (spe genes) were underex-pressed in the codY mutant.

The decision to include in our analysis only those genes forwhich the codY mutation caused a 4-fold or greater change inexpression was arbitrary. We sought to restrict our attention togenes for which regulation by CodY has a significant biologicalimpact. As a result, we may have underestimated the numberof genes whose expression responds to CodY activity.

To confirm the gene expression results obtained from themicroarray, both quantitative (Table 3) and semiquantita-tive (see Fig. S1 in the supplemental material) reverse tran-scription-PCR (RT-PCR) analyses were performed to de-termine transcript levels for selected genes in cells of strainsJIR8094::pSD21 and JIR8094(pSD21) grown in TY mediumto mid-exponential phase. The results of similar experimentswere previously reported for the genes of the PaLoc (7). Con-sistent with the microarray results, transcript levels for ilvC,CD2344 (which encodes a putative membrane protein involvedin �-aminobutyrate metabolism), glgC, hisZ, CD1476 (encod-ing a putative cyclic di-GMP metabolism enzyme), andCD1803 (putatively encoding a cell surface protein) werefound to be higher in the codY mutant (JIR8094::pSD21) thanin the strains that expressed wild-type levels of CodY [JIR8094and JIR8094(pSD21)] (Table 3 and Fig. S1). Also in accor-dance with the microarray results, speA transcript levels werefound to be lower in the codY mutant than in the strains thatexpressed wild-type levels of CodY (Table 3 and Fig. S1). Theapparent differential expression of the spoIIE gene seen in themicroarray experiment, however, was not confirmed by qRT-PCR (Table 3).

Genome-wide identification of direct CodY targets. To iden-tify genes that are directly activated or repressed by CodY, weperformed a genomic DNA pulldown experiment using puri-fied His6-tagged CodY as bait, based on a method developedby Majerczyk et al. (27) (see Materials and Methods). A con-trol experiment used as bait His10-tagged B. subtilis aconitase,a protein that does not bind to DNA. The DNA fragments thatbound to the His-tagged proteins were sequenced en masse.Using the sequencing results for unselected gDNA fragmentsas a baseline, we then determined a fold-enrichment value foreach position on the chromosome by dividing the coveragemap of each pulldown by the coverage map of the unselectedgDNA. The coverage maps for the aconitase-containing sam-



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TABLE 2. Selected genes whose expression was altered in the codY mutant

Gene group, function, and namea Predicted functionb Foldchangec Enriched gened

CodY-repressed genesAmino acid biosynthesis

CD0989-CD0992 (leuACDB) Leucine biosynthesis 21–99.5 leuACD1547-CD1554 (hisZGCBHAF) Histidine biosynthesis 7.9–15.1 hisZCD1565-CD1566 (ilvCB) Isoleucine-valine biosynthesis 58.1–154.8 ilvCCD2014 (ilvD) Isoleucine-valine biosynthesis 31.6CD2030-CD2034 (argFJC) Arginine biosynthesis 2.5*–10.6 argF, argCCD2502 Histidine biosynthesis 34.6 CD2502CD2726 (glyA) Glycine biosynthesis 4.7 glyACD2828 Aromatic amino acid biosynthesis 18.6 CD2828

TransportCD0165 Amino acid transporter 31.9 CD0165CD0324 (cbiM) Cobalt transporter 5.16CD0388-CD0390 (bglPAG) -Glucoside PTS transporter 14.6–19.2 bglPCD0449 Na�/H� antiporter 7.5CD0853-CD0857 (oppBCADF) Oligopeptide ABC transporter 12.6 oppBCD0875-CD0873 ABC transporter 3.1*–4.9 CD0875CD0878-CD0876 ABC transporter 4.5–15.6 CD0878CD0879 Carbohydrate kinase 7.5 CD0879CD1259-CD1260 Branched-chain amino acid transporter 4.9–23.7 CD1259, CD1260CD2511-CD2508 Sugar transport system 3.6*–10.1 CD2511CD2512 PTS system, IIa component 4.0CD2702 (brnQ) Branched-chain amino acid transporter 6.8 brnQCD3615 MerR-family transcriptional regulator 5.5CD3616 Na�/H� exchanger 7.0CD3628 Sugar transporter 6.2 CD3628

SporulationCD0770-CD0772 (spoIIAA-AB-AC) Anti-sigma F factor antagonist 7.9–12.7CD3490 (spoIIE) Stage II sporulation protein 17.1CD3516 (spoVG) Sporulation protein VG 4.87

Protein degradationCD1086 Peptidase 57.1CD2822 Peptidase 89.5 CD2822CD2862 Dipeptidase 21.6

Fatty acid and membrane synthesisCD1054-CD1059 (bcd2 etfB2 etfA2 crt2 hbd thlA1) Butyrate metabolism 21.9–67.1 bcd2CD1327 (pgsA) Phosphatidylglycerophosphate synthase 607.7CD2426-CD2425 Butyrate kinase 4.6–6.6 CD2426

Energy metabolismCD2960-CD2954 (ntpIKECABD) V-type sodium ATP synthase 14.4–89.3 ntpC, ntpI

Other metabolismCD0444 Oxidoreductase 10.4CD0445-CD0447 D-Ornithine utilization 3.9*–15.4 CD0445CD0849 (abgB2) Aminobenzoyl-glutamate utilization 7.7 abgBCD0882-CD0886 (glgCDAP) Glycogen biosynthesis 6.1–17.8 glgCDCD1263 FMN-dependent dehydrogenase 4.1 CD1263CD1389 Chloromuconate cycloisomerase 160.3CD1612 Amidohydrolase 18.9CD2158 (gabT) 4-Aminobutyrate aminotransferase 31.9 CD2158CD2164 (ldh) L-Lactate dehydrogenase 6.8 CD2164CD2344-CD2340 Gamma-aminobutyrate/hydroxybutyrate metabolism 26.7–95.1 CD2344CD2819 Amino acid racemase 5.6 CD2189CD2859 D-Aminoacylase 4.3 CD2859

Chromosome replication and cell divisionCD1328 (recA) RecA protein (recombinase A) 211.6CD1329 Nucleic acid-binding protein 103.8

VirulenceCD0440 Cell surface protein 4.46 CD0440CD0659-CD0663 (tcdRBEA) Sigma factor, toxin B, holin, toxin A 51.7–174.8 tcdR, tcdB

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ple and the unselected gDNA were not significantly different.Regions of DNA that were enriched more than 5-fold in theCodY-selected samples were compiled. Of the 628 regionsidentified, we selected the 350 most highly enriched CodY-binding regions for further characterization (see Table S3 inthe supplemental material). Each of these regions had an en-richment factor of at least 17.

To assess the potential effects of CodY-DNA binding, wefirst surveyed the area of the chromosome surrounding eachregion of enrichment and determined the most likely regula-tory targets of CodY. For example, the enriched region span-ning coordinates 786094 to 786237 (based on NCBI sequenceNC_009089.1) is located in the intergenic region upstream ofthe tcdR coding sequence. As no other transcripts are directed

TABLE 2—Continued

Gene group, function, and namea Predicted functionb Foldchangec Enriched gened

CD1803 Cell surface protein 23.79 CD1803

Signaling and regulationCD0616 MerR-family transcriptional regulator 7.32CD0618 Transcriptional regulator 53.72CD0757 c-di-GMP signaling protein 4.49CD1265 Regulatory protein 4.48CD1476 c-di-GMP signaling protein 4.12 CD1476

CodY-activated genesCD0106 (cwlD) Germination-specific N-acetylmuramoyl-L-alanine

amidase0.21

CD0714 Conserved hypothetical protein 0.07CD0758-CD0759 (plfAB) Pyruvate formate-lyase 0.13–0.21CD0888-CD0891 (speADEB) Spermidine biosynthesis 0.08–0.12CD0893 Iron-dependent hydrogenase 0.11CD1695 Arsenical pump membrane protein 0.22CD1768 Membrane protein 0.19CD1893 Regulatory protein 0.19CD2102 Na(�)/H(�) antiporter 0.05CD2107 Permease 0.19 CD2107CD2305 Pilin/general secretion pathway protein 0.23CD2380 (iorB) indolepyruvate oxidoreductase 0.22CD2593 Antibiotic resistance ABC transporter, ATP-

binding protein0.23

CD3006 Probable alcohol dehydrogenase 0.17CD3158 Conserved hypothetical protein 0.20 CD3158

a A selection of genes, representing major functional classes, whose expression was affected �4-fold by a codY mutation. (All genes whose expression was affected�4-fold by a codY mutation are listed in Table S2 in the supplemental material.) Gene names reflect their positions in the strain 630 genome sequence, accompaniedin some cases by the names of similar genes of known function in other organisms. Genes that lie in apparent operons are grouped.

b PTS, phosphotransferase system; FMN, flavin mononucleotide.c Fold change is the ratio of the hybridization signal for codY mutant RNA relative to codY� RNA. The cutoff for significant change was set at 4-fold. Some genes,

indicated by asterisks, had a fold change less than 4-fold but were included because they appeared to be in the same transcription unit with CodY-regulated genes.d Genes or transcription units that were identified by affinity purification of CodY-binding DNA fragments are indicated in the rightmost column.

TABLE 3. Quantitative RT-PCR analysis of transcript levels

GeneJIR8094 (pSD21) transcript data JIR8094 codY::pSD21 transcript data

Expression ratioa P valueb Expression ratioa P valueb

ilvC 1.0 � 0.3 0.86 85.9 � 7.5 0.004CD2344 1.3 � 0.7 0.65 194.4 � 103.2 0.11glgC 1.7 � 0.1 0.009 24.0 � 5.4 0.027hisZ 1.2 � 0.2 0.33 17.7 � 1.3 0.003CD1276 1.5 � 0.6 0.36 0.008 � 0.0005 10�7

CD1476 0.9 � 0.06 0.07 3.7 � 0.9 0.048CD1803 1.1 � 0.07 0.39 53.6 � 11.7 0.023speA 1.3 � 0.4 0.42 0.07 � 0.04 0.0008spoIIE 0.9 � 0.17 0.40 1.5 � 0.4 0.21

a RNA from the codY� and codY mutant strains was used as a template for cDNA synthesis. The cDNA was then amplified by PCR. The production ofdouble-stranded DNA was monitored by detecting the binding of SYBR green by increased fluorescence. The amount of PCR product for each gene in each strainwas normalized to the amount of an internal control gene, rpoC, in that strain and then compared to the ratio of rpoC to the gene in question in strain JIR8094 (setto 1.0 for all genes and not shown in the table). Each data point represents the average of triplicate assays of at least two RNA preparations.

b The P value was determined by a two-tailed Student’s t test as a measure of the significance of the difference between expression in the tested strain and expressionin strain JIR8094.



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TABLE 4. Selected CodY-binding regions containing CodY consensus sequences

Genetic regiona Coordinatesb Potential CodY boxc MMd Potential CodYtargete Putative target function

Foldchange in

microarrayf

CD0013-CDs001 17426–17574 AAATTAAAGAAAATT/AATTCTAAAAAAATT

3/3 CDs001 Structural RNA ND

CD0020-CD0021 34538–34706 AATTTTCACATAAAT 3 CD0021* Pyruvate metabolism 0.74CD0044-CD0045 69469–69716 AATTTTTAGAATTTT 3 CD0045 Carbohydrate metabolism 11.01CD0107 136535–136773 ATTCTTCTAAAAATT 3 CD0108* Nucleotide metabolism 0.87CD111A-CD0112 155835–156107 AATATTTCGAATATT 3 CD0112* Butanoate metabolism 0.65CD0178 236362–236609 AATTTTATGAATATT 2 CD0179* Glutamate metabolism 0.27CD0214-CD0215 278547–278714 AATTATTTGAATATT 3 CD0214/CD0215* Unknown/transposase ND/0.49CD0294-CD0295 356131–356235 AATATACTAAAAATT 3 CD0295* Fe-S binding protein 1.25CD0334-CD0335 407350–407557 AATATTCAGAAAATT 1 UCD0351-CD0352 426229–426429 CATTTTCACAAAATT/AAATTTCA

GAAAACT2/2 CD0352* Transcriptional regulator 7.47

CD0387-CD0388 457083–457305 TAGTATCAGAAAATT 3 CD0388* Sugar transport 19.22CD0440-CD0440A 523368–523530 AAGTTCCAGAAAAGT/AATTTTG

TGAATATA3/3 CD0440A Transcriptional regulator ND

CD0444-CD0445 528832–528971 AAAATTCAGAAAATT 2 CD0445* Ornithine aminomutase 13.09CD0536 640661–640900 AATATTCGGATTATT 3 CD0537* Chemotaxis 0.81CD0549-CD0550 654816–655019 AATATTCTGAAAAAT 2 CD0550 Unknown 0.88CD0577-CD0578 690428–690792 AATTTTTATAAAGTT/AAATTAG

AGAAAATT/TATTTTCTTAATATT

3/3/3 CD0577* Phosphohydrolase 39

CD0658-CD0659 786357–786612 AATGTTGTCAAAATT/AATTTTCAAATAAAT

3/3 CD0659* Transcriptional regulator 104.52

CD0664-CD0664A 805028–805140 TATTTTCCTAAAATA 3 CD0664 Toxin production 0.59CD0669-CD0670 811707–811932 AATATTTTGAAAATA 3 CD0670 Transcriptional regulator 0.97CD0678-CD0679 821392–821561 AATATTTAGAAAAAT 3 CD0679* Unknown 0.76CD0738-CD0739 901188–901315 TATTTTTAGAATATT/AATATTCA

GTAAAAT3/3 CD0738/CD0739 Unknown/unknown 0.80/1.16

CD0739-CD0740 902366–902610 TATTATAGGAAAATT 3 UCD0781-CD0782 954316–954498 AATATTCTGTACATT 3 CD0781/CD0782 Penicillin binding protein/unknown 0.61/0.67CD0799-CD0800 972046–972245 AATTTAGTGAAATTT 3 CD0800* Crotonase 2.77CD0812 985543–985711 AAAATTCTGAAAATT 2 CD0813 Sugar transport 0.89CD0848-CD0849 1025023–1025198 AATTAACAAAAAATT 3 CD0849* Unknown 7.69CD0852-CD0853 1028194–1028302 AATTTGATGAAATTT 3 CD0852/CD0853* Oligopeptide transport 0.95/12.59CD0921 1100828–1101048 TATTCTTTGAAAATT 3 CD0921* Phage protein 2.94CD0988-CDs021 1152458–1152735 AATATTTAGAAAATT 2 CD0989* Leucine biosynthesis 67.65CD0989 1153010–1153269 ATTTTTAAGAAAATT 2 CD989* Leucine metabolism 67.65CD1031 1204553–1204718 GATATTCTGACAATT 3 CD1031* Cellulose metabolism 0.53CD1053-CD1054 1246326–1246760 AATTATTGAAAAATT 3 CD1054* Butanoate metabolism 33.53CD1071-CD1072 1265373–1265693 AATATTTAGAAAACT 3 CD1071/CD1072 Unknown/aminopeptidase 0.54/0.43CD1084-CD1085 1277778–1277975 AATTTTATGAAAAAA/TATTTTTT

GCAAATT3/3 CD1085* Glutamate metabolism 29.61

CD1120 1316544–1316724 CAGTTTCAGAAAATA 3 CD1120* Glycerol metabolism 2CD1211 1410549–1410682 AATATTCAGTAAATA 3 CD1211 Acetyltransferase 1.38CD1259-CD1260 1463178–1463324 AATTTTTAGTTAATT/AATTTTCA

GAAA3/3 CD1260 Amino acid transport 23.7

CD1259-CD1260 1463631–1463859 AATATTGTGAAGATT 3 CD1260 Amino acid transport 23.7CD1266 1471874–1472213 AATTTTCTTTTAATT 3 CD1266* ABC transporter 9.27CD1274-CD1275 1481454–1481685 AATTTTCATTAAATG 3 CD1275 *CodY* 1.15CD1352 1569302–1569430 AATTCTGTGAAAACT 3 CD1352 ABC transporter system 0.49CD1353 1569853–1570210 ATTTTTCCAAACATT 3 CD1353 Vitamin B6 metabolism 0.67CD1378A-

CD1378B1595378–1595585 AATATTCAGAAGATG 3 CD1378, CD1378A-B Transcriptional regulator/unknown 0.73/ND

CD1403 1624922–1625131 AATTTTCTGTATATA 3 CD1404* Oligopeptide transport 0.36CD1424-CD1425 1653848–1654028 ACTTGTCAGAAAATT 2 CD1424 Unknown 1.49CD1511-CD1512 1749752–1749968 AATATTCAGTAAATA 3 CD1511* Unknown 0.33CD1546-CD1547 1795436–1795540 AATTTTTGAAAAATA 3 CD1547* Histidine metabolism 15.06CD1562-CDs028 1810325–1810537 AATAATCTGAAAATT 2 CD1563* Structural RNA/BCAA metabolism 9.77CDs031-CD1580 1831392–1831606 AAAAATCGGAAAATT 3 CDs031, CD1580* Structural RNA/Amino acid metabolism ND/1.99CD1631 1890125–1890375 AATTTTGCAATAATT 3 CD1631* Superoxide dismutase 0.8CD1654 1918265–1918428 CATTTCCAGAAAATT 2 CD1654* Lipoic acid metabolism 0.83CD1663-CD1664 1936692–1936844 AAATTTATGAATATT 3 CD1663 Guanine deaminase 1.17CD1703-CD1704 1978612–1978866 AATATTAAAAAAATT 3 CD1704* Thiamine biosynthesis 2.29CD1763-CD1764 2041381–2041551 AAATTTAAAAAAATT 3 CD1763/CD1764* Unknown/unknown 2.21/1.78CDs039-CD1774 2055675–2055882 AATATTTTAAAAATT/AATATTAT

GAAAAAT3/3 CD1774* Amino acid transport 3.69

CD1779 2060212–2060352 AATTTAAAGAAAATG/AATATACTCAAAATT

3/3 CD1779* Unknown 1.27

CD1806-CD1807 2089882–2090196 AATTTTCTAATTATT/AATTTATAAAAAATT

3/3 CD1806 Fructokinase 0.77

CD1988-CDs044 2294168–2294473 AATTTTCAGTAAAAT 2 CD1988 Tryptophan transport 0.79CD2007-CD2007A 2317619–2317709 AGTTTTGAGAATATT 3 CD2007 Erythromycin resistance 0.74CD2010-CD2010A 2319981–2320126 AGTTTTGAGAATATT 3 CD2010* Erythromycin resistance 0.51CD2029-CD2030 2341391–2341584 AATTTTCATTAAAGT 3 CD2029* Lipoprotein 0.57

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TABLE 4—Continued

Genetic regiona Coordinatesb Potential CodY boxc MMd Potential CodYtargete Putative target function

Foldchange in

microarrayf

CD2030-CD2031 2342425–2342606 TATTTTTTGAATATT 3 CD2030* Ornithine carbamoyltransferase 10.58CD2038-CD2039 2351975–2352131 AATTATCTGACAACT 3 CD2038* Drug/Na� antiporter 0.86CD2108 2436840–2437027 TATTTTCTGATAATA 3 CD2108* Antiporter 0.67CD2109 2438222–2438382 TATTTACAGAAAATT 2 CD2109 Symporter 1.25CD2123-CD2124 2459489–2459651 TTTTGTCAGAAAATT 3 CD2123/CD2124 Multi-protein complex

assembly/transporter0.67/0.89

CD2127-CD2128 2464269–2464411 AATTTTCACTATATT 3 CD2127 Unknown 10.72CD2151-CD2152 2490473–2490640 AGTTTTCAGAACATT 2 CD2151/CD2152 Unknown/glutamyl aminopeptidase 2.64/38.05CD2158-CD2159 2497849–2498061 AATTTTCAAAATATT 2 CD2158 Amino acid metabolism 31.98CD2163-CD2164 2502982–2503060 AATTTTCTGAAATAA 3 CD2164 Metabolic pathways 6.78CD2201-CD2202 2550642–2550769 TATATTCTCAAAATT 3 CD2201/CD2202 Sugar transport/symporter 0.47/1.30CD2213-CD2214 2566366–2566491 AATTTTCAAAATATA 3 CD2213/CD2214 Nitrogen metabolism/transcriptional

regulator2.87/0.90

CD2217-CD2218 2569491–2569598 AATTTTCATAATATT 2 CD2217 Amino acid metabolism 1.43CD2336-CD2337 2703228–2703567 TTTTTTCCGCAAATT 3 CD2336 Toxin anion resistance 0.84CD2357-CD2358 2725674–2725854 TTTTTTCAGAAAATT 2 CD2357* Pyrimidine metabolism 1.34CD2418-CD2419 2791999–2792145 AATTTTCAGAAATAT 2 CD2418* Sugar transport 0.48CD2424 2799778–2800030 AATTTTCAGATAATA 2 CD2424* Aminotransferase 27.37CD2429A-CD2430 2805212–2805356 AATTTTAAGAATATT 2 CD2429* Oxidoreductase 4.03CD2442 2818120–2818383 AATTTTAGCAATATT 3 CD2441A Phosphate starvation NDCD2450-CD2451 2825411–2825637 AATAGTCAGAATATT 3 CD2450* Ribosomal methyltransferase 0.99CD2459-CD2460 2838224–2838401 ATTTTTCAGAAAATT 1 CD2459* Carbohydrate metabolism 0.7CD2491-CD2492 2874398–2874563 ATATATCAGAAAATT 3 CD2492 Transcriptional regulator 0.93CD2501-CD2502 2887393–2887548 TATTTTCATAATATT 3 CD2502 Amino acid metabolism 34.6CD2510-CD2511 2899716–2899959 ACTTTTCTAAATATT 3 CD2511* Transcription antiterminator/sugar

transport4.11

CD2517-CD2517A 2907057–2907203 AATTTTCAAACAATC 3 CD2517 Transcriptional regulator 0.99CD2568-CD2569 2970536–2970719 AATTTTGGGAGGATT 3 CD2569 Carbohydrate metabolism 1.16CD2617-CD2618 3024304–3024454 AAATTTTGGAATATT 3 CD2617* Unknown 2.67CDs055-CD2619 3027882–3028108 AATTCTCTGTAAAAT 3 CD2618 tRNA biosynthesis 1.36CD2627-CD2628 3035283–3035389 ACTATTTAGAAAATT 3 CD2627* Unknown 7.34CD2675-CD2676 3091893–3092131 TTTATTCAGAAAATT 3 CD2675/CD2676 Transcriptional regulator/metabolism 0.70/22.89CD2702-CD2703 3124233–3124541 TATTTTCAGAAAAAT 2 CD2702* BCAA metabolism 6.78CD2728-CD2729 3165395–3165598 AATCTTTTGAAAATC 3 UCD2755-CD2756 3203460–3203634 ATTTTTTATAAAATT 3 CD2755* Carbohydrate metabolism 1.34CD2797 3261176–3261408 AATCTTCTGCTAATT 3 CD2796 Cell surface protein 0.72CD2818-CD2819 3291095–3291200 ACTTTGCATAAAATT/AACTTATT

GAAAATT3/3 CD2819 Amino acid racemase 5.57

CD2827-CD2828 3300000–3300134 AATTTTTCGAAAATT 1 CD2827*/CD2828 Transcriptional regulator/aspartateaminotransferase

0.74/18.58

CD2836 3314798–3315060 AATTTTCTGATATTT 2 CD2835* Aminobenzoyl-glutamate transport 29.28CD2837-CD2828 3317022–3317154 AACTCTCAGTAAATT/AATTAAA

TGAAAATT3/3 CD2837* Unknown 3.99

CD2859 3343496–3343735 AATATTCTGTAAAAT/CATTTTCAGAAGAAT

3/3 CD2859* D-Aminoacylase 4.28

CD2869 3356859–3357058 AATTATCAGAATTTT 3 CD2870 Sugar transport 2.7CD2938-CD2939 3420861–3421088 AATTTTCAAAGAATA 3 CD2938* Unknown 2.04CD2947-CD2948 3425099–3425259 TATTTTCAAAATATT 3 CD2947* Unknown 1.19CD2957 3435338–3435581 AATATTCTCAAAATT 2 CD2957* Electron transport 27.73CD2960-CD2961 3438638–3438889 AATTTTCAGACATTC 3 CD2960* Electron transport 28.32CD2965 3443594–3443803 AGTTTTCTTATAATT 3 CD2965* Signaling protein 6.79CD2965 3444451–3444615 AGTTTTTGAAAAATT 3 CD2965* Signaling protein 6.79CD2983 3466329–3466483 AATTTTTTTATAATT 3 CD2983* Unknown 2.33CD3004-CD3005 3489854–3490056 AATTTTTTCAAACTT/AACTTTCT

GATAATA3/3 CD3004*/CD3005 Sugar transport/transcriptional regulator 0.19/1.38

CD3048 3543094–3543330 AAAATTCTGTAAATT/AATTTTTATAAAATC

3/3 CD3048* Sugar transport 4.31

CD3138 3660297–3660539 AGATTTCATAAAATT 3 CD3138* Transcriptional antiterminator 1.04CD3157-CD3158 3688291–3688490 AATTTACTAAATATT 3 CD3157*/CD3158 SsrA-binding protein/unknown 1.08/0.20CD3193 3736711–3736834 AATTTTCTTATAATA 3 CD3193 Phage-related protein 19.34CD3223-CDs061 3772940–3773047 AATTTTAGCAATATT 3 CD3224* Amino acid metabolism 0.91CD3317-CD3319 3884259–3884475 AATTTTCTCACAATT 2 CD3317* Metabolic pathways 3.12CD3458-CD3459 4052622–4052839 ATTTTTCTGAATAGT 3 CD3458* Unknown 12.05CD3491 4082859–4083107 ACTTATCAGAAAATT 2 CD3491* Exopolyphosphatase 0.71CD3602 4209798–4209998 AAATTTCAAAAGATT 3 CD3602 ATPase 0.62CD3628-CD3629 4236842–4237066 TATTATCTAAAAATT 3 CD3628* Carbohydrate metabolism 6.17CD3650 4258531–4258683 AATTTTTTGAAAGAT 3 CD3650* Signaling protein 0.99

a Region of the strain 630 genome enriched by binding to CodY, as indicated by gene names. Of the 350 most-enriched regions, only those that contain CodYconsensus sequences (�3 mismatches) are included in this table. All of the 350 most-enriched regions are shown in Table S3 in the supplemental material.

b Coordinates of the enriched region.c Putative CodY binding sequences in the enriched region, as defined by the consensus sequence (AATTTTCWGAAAATT) determined for B. subtilis and L. lactis.

Up to three mismatches were allowed. Slashes separate multiple potential CodY boxes in the same region.d Number of mismatches relative to consensus CodY box.e Most likely target of CodY binding based on location and orientations of neighboring genes. Genes with asterisks appear to be organized in multigene transcription units.f Expression ratio (codY mutant/wild type) for putative target genes.



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outwardly from this region, the most probable transcriptionaltarget of CodY binding is tcdR, which encodes the sigma factorthat regulates transcription of toxin genes (30). This region waspreviously shown to be a site of CodY binding by electro-phoretic mobility shift and DNase I footprinting assays (7).Enriched regions near the tcdB and tcdC genes were alsodetected. The tcdB promoter region is a weak site of binding ofCodY in gel shift assays (7), but the region upstream of tcdCincludes a sequence to which CodY binds tightly in gel shiftand DNase I footprinting experiments (data not shown). Otherenriched regions correspond to many of the operons whoseexpression is affected by a codY mutation, including those forbiosynthesis of the BCAAs (histidine, arginine, and glycogen),peptide, sugar and amino acid transport, ATP synthase, andbutyrate metabolism. Interestingly, the spe operon, which ispositively regulated by CodY, was not enriched in the pulldownexperiment. Notably, 37 regions of enrichment were in or nearregulatory genes, including those that encode transcriptionantiterminators, transcription activators, repressors, and two-component systems. Thus, it is likely that CodY influences thetranscription of many transcripts indirectly by regulating thesynthesis of other transcription factors.

Of the enriched regions, 118 contained one or more se-quences with three or fewer mismatches to the consensusCodY-binding motif (Table 4). (The A�T-rich C. difficile ge-nome contains only one site with the perfect consensus se-quence, seven sites with one mismatch, 137 sites with twomismatches and 1,337 sites with three mismatches. Althoughthe consensus sequence is clearly important for CodY binding[2], it is not usually sufficient [B. Belitsky, personal communi-cation].)

The enriched sequences were also searched for a commonmotif as a means of testing the applicability of the CodYconsensus binding sequence previously proposed for L. lactisand B. subtilis. As shown in Fig. 1, a motif found in 280 se-quences had an overall E value of 5.2E�68 and included al-most the entire previously derived consensus sequence, as wellas an upstream sequence very reminiscent of the correspond-ing motif found for S. aureus (27).

Of the 82 transcription units that were overexpressed in acodY mutant, 52 had enriched regions near the 5� end of thetranscription unit (those genes are indicated in Table 2; seealso Table S2 in the supplemental material), suggesting thatthese genes are near sites of direct repression by CodY. Onlytwo of the transcription units that were underexpressed in acodY mutant were associated with enriched regions (Table 2),suggesting that most CodY-activated genes are influenced byCodY indirectly.

Interaction of CodY with target promoter regions. To con-firm that CodY plays a direct role in the regulation of some ofthe genes identified as putative targets by both microarray andCodY pulldown screens, in vitro DNA binding experimentswere performed. Radioactive double-stranded DNA probescorresponding to the upstream regions of the ilvC, CD2344,glgC, and hisZ genes were subjected to gel mobility shift anal-ysis using purified C. difficile CodY (7). All four genes werehighly overexpressed in the codY mutant strain (Table 2). C.difficile CodY bound to the DNA region upstream of all fourgenes, and this binding was enhanced in the presence of theknown effectors of C. difficile CodY binding, GTP and a mix-ture of BCAAs (Fig. 2). The estimated KD (equilibrium disso-ciation constant) for binding (approximated as the concentra-tion of CodY that shifted 50% of the DNA [5]) in the presenceof both effectors was 50 nM, 50 nM, �12.5 nM, and 12.5 nMCodY for the ilvC, CD2344, glgC, and hisZ DNA fragments,respectively. Based on previous analyses of the interaction ofC. difficile CodY with DNA targets, the high binding affinitiessuggest that these DNA fragments are likely to contain directtargets of CodY in vivo (7). In contrast, only weak interactions(estimated KD of �200 nM) were observed between C. difficileCodY and a DNA fragment containing the speA promoterregion even in the presence of GTP and BCAAs (data notshown). These results confirm the pulldown results and indi-cate that the speADEB transcription unit is unlikely to bedirectly regulated by CodY.

To identify specific binding sites for CodY, a DNase I foot-printing assay was used to analyze the interaction of CodY withthe DNA fragments for which CodY had high affinity. Sincethe specific interaction of CodY with ilv gene promoters hasbeen extensively studied in other Gram-positive bacteria (2, 36,

FIG. 1. Motif search in genomic regions enriched by binding toCodY. Using the MEME Suite, 293 regions of the C. difficile genomethat had been enriched by affinity purification with His6-tagged CodYwere searched for a common motif. The resulting motif was found in280 regions and proved to be similar to a motif discovered using thesame methods for the S. aureus genome (27). Both motifs are com-pared to the CodY box defined for L. lactis (6, 10) and B. subtilis (2).

FIG. 2. Gel mobility shift assays for binding of CodY to selectedgene promoter regions. DNA fragments containing the ilvC (423 bp),CD2344 (494 bp), glgC (780 bp), and hisZ (861 bp) promoter regionswere incubated with increasing concentrations of CodY with and with-out the effectors GTP and branched-chain amino acids (BCAA), asdescribed in Materials and Methods.



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45, 50), we focused on the CD2344, glgC, and hisZ regulatoryregions. The binding site in the hisZ region with the highestaffinity was located at positions �588 to �625 relative to theATG start codon (Fig. 3). A 15-bp sequence, AATTcTCTGAAAcac, with four mismatches (indicated by lowercase letters)compared to the consensus CodY-binding motif, overlaps withthe protected region. The transcription start point of the hisZgene, as determined by 5� RACE analysis, however, lies about300 bp downstream of the high-affinity CodY binding site. HowCodY regulates hisZ from the observed site is unclear.

For the fragment of DNA containing the CD2344 promoterregion, a region that spans the sequence from positions �310to �350 relative to the ATG start codon was protected byCodY from DNase I digestion in the presence of GTP andBCAAs, with protection appearing at 100 nM CodY (Fig. 3).This region of protection was fully contained within the region

between CD2344 and CD2345 that was highly enriched in thepulldown experiment (Fig. 4). To correlate the location of theCodY binding site with the transcription start site, a primerextension experiment was performed. Primer extension analy-sis revealed an apparent transcription start located 300 bpupstream of the CD2344 initiation codon (Fig. 5). The pre-dicted �10 and �35 promoter sequences would lie within theCodY binding region as defined by DNase I protection, fittinga model in which CodY acts as a direct repressor of CD2344transcription. It is worth noting that the primer extension prod-uct was detected only when RNA from the codY mutant strainwas used, affirming that the transcript in question is regulatedby CodY (Fig. 5). A 15-bp sequence reminiscent of the CodYconsensus site, AAgTTTtTGAtAtTT, with four mismatches(indicated by lowercase letters), was identified within theCodY protected region and overlapping with the putative pro-

FIG. 3. DNase I protection assays for binding of CodY to selected gene promoter regions. Binding reaction mixtures contained 2 mM GTP,10 mM BCAA, and various concentrations of CodY (nM) as described in Materials and Methods. Protected regions are marked by vertical bars.The location of the transcription start site for each gene is indicated by a bent arrow that also indicates the direction of transcription.

FIG. 4. Regulatory site architecture. The locations of transcription start sites (�1), promoter �10 and �35 elements, start codons (ATG),CodY binding sites determined by DNase I protection assays (PR; gray blocks), and regions enriched in CodY affinity chromatography experiments(hatched blocks) are indicated for three C. difficile regulatory regions. Coordinates are given with respect to the transcription start site. The regionsof the tcdR locus protected by CodY were determined in previous work (7).



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moter site. Interestingly, CD2345, the gene immediately up-stream of CD2344 (Fig. 4), is transcribed divergently withrespect to CD2344 and encodes a protein similar to LysRfamily regulatory proteins. LysR regulator genes are frequentlytranscribed divergently from the first genes of the operons theycontrol. We found no evidence that CD2345 is regulated byCodY.

In a DNase I footprinting experiment for the glgC pro-moter region, CodY protected a region from positions �595to �629 relative to the ATG start codon, with protectionbeginning to appear at 25 nM CodY (Fig. 3). The region ofprotection overlapped with the region that was enriched inthe pulldown experiment. A primer extension experimentestablished that the glgC transcription start point appears tobe 560 bp upstream of the initiation codon (Fig. 5). The �35promoter sequence for this transcript would be predicted tooverlap with or be directly downstream of the CodY bindingregion (Fig. 4), fitting a model in which CodY would act asa repressor of glgC gene expression. As seen for the CD2344primer extension experiment, the glgC primer extensionproduct was detected only when codY mutant strain RNAwas used as a template, indicating that the transcriptionstart site identified is for a transcript regulated by CodY(Fig. 5). Two 15-bp sequences, AATTaaCAGAAtcTT andAATcTTtAtAcAATT, each with four mismatches comparedto the consensus CodY-binding motif, were identified withinthe CodY protected region.

The role of CodY in virulence gene regulation. In previouswork, we showed that CodY is a repressor of tcdR, the genethat encodes the sigma factor that is needed for expression ofthe major virulence factors of C. difficile. We found threebinding sites for CodY in the region upstream of the tcdR gene(7) but were unable to confidently predict the effect of CodYbinding on gene expression because we had yet to reliably mapthe transcription start site for the tcdR gene. By integratingnew information from the pulldown experiment and 5� RACE,we were able to orient the binding sites with respect to thetranscription start point. For 5� RACE analysis, RNA wasisolated from mid-exponential-phase cells of the codY mutant

strain JIR8094::pSD21, a strain that expresses high levels oftcdR transcript. The 5� end of the tcdR transcript mapped to anA residue located 16 bp upstream of the ATG start codon(data not shown). The �35 promoter sequence for this tran-script would be predicted to fall within the previously deter-mined CodY-protected region III (Fig. 4) (7). Two 15-bp se-quences, AATgTTgTcAAAATT and AATTTTCAaAtAAaT,each with three mismatches compared to the consensus CodY-binding motif, lie within or overlap with the CodY-protectedregion. Two other CodY-protected regions (I and II) lie fur-ther upstream (7). These latter protected regions fall within oroverlap with the region enriched in the pulldown experiment(Fig. 4).

The tcdR gene is very likely to be transcribed from twopromoters, one recognized by the A-containing form ofRNA polymerase and a second promoter that is recognizedby the TcdR-containing RNA polymerase. The A-depen-dent promoter should be active at least at a low level duringthe exponential growth phase to allow basal expression oftcdR (TcdR cannot be totally dependent on itself for itssynthesis), whereas the TcdR-dependent promoter shouldbe activated only in stationary phase when toxin synthesis ismaximal. It would be reasonable to suggest that the CodY-protected regions I and II correspond to one of the promot-ers and the protected region III corresponds to the other.But which is which? The CodY binding sites I and II and theregion of enrichment in the pulldown experiment lie withinor downstream of a TcdR-type promoter sequence (31),whereas site III overlaps with a A-type promoter sequence.Thus, the most likely scenario is that CodY binds to bothpromoters, preventing transcription from both during rapidexponential growth. Note that the 5� RACE results revealedonly one transcription start, corresponding to the A-typepromoter. When two transcripts overlap, however, 5� RACEpreferentially amplifies the shorter transcript.

Whereas the synthesis of the major toxins A and B isregulated by CodY, the synthesis of the third toxin, CDT, isnot. We found no evidence for altered expression of thecdtAB operon in a codY mutant and no enrichment for thecdt locus in the pulldown experiment. Other potential viru-lence factors do appear to be under CodY control, however.CD0440 and CD1803 encode putative surface proteins thatmight interact with host cells. The genes CD0757 andCD1476 were highly expressed in the codY mutant; theCD1476 region was also identified as a site of CodY binding,as were the genes CD2385, CD2873, CD2965, and CD3650.All of these genes are predicted to have EAL and/or GG-DEF domains, which are associated with the synthesis andbreakdown of the second messenger molecule, cyclic di-GMP (39). In other bacteria, cyclic di-GMP has been im-plicated in control of motility, biofilm formation, and viru-lence gene expression (49). If CodY affects signalingthrough cyclic di-GMP, its influence on virulence could beconsiderably broader than we currently know.

The coregulation of metabolic genes and virulence genes byCodY underscores the connection between nutritional suffi-ciency and pathogenesis in C. difficile. This bacterium hasevolved to express its most potent virulence factors at highlevel only after experiencing nutrient deprivation. Given thewide occurrence of CodY homologs in Gram-positive patho-

FIG. 5. Primer extension analysis to map the start sites for theCD2344 and glgC transcripts. Oligonucleotide primers radioactivelylabeled with 32P at the 5� end were annealed to total RNA extractedfrom the codY mutant, JIR8094::pSD21, and the codY� strain,JIR8094 (pSD21), as described in Materials and Methods. Arrowsindicate the positions of the major CodY-regulated primer extensionproducts.



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gens and their implication in virulence in several such species,this strategy appears to be widespread.

ACKNOWLEDGMENTS

We thank Charlotte Majerczyk for providing advice and a detailedprotocol for the CodY pulldown experiment, Boris Belitsky for helpfuldiscussions, Chris Parkin and Kip Bodi for bioinformatics analysis, andLaurent Bouillaut for oLB51 and oLB52.

This work was supported by grants from the U.S. Public HealthService to A.L.S. (R01 AI057637) and to the Tufts Center for Neuro-science Research (P30 NS047243). S.S.D. and S.M.M. were NationalResearch Service Award postdoctoral trainees supported by awardsT32 DK007542 and F32 DK082156, respectively.

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integration of metabolism and virulence by clostridium difficile cody â€

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