Identification of Direct Transcriptional Target Genes of ExoS/ChvI Two ...

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JOURNAL OF BACTERIOLOGY, Nov. 2009, p. 6833–6842 0021-9193/09/$12.00 doi:10.1128/JB.00734-09 Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Vol. 191, No. 22

Identification of Direct Transcriptional Target Genes of ExoS/ChvI Two-Component Signaling in Sinorhizobium meliloti䌤 Esther J. Chen,1* Robert F. Fisher,2 Virginia M. Perovich,1 Erich A. Sabio,1 and Sharon R. Long2 Department of Biological Science, Center for Applied Biotechnology Studies, College of Natural Sciences and Mathematics, California State University Fullerton, Fullerton, California 92834-6850,1 and Department of Biological Sciences, Stanford University, 371 Serra Mall, Stanford, California 94305-50202 Received 4 June 2009/Accepted 1 September 2009

The Sinorhizobium meliloti ExoS/ChvI two-component signaling pathway is required for the development of a nitrogen-fixing symbiosis between S. meliloti and its plant hosts. ExoS/ChvI also has important roles in regulating succinoglycan production, biofilm formation, motility, nutrient utilization, and the viability of free-living bacteria. Previous microarray experiments with an exoS96::Tn5 mutant indicated that ExoS/ChvI influences the expression of a few hundred genes, complicating the investigation of which downstream genes respond directly or indirectly to ExoS/ChvI regulation. To focus our study of ExoS/ChvI transcriptional target genes, we performed transcriptional profiling with chvI gain-of-function and reduced-function strains. The chvI gain-of-function strain that we used contains a dominant gain-of-function chvI allele in addition to wild-type chvI. We identified genes that, relative to their expression level in the wild type, are both upregulated in the chvI gain-of-function strain and downregulated in the reduced-function strain or vice versa. Guided by this focused set of genes, we performed gel mobility shift assays and demonstrated that ChvI directly binds the intergenic regions upstream of ropB1, SMb21440, and SMc01580. Furthermore, DNase I footprint analysis of the region upstream of SMc01580 identified a specific DNA sequence bound by ChvI and allowed the discovery of a possible motif for ChvI binding. Our results provide insight into the mechanism of how ExoS/ChvI regulates its downstream targets and lay a foundation for studying this conserved pathway with critical roles in free-living and symbiotic bacteria. tose, with acetyl, succinyl, and pyruvyl modifications (43). The symbiotically active form of succinoglycan is the trimer form (52). Mutants that fail to synthesize succinoglycan or that fail to synthesize succinoglycan with the proper modifications have defects in infection thread initiation and elongation (10). The ExoS/ChvI two-component system positively regulates the transcription of exo genes that encode enzymes for succinoglycan biosynthesis (11, 14, 54). ExoS is a periplasmic sensing histidine kinase that controls the phosphorylation of ChvI, which regulates the transcription of downstream genes (11, 36). ExoS/ChvI is negatively regulated by the periplasmic inhibitor protein ExoR (9, 53). Symbiotic defects can result both from mutations that increase ExoS/ChvI activity [such as exoR95::Tn5, exoR(G76C), or exoS(G268S)] and from mutations that decrease ExoS/ChvI activity [such as chvI(K214T)] (9, 14, 40, 53, 55). In addition to its role in succinoglycan synthesis, ExoS/ChvI signaling is critical for the viability of free-living S. meliloti strains. Attempts to construct null alleles of exoS or chvI in S. meliloti were unsuccessful (11, 39), suggesting that exoS and chvI are essential genes. Besides its roles in viability and symbiosis, ExoS/ChvI is important for biofilm formation, motility, and nutrient utilization (20, 53, 55). Furthermore, orthologs of ExoS/ChvI in other alphaproteobacteria, BvrS/BvrR in the mammalian pathogen Brucella abortus and ChvG/ChvI in the plant pathogen Agrobacterium tumefaciens, are required for virulence (8, 50). Despite the importance of ExoS/ChvI signaling in S. meliloti, many of the downstream genes that mediate its various functions have not been identified. Transcriptional profiling of exoR95::Tn5 and exoS96::Tn5 mutants demonstrated that, in

During symbiosis, root-associated bacteria (rhizobia) supply legume plants with the fixed nitrogen necessary for plant growth in exchange for carbon from the plant. These nitrogenfixing symbioses result in the formation of root nodules that enable plant growth in nitrogen-poor soil (2). The earliest steps in the development of nitrogen-fixing nodules involve communication via secreted chemical signals. Plant flavonoids induce the rhizobia to synthesize Nod factor, which induces root hair curling and root cortical cell division. Rhizobia colonize curled root hairs and invade via infection threads that extend through root hairs into cells within the nodule. The rhizobia are then released into the cytoplasm of nodule cells in plant membranebounded symbiosomes, where they differentiate into nitrogenfixing bacteroids (21, 30). Bacterial polysaccharides play an important role in nodulation (18). Three polysaccharides that allow Sinorhizobium meliloti to invade nodules in alfalfa are succinoglycan, galactoglucan, and K antigen; of these three polysaccharides, succinoglycan is most efficient at mediating infection thread initiation and elongation (41). The sequenced S. meliloti strain Rm1021 must produce succinoglycan to invade plant roots, since Rm1021 does not normally produce galactoglucan or symbiotically active K antigen (24, 32, 44). Succinoglycan is a polymer of an octasaccharide containing seven glucoses and one galac-

* Corresponding author. Mailing address: Department of Biological Science, Center for Applied Biotechnology Studies, College of Natural Sciences and Mathematics, California State University Fullerton, 800 N. State College Blvd., Fullerton, CA 92834-6850. Phone: (657) 2782543. Fax: (657) 278-3426. E-mail: [email protected]. 䌤 Published ahead of print on 11 September 2009. 6833

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addition to exo genes, the expression of hundreds of genes was altered (53, 55). The sheer number of potential transcriptional targets from these previous studies made it difficult to begin to investigate the mechanism of ExoS/ChvI regulation. Genes subject to ExoS/ChvI transcriptional control could not be distinguished easily from genes whose expression was altered as an indirect consequence of other ExoS/ChvI mutant phenotypes. In an attempt to identify new ExoS/ChvI transcriptional target genes, we also tried a genetic screen for suppressors of the chvI(K214T) mutation, but this screen yielded only additional mutations in exoR and exoS (9). To identify a focused set of genes that likely to represent true transcriptional targets of ExoS/ChvI, we performed microarrays with gain-of-function and reduced-function chvI strains. The streamlined set of candidate downstream genes revealed by these studies allowed us to identify direct ExoS/ ChvI transcriptional target genes and a binding site for ChvI. MATERIALS AND METHODS Strains, media, growth conditions, and genetic techniques. All strains in this study (Table 1) are derived from Rm1021 (streptomycin [Sm]-resistant derivative of wild-type strain SU47 used for genome sequencing [23]) and were grown at 30°C in LB medium. Calcofluor white M2R (Sigma) was filter sterilized and added to a final concentration of 0.02% in LB agar medium (32). Antibiotics were used at the following concentrations: Sm, 500 ␮g/ml; neomycin (Nm), 50 ␮g/ml; hygromycin (Hy), 50 ␮g/ml; spectinomycin (Sp), 50 ␮g/ml; ampicillin (Ap), 100 ␮g/ml; kanamycin (Km), 30 ␮g/ml; and chloramphenicol (Cm), 50 ␮g/ml. All Escherichia coli plasmids were maintained in DH5␣ cells. Plasmids were transferred from E. coli to S. meliloti by triparental conjugation using helper plasmid pRK600 (15). N3 phage transduction was performed as described previously (35). Construction of strains used for transcriptional profiling. The chvI(K214T) (EC69) and wild-type (EC176) strains used for microarray analysis were previously described (53). Both strains are marked with Hyr by the integration of pDW181 (which is PhisB in pDW33) at hisB, about 10 kb upstream of the chvI locus. The chvI(D52E)/chvI⫹ strain (EC220) was constructed as follows. The complete chvI open reading frame (ORF), plus 450 bp upstream, was PCR amplified and TA cloned into pCR2.1-TOPO (Invitrogen), generating pEC78. To generate the D52E mutation, site-directed mutagenesis (QuikChange; Stratagene) of pEC78 was used to replace the GAC codon with GAG at amino acid 52, generating pEC97. Both pEC78 and pEC97 were verified by sequencing. The SpeI/XhoI fragment with the chvI upstream region and the ORF from pEC97 was subcloned into the suicide vector pDW33, generating pEC177. pEC177 was introduced into Rm1021 by triparental mating, resulting in a strain with both chvI(D52E) and wild-type chvI. The chvI(D52E)/chvI⫹ allele from the transconjugant was transduced into Rm1021 once more, generating EC220. Construction of uidA transcriptional fusion strains and GUS assays. ␤-Glucuronidase (GUS) fusion plasmids were constructed by PCR amplifying gene regions with flanking SpeI/XhoI sites and ligating them into pVO155. The specific regions cloned into each plasmid are as follows: pEC340 contains the 773-bp intergenic region upstream of the exoY ORF, pEC573 contains the 361-bp intergenic region upstream of the ropB1 ORF, pEC571 contains the 353-bp intergenic region upstream of the SMb21188 ORF, pEC616 contains the intergenic region from 309 bp upstream through the first 91 bp of the SMb21440 ORF, pEC617 contains bp 110 to 442 of the SMb21491 ORF, pEC618 contains 215 bp of the upstream intergenic region and the entire ORF through the stop codon of SMc01580, pEC565 contains bp 116 to 425 of the SMc01855 ORF, and pEC572 contains 191 bp of the intergenic upstream region through the first 160 bp of the SMc00159 ORF. All plasmids were verified by sequencing. None had any mutations except pEC565, which had a silent mutation, Gln(CAA3CAG), at amino acid 58 of the SMc01855 ORF. Conjugation into Rm1021 resulted in the integration of each transcriptional fusion plasmid into the S. meliloti genome via a single crossover event. For exoY, ropB1, and SMb21188, the integration event resulted in the duplication of the upstream intergenic region; for SMb21440, SMc01580, and SMc00159, the integration event resulted in the duplication of the upstream region as well as the duplication of all (for SMc01580) or part (for SMb21440 and SMc00159) of the ORF; and for SMb21491 and SMc01855, the integration event resulted in the inter-

J. BACTERIOL. TABLE 1. Strains and plasmids a

Strain or plasmid

Strains Rm1021 Rm7095 Rm7096 EC69 EC176 EC220 EC407 EC409 EC412 Plasmids pDW33 pDW181 pEC78 pEC97 pEC177 pEC340 pEC405 pEC406 pEC565 pEC571 pEC572 pEC573 pEC616 pEC617 pEC618 pMB439 pRF1206 pRF1211 pRF1212 pRF1218 pRK600 pVO155 a

Genotype or relevant characteristics

Reference or source

Derivative of RCR2011; Sm exoR95::Tn5; Sm Nm exoS96::Tn5; Sm Nm chvI(K214T) (integrated pDW181); Sm Hy Wild type (integrated pDW181); Sm Hy chvI(D52E)/chvI⫹ (integrated pEC177); Sm Hy Wild type (integrated pEC406); Sm Sp chvI(D52E)/chvI⫹ (integrated pEC405); Sm Sp chvI(K214T) (integrated pEC406); Sm Sp

37 14 14 53 53 This study

Identical to pVO155; Ap Hy PhisB in pDW33, Ap Hy PchvI and chvI in pCR2.1 TOPO; Ap PchvI and chvI(D52E) in pCR2.1 TOPO; Ap chvI(D52E) in pDW33; Ap Hy PexoY in pVO155; Ap Nm/Km chvI(D52E) in pMB439; Ap Sp PhisB in pMB439; Ap Sp SMc01855 internal fragment (bp 116-425) in pVO155; Ap Nm/Km Psmb21188 in pVO155; Ap Nm/Km Psmc00159 in pVO155; Ap Nm/Km PropB1 in pVO155; Ap Nm/Km Psmb21440 in pVO155; Ap Nm/Km SMb21491 internal fragment (bp 110-442) in pVO155; Ap Nm/Km Psmc01580 and SMc01580 in pVO155; Ap Nm/Km pBluescript SK(⫺) derivative with Spr cassette; Ap Sp Position ⫺359 through 5 bp of ropB1 ORF in pCR2.1 TOPO; Ap Km Position ⫺143 through 11 bp after stop codon of SMc01580 in pCR2.1 TOPO; Ap Km Position ⫺297 through start codon of SMb21440 in pCR2.1 TOPO; Ap Km Position ⫺390 through ⫺96 before start codon of SMc01580 in pCR2.1 TOPO; Ap Km Conjugal transfer helper plasmid; Cm Terminator and polylinker preceding uidA in pUC119 derivative; Ap Nm/Km

13 53 This study This study

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All strains are derived from Rm1021.

ruption of the ORF. Each transcriptional fusion allele was transduced into chvI(K214T) (EC412), chvI(D52E)/chvI⫹ (EC409) and wild-type (EC407) strains. Note that EC69, EC220, and EC176 already contain pDW33-derived plasmids (with uidA) and thus could not be used for these GUS assays. Two independent transductants were each assayed in duplicate for GUS activity as previously described (51). Affymetrix GeneChip analysis. Three biological replicates of each strain were cultured in separate flasks of LB medium with Sm selection to an optical density at 600 nm of 0.5 to 0.7. RNA was isolated; cDNA was synthesized, labeled, and hybridized; and data were analyzed using Affymetrix software as previously described (5). Gel mobility shift assays. Purified recombinant wild-type and D52E ChvI proteins were prepared as follows. N-terminal S-tagged wild-type and S-tagged D52E ChvI were constructed by PCR amplifying full-length wild-type or chvI(D52E) strains with flanking EcoRI/HindIII restriction sites and cloning into pET29a (Novagen), creating pDW231 and pRF1173, respectively. Plasmids were

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DIRECT TRANSCRIPTIONAL TARGETS OF ExoS/ChvI

RESULTS The D52E mutation results in constitutively active chvI. After performing genetic screens to identify new components of the ExoS/ChvI pathway and repeatedly isolating suppressor mutations in exoR and exoS (9), we decided to pursue a genomic approach. Previously, transcriptional profiling indicated that the exoR95::Tn5 and exoS96::Tn5 mutations each affected the expression of a few hundred genes (53, 55). To increase the stringency of our approach, we chose to perform transcriptional profiling with a chvI partial-loss-of-function strain, a chvI gain-of-function strain, and the wild type. We reasoned that genes that were transcriptionally regulated by ExoS/ChvI should show both decreased expression levels in the partial-loss-of-function strain and increased expression levels in the gain-of-function strain or vice versa. Conversely, false positives and genes that were affected only after physiological adaptation to ExoS/ChvI mutant phenotypes were unlikely to show opposite changes in gene expression in the chvI mutants. In this experiment, we used the chvI(K214T) partial-loss-offunction mutant (EC69) that was described previously (53). We constructed a strain with increased chvI function through site-directed mutagenesis. A mutation of the conserved aspartate to glutamate at the site of phosphorylation was previously shown to result in the constitutive activation of the NtrC response regulator (31). We generated the analogous chvI (D52E) mutation and integrated a plasmid with this allele into the genome of wild-type S. meliloti by a single crossover event. The chvI(D52E) allele behaved like a dominant gain-of-function allele, since this strain (EC220) with one wild-type chvI and one chvI(D52E) allele strongly overproduced succino-

ch vI (K

W T

W T

(H y )r

21 4T ch ) vI (D 52 E) ex /c oR hv I+ 95 ::T ex n5 oS 96 ::T n5

A

UV

visible

B

15 (Miller units)

PexoY::GUS activity

verified by sequencing and transformed into BL21(DE3) cells for protein expression. S-tagged ChvI proteins were purified using the S-Tag thrombin purification kit (Novagen). Briefly, cultures were induced with 1 mM IPTG (isopropyl-␤-D-thiogalactopyranoside) for 3 h at 37°C, and cells were lysed by freeze-thaw and sonication. The S-tagged ChvI protein was bound to S-protein agarose by incubation at 4°C and then cleaved from the agarose beads by incubation with biotinylated thrombin for 90 min at room temperature. The eluate was concentrated with a BioMax 10-kDa Ultrafree-15 centrifugal filter device (Millipore) and stored as aliquots at ⫺80°C in ChvI storage buffer (20 mM HEPES [pH 7.4], 50 mM KCl, 5 mM dithiothreitol, 50% glycerol). The regions upstream of ropB1, SMb21440, and SMc01580 were 3⬘ end labeled by digesting pRF1206, pRF1211, pRF1212, and pRF1218 with EcoRI (which flanks the TA-cloned inserts in all four plasmids) and filling in with [␣-32P]dATP and unlabeled dCTP, dGTP, and dTTP with the Klenow fragment of DNA polymerase I. The labeled DNA fragments were purified following electrophoresis on 5% polyacrylamide gels. Any secondary digests to localize the ChvI binding site were performed with these gel-purified fragments. Labeled DNA fragments were incubated with purified ChvI(D52E) protein at room temperature in binding buffer (10 mM Tris-HCl [pH 7.5], 10 mM KCl, 0.1 mM dithiothreitol, 2.5% glycerol, and 25 ␮g/ml bovine serum albumin) for 20 min and then run on 5% Tris-borate-EDTA gels at 4°C as described previously (16). Gels were dried, and DNA bands were visualized by autoradiography. DNase I footprinting. The EcoRI-NruI fragment from pRF1211 and the SalIEcoRI fragment from pRF1218 were end labeled at the EcoRI ends as described above. The labeled fragments were incubated in a final volume of 7 ␮l with purified ChvI or ChvI(D52E) protein as described above. A total of 150 mU RQ1 DNase (Promega) in a solution containing 25 mM Tris-HCl (pH 7.5), 30 mM MgCl2, and 30 mM CaCl2 was added, and the reaction mixture was incubated for 30 s before stopping by the addition of 12 ␮l DNase stop solution (90% formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol FF). Samples were heated for 3 min at 95°C, quick cooled on ice, and loaded onto sequencing gels as described previously (17). Maxam-Gilbert DNA sequencing ladders (G and G⫹A) of the same fragments were run next to the DNase I footprint samples.

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chvI chvI (K214T) (D52E)/ chvI+

FIG. 1. chvI(D52E) is a gain-of-function mutation. (A) The chvI(D52E) mutation results in the overproduction of succinoglycan. Serial dilutions of S. meliloti strains [wild type (WT) (Rm1021), WT marked with Hyr (EC176), chvI(K214T) (EC69), chvI(D52E)/chvI⫹ (EC220), exoR95::Tn5 (Rm7095), and exoS96::Tn5 (Rm7096)] were spotted onto LB medium with 0.02% calcofluor plates, incubated at 30°C for 3 days, and photographed with UV or visible light. (B) The chvI(D52E) mutation results in increased exoY transcription. GUS activity was measured in strains [wild type (EC407), chvI(K214T) (EC412), and chvI(D52E)/chvI⫹ (EC409)] with an integrated PexoY:: uidA fusion (pEC340). Two independent transductants were assayed in duplicate, and standard deviations are shown. The experiment was repeated twice, with similar results.

glycan (Fig. 1A). Succinoglycan overproduction in this chvI(D52E)/chvI⫹ strain was due to the increased transcription of succinoglycan biosynthesis genes such as exoY (Fig. 1B). We attempted to replace wild-type chvI with the chvI(D52E) allele via double recombination but failed to recover strains that contained only the chvI(D52E) allele (not shown). We also tried constructing a constitutively inactive chvI allele by making a chvI(D52N) mutation, but we were unable to recover any strains that had wild-type chvI replaced by chvI(D52N) (not shown). These results suggest that the expression of a constitutively active or inactive chvI allele on its own may be lethal for S. meliloti. Thus, we used strain EC220, containing both the

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wild-type and D52E chvI alleles, as the gain-of-function chvI strain for transcriptional profiling. Identification of putative chvI target genes via microarray analysis. We performed microarray analysis with three S. meliloti strains: the partial-loss-of-function chvI(K214T) mutant (EC69), the gain-of-function chvI(D52E)/chvI⫹ mutant (EC220), and a wild-type control (EC176). Three biological replicates of each strain were cultured in LB medium and analyzed on Affymetrix GeneChips, allowing for nine pairwise comparisons of gene expression between each mutant and wild-type data set. We found 59 genes that showed decreased levels of expression in the chvI(K214T) strain and increased levels of expression in the chvI(D52E)/chvI⫹ strain, or vice versa, compared to their levels of expression in the wild-type strain (Table 2). Fifty-five of these genes were downregulated in the chvI (K214T) strain and upregulated in the chvI(D52E)/chvI⫹ strain, suggesting that chvI functions mainly as an activator of transcription. As a validation of our approach, 15 of the putative ChvI target genes are exo genes involved in succinoglycan production, a process known to be regulated by ExoS/ChvI. Also, the transcription of ropB1 appears to be controlled by ExoS/ChvI. RopB1 is an outer membrane protein that influences the phage sensitivity of S. meliloti (7), whose orthologs in B. abortus (Omp3A) and A. tumefaciens (AopB) were previously shown to be regulated by ExoS/ChvI orthologs in those respective bacteria (27, 33). Only one putative target gene has homology to a transcriptional regulator, SMa1705 (3). Many of the putative chvI target genes encode hypothetical proteins with predicted signal sequences or transmembrane domains (Table 2), according to annotations in the S. meliloti 1021 genome database (http://iant.toulouse.inra.fr/bacteria/annotation /cgi/rhime.cgi) based on the signalp and tmhmm prediction programs. Other polysaccharide-related genes that appear to be controlled by ChvI include the msbA2 operon, eglC, prsD, and opgC. MsbA2 is required for symbiosis, and the msbA2 operon (SMb21188 to SMb21191) is involved in polysaccharide synthesis (6, 25). EglC is an endoglycanase that contributes to the depolymerization of high-molecular-weight succinoglycan (49). PrsD is a component of an ABC transporter that contributes to the secretion of low-molecular-weight succinoglycan (56). OpgC has homology to a cyclic ␤-1,2-glucan succinyltransferase from Brucella species (45). Genes involved in inositol catabolism also appear to be positively regulated by ChvI. The iolB, iolC, iolD, and iolE genes are predicted to have roles in inositol catabolism based on homology to genes in Bacillus subtilis (57). Some S. meliloti strains can catabolize rhizopines using the myo-inositol catabolic pathway (22), but most S. meliloti strains, including Rm1021, lack the ability to synthesize and catabolize rhizopines (46). For Rm1021, the role of inositol catabolism in symbiosis is unclear, but defects in inositol utilization pathways were previously shown to decrease the symbiotic competitiveness of Rhizobium leguminosarum bv. viciae (19) and Sinorhizobium fredii (29) and were suggested to decrease the symbiotic competitiveness of an engineered S. meliloti strain in a field study (48). Verification of putative chvI target genes. To confirm results from transcriptional profiling, we made GUS transcriptional

J. BACTERIOL.

fusions for seven genes in Table 2. The seven genes that we tested were ropB1 and SMb21188 (the first gene in the operon with msbA2) as well as some of the hypothetical genes with the highest average signal log ratios (SLRs) in Table 2 (SMb21440, SMb21491, SMc01580, SMc01855, and SMc00159). Each transcriptional fusion was integrated into the S. meliloti genome by a single crossover event and transduced into the wild-type, chvI(K214T), and chvI(D52E)/chvI⫹ strains to measure GUS activity. GUS activity assays with these strains confirmed the microarray results for these seven genes (Fig. 2). GUS fusions to the genes that appeared to be positively regulated by ChvI in the microarray showed lower levels of expression in the chvI(K214T) background and higher levels of expression in the chvI(D52E)/chvI⫹ background than in the wild type. Conversely, the GUS fusion to SMc00159, which appeared to be negatively regulated by ChvI in the microarray, showed higher levels of expression in the chvI(K214T) strain and lower levels of expression in the chvI(D52E)/chvI⫹ strain than in the wildtype strain. These data validate the results of the microarray experiment and indicate that ropB1, SMb21188, SMb21440, SMb21491, SMc01580, SMc01855, and SMc00159 are indeed transcriptionally regulated by ChvI. Identification of direct chvI transcriptional target genes. To understand how ExoS/ChvI regulates gene expression, we wanted to identify genes whose transcription is directly controlled by ChvI binding to DNA. We used gel mobility shift assays to test 19 upstream regions that correspond to 31 of the 59 genes (Table 2), some of which are in operons. We tested upstream regions for most of the exo genes, the seven genes confirmed to be ChvI regulated in Fig. 2, and some genes from Table 2 with the highest average SLRs. We used purified recombinant ChvI(D52E) for the gel shift assays, reasoning that this mutant protein might bind DNA more tightly in vitro. Of the 19 upstream regions tested, those upstream of ropB1, SMb21440, and SMc01580 showed a mobility shift after incubation with the ChvI(D52E) protein (Fig. 3). Since the intergenic region upstream of SMc01580 was relatively large, overlapping fragments of that region were cloned into two different plasmids, pRF1218 and pRF1211. Both of these fragments showed a mobility shift (Fig. 3C), suggesting that a sequence bound by ChvI upstream of SMc01580 is within the overlapping region. Alternatively, there could be two independent ChvI binding sites on the inserts in pRF1218 and pRF1211. To refine the localization of the ChvI binding site, we performed restriction digestion of the full-length fragments. Gel shift assays with products of the restriction digests allowed us to determine which fragments contained the DNA region bound by ChvI. From these gel shift assays, we found that ChvI binding sites are within the 116-bp SspI-Sau3AI fragment upstream of ropB1, the 97-bp Sau96I-AvaI fragment upstream of SMb21440, and a 48-bp region in the SalI-NruI fragment upstream of SMc01580 (Fig. 3). Many of the restriction digests shown in Fig. 3 are partial digests, so the full-length fragment is still present. The presence of the full-length fragment serves as an internal control for the gel shift assays; had neither of the smaller fragments shifted, it would have implied that the ChvI binding site was cleaved by the restriction enzyme. In sum, we

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DIRECT TRANSCRIPTIONAL TARGETS OF ExoS/ChvI

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TABLE 2. ChvI-regulated genes from microarray analysis with chvI(D52E)/chvI⫹ and chvI(K214T) strains Avg SLR (SD)a chvI(D52E)

Geneb

Gene product and description

EglC; endo-1,3-1,4-␤-glycanase Transcriptional regulator, MucR family ABC transporter, periplasmic solute binding protein; induced by myo-inositol ThuA; trehalose catabolism protein ExoZ; acetyltransferase ExoQ; Putative polysaccharide polymerase, similar to the Wzy protein ExoF1; periplasmic protein involved in polysaccharide export ExoY; exopolysaccharide production protein ExoU; glucosyltransferase ExoV; pyruvyltransferase ExoH; succinyltransferase ExoK; endo-␤-1,3-1,4-glucanase ExoL; putative glucosyltransferase ExoA; glucosyltransferase ExoM; glucosyltransferase ExoO; glucosyltransferase ExoN; UDPglucose pyrophosphorylase ExoP; protein-tyrosine kinase Hypothetical protein Putative acyltransferase, possibly surface saccharide-specific acetyltransferase Putative glycosyltransferase Putative glycosyltransferase MsbA2; polysaccharide-exporting ABC transporter, ATP binding and permease Conserved hypothetical protein, signal peptide PrsD; ABC transporter, ATP binding component, for low-molecular-wt succinoglycan biosynthesis Hypothetical protein Hypothetical exported protein ExoW; glucosyltransferase Hypothetical protein Conserved hypothetical protein signal peptide Hypothetical protein Conserved hypothetical protein, signal peptide SinI; N-acyl-L-homoserine lactone synthetase Putative signal transduction histidine kinase, phosphotransfer (Hpt) region Hypothetical protein IolB; putative myo-inositol catabolism protein IolE; putative myo-inositol catabolism protein RopB1; outer membrane protein Putative oxidoreductase IolC; putative sugar kinase IolD; putative malonic semialdehyde oxidative decarboxylase Hypothetical protein signal peptide Conserved hypothetical protein Hypothetical transmembrane protein Hypothetical protein signal peptide Putative membrane-bound lytic transglycosylase, signal peptide Hypothetical transmembrane protein Hypothetical protein signal peptide Hypothetical protein Hypothetical protein signal peptide Hypothetical transmembrane protein Calcium binding protein Putative glycine-rich cell wall structural transmembrane protein Hypothetical protein signal peptide OpgC; putative glucan succinyltransferase ABC transporter periplasmic solute binding protein for mannose, L-xylose, glucose, and sorbose Conserved hypothetical protein, transthyretin-like protein Conserved hypothetical protein, signal peptide Rnd1; RNase D

chvI(K214T)

1.60 (0.16) 0.75 (0.34) 1.48 (0.38) 0.88 (0.32) 0.90 (0.13) 1.17 (0.23) 1.09 (0.18) 1.30 (0.20) 0.70 (0.20) 0.80 (0.16) 1.49 (0.27) 1.36 (0.26) 1.19 (0.13) 1.30 (0.14) 1.40 (0.13) 1.10 (0.16) 1.12 (0.22) 1.22 (0.20) 0.93 (0.58) 1.39 (0.26) 1.41 (0.24) 0.79 (0.14) 1.32 (0.34) 2.44 (0.27) 0.54 (0.17)

⫺0.85 (0.13) ⫺1.09 (0.31) ⫺0.96 (0.30) ⫺0.63 (0.08) ⫺0.89 (0.18) ⫺1.33 (0.20) ⫺1.41 (0.18) ⫺1.36 (0.21) ⫺0.79 (0.17) ⫺1.58 (0.35) ⫺2.00 (0.29) ⫺1.52 (0.34) ⫺1.07 (0.16) ⫺1.58 (0.13) ⫺2.10 (0.41) ⫺1.77 (0.40) ⫺2.09 (0.17) ⫺1.80 (0.36) ⫺1.26 (0.27) ⫺1.26 (0.16) ⫺1.46 (0.22) ⫺1.23 (0.19) ⫺2.86 (0.99) ⫺3.22 (0.32) ⫺0.70 (0.16)

SMa1587 SMa1705 SMb20072 SMb20329 SMb20943 SMb20944 SMb20945 SMb20946 SMb20948 SMb20949 SMb20954 SMb20955 SMb20956 SMb20957 SMb20958 SMb20959 SMb20960 SMb20961 SMb21026 SMb21188 SMb21189 SMb21190 SMb21191 SMb21440 SMb21466

0.89 (0.12) 1.23 (0.07) 0.98 (0.18) 1.11 (0.20) 1.11 (0.20) 0.82 (0.16) 0.77 (0.23) 0.52 (0.27) 0.82 (0.24) 0.81 (0.23) 1.17 (0.15) 1.10 (0.22) 0.60 (0.10) 1.35 (0.20) 1.22 (0.18) 1.26 (0.07) 0.85 (0.19) 2.08 (0.33) 1.89 (0.32) 0.96 (0.38) 1.72 (0.21) 0.66 (0.11) 0.77 (0.09) 0.60 (0.10) 1.26 (0.31) 0.53 (0.13) 1.13 (0.22) 2.10 (0.50) 1.55 (0.15) 0.72 (0.10) ⫺0.60 (0.24)

⫺1.28 (0.25) ⫺1.27 (0.06) ⫺1.71 (0.14) ⫺1.33 (0.15) ⫺0.93 (0.06) ⫺1.34 (0.24) ⫺1.80 (0.30) ⫺1.02 (0.27) ⫺0.88 (0.16) ⫺2.18 (0.15) ⫺1.01 (0.10) ⫺0.95 (0.23) ⫺0.60 (0.05) ⫺1.45 (0.20) ⫺0.63 (0.31) ⫺0.72 (0.26) ⫺0.71 (0.12) ⫺2.76 (0.35) ⫺2.49 (0.73) ⫺1.15 (0.54) ⫺2.41 (0.20) ⫺0.52 (0.14) ⫺0.98 (0.15) ⫺0.67 (0.09) ⫺0.63 (0.48) ⫺0.61 (0.15) ⫺1.32 (0.23) ⫺2.40 (0.88) ⫺1.22 (0.40) ⫺0.69 (0.10) 0.71 (0.32)

SMb21467 SMb21491 SMb21690 SMc00062 SMc00070 SMc00084 SMc00096 SMc00168 SMc00191 SMc00404 SMc00432 SMc00433 SMc00604 SMc01163 SMc01165 SMc01166 SMc01556 SMc01580 SMc01581 SMc01774 SMc01855 SMc02242 SMc02317 SMc02552 SMc02854 SMc02986 SMc03108 SMc04236 SMc04276 SMc04381 SMb20902c

⫺0.94 (0.44) ⫺2.10 (0.28) ⫺0.71 (0.11)

0.53 (0.35) 1.04 (0.31) 0.69 (0.14)

SMb21285c SMc00159c SMc00622c

a Pairwise comparisons between the mutant and wild type yielded average SLR values for each gene. SLR is the log2 ratio of the change, so an SLR of 1 is equivalent to a twofold change. Genes listed here have an average SLR with an absolute value of 0.5 or greater for both the chvI(D52E)/chvI⫹ and chvI(K214T) strains compared to the wild type and an increase or decrease in expression levels in at least six of the nine pairwise comparisons with the wild type. b The intergenic upstream regions for the genes with both the gene and gene product in boldface type were tested for binding to ChvI in gel mobility shift assays; those with only the gene product in boldface type appear in an operon whose upstream region was tested for binding to ChvI in gel mobility shift assays. c Negatively regulated by ChvI. All other genes are positively regulated by ChvI.

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GUS activity (normalized to WT)

16

WT chvI(K214T) chvI(D52E)/ chvI+

12

8

4

0

ropB1

SMb21188

SMb21440

SMb21491

SMc01580

SMc01855

SMc00159

FIG. 2. ChvI regulates the transcription of ropB1, SMb21188, SMb21440, SMb21491, SMc01580, SMc01855, and SMc00159. GUS activity was measured in strains [wild type (WT) (EC407), chvI(K214T) (EC412), and chvI(D52E)/chvI⫹ (EC409)] with an integrated uidA fusion to each indicated gene (ropB1 [pEC573], SMb21188 [pEC571], SMb21440 [pEC616], SMb21491 [pEC617], SMc01580 [pEC618], SMc01855 [pEC565], and SMc00159 [pEC572]). Two independent transductants were assayed in duplicate, and standard deviations are shown. The experiment was repeated twice, with similar results. The GUS activity of mutant strains was normalized to the GUS activity of the wild type. The absolute GUS activity in Miller units for each fusion in the wild type was as follows: ropB1, 1.86 ⫾ 0.04; SMb21188, 0.118 ⫾ 0.003; SMb21440, 1.77 ⫾ 0.17; SMb21491, 0.110 ⫾ 0.002; SMc01580, 0.32 ⫾ 0.01; SMc01855, 12.89 ⫾ 0.16; and SMc00159, 0.45 ⫾ 0.06.

identified three genes, ropB1, SMb21440, and SMc01580, whose upstream regions are directly bound by ChvI. Identification of a DNA sequence bound by ChvI. To pinpoint the nucleotide sequence directly bound by ChvI, we performed DNase I footprint analysis with a region upstream of SMc01580, the small NruI fragment from pRF1211 (Fig. 3C). A 25-bp region corresponding to positions ⫺118 to ⫺94 before the start codon of SMc01580 was protected in samples with ChvI compared to samples without ChvI (Fig. 4A). Adjacent to this region were two sites that were hypersensitive to DNase I cleavage, likely due to a conformational change induced by nearby ChvI binding. Footprinting using wild-type recombinant ChvI and the small SalI fragment of pRF1218 (Fig. 3C) radiolabeled on the opposite strand showed essentially the same protection patterns (Fig. 4B). Vector sequences are visible at the bottom of Fig. 4A (right) and B. Figure 4C summarizes the DNA sequence protected by the direct binding of ChvI in vitro. Using the MEME algorithm (http://meme.sdsc.edu), we searched for a ChvI binding motif in the following 47 sequences: the footprinted region upstream of SMc01580, the gel-shifted regions upstream of ropB1 and SMb21440, and the 44 upstream intergenic sequences (ⱖ50 bp in length) corresponding to all of the remaining genes in Table 2. We found an 11-bp-long motif that was present at least once in each of the 47 sequences analyzed, with an overall E value of 8.0e⫺006 (Fig. 5). Visual inspection of the intergenic region upstream of SMc01580 revealed the presence of another of these motifs; besides the motif within the footprinted region, there is a second motif about 170 bp upstream of the second one (not shown). Our finding that ChvI binding was detected at only one of these motifs in gel shift assays indicates that ChvI binding is highly sequence specific in vitro. In vivo, it is possible that additional factors contribute to the specificity of ChvI binding to DNA. DISCUSSION To identify transcriptional target genes of ExoS/ChvI, we used Affymetrix GeneChip microarrays with strains with in-

creased chvI function and reduced chvI function and identified 59 putative ExoS/ChvI target genes (including 15 exo genes). Fifty-five of these genes are both upregulated in the chvI gainof-function strain and downregulated in the chvI reduced-function strain compared to the wild type; the remaining four genes were downregulated in the gain-of-function strain and upregulated in the reduced-function strain. Many of these genes encode hypothetical proteins with predicted signal sequences or transmembrane domains. Using transcriptional fusions, we confirmed that ropB1, SMb21188 (the first gene in the msbA2 operon), and five uncharacterized genes are indeed transcriptionally regulated by ExoS/ChvI. Furthermore, we identified three direct transcriptional targets of ChvI: ropB1, SMb21440, and SMc01580. Finally, we identified a nucleotide sequence bound by ChvI with DNase I footprinting and found a conserved motif in the intergenic sequences upstream of the 59 putative ExoS/ChvI genes. Since ExoS/ChvI is a key signaling pathway in both free-living and symbiotic S. meliloti strains, our study provides an important foundation for future investigations of ExoS/ChvI target genes and the molecular mechanism of ExoS/ChvI regulation. Previous microarray experiments indicated that the expression of hundreds of genes was altered in exoR95::Tn5 and exoS96::Tn5 mutants (53, 55), making it difficult to begin to distinguish which genes were truly regulated by ExoS/ChvI and which genes were affected only indirectly due to physiological adaptations to an ExoS/ChvI mutation. Identifying the set of genes that were both upregulated in a strain with increased chvI function and downregulated in a strain with reduced chvI function compared to the wild type, or vice versa, allowed us to focus on a shorter list of genes that were likely to be true ExoS/ChvI targets and facilitated our search for direct ExoS/ ChvI transcriptional target genes (Fig. 3). Furthermore, we can now begin to characterize these genes with respect to the many functions regulated by ExoS/ChvI. Due to the stringency of our approach, the 59 genes shown in Table 2 likely represent an underestimate of the true number of genes transcriptionally regulated by ExoS/ChvI. From their genomic context, five of the genes in Table 2 (prsD, thuA,

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FIG. 3. ChvI binds to DNA upstream of ropB1, SMb21440, and SMc01580. Gel mobility shift assays were performed with purified recombinant ChvI(D52E) protein (40 ng) and the upstream intergenic regions of ropB1 (A), SMb21440 (B), and SMc01580 (C). Gel shift assays were first performed with the indicated full-length intergenic regions (for A and B) or full-length plasmid inserts (for C). As described in Materials and Methods, these full-length inserts were TA cloned with flanking EcoRI sites, which were used to end label the fragments. Gel shifts were also performed with fragments digested with the indicated restriction enzymes to narrow down the DNA region bound by ChvI. Below each gene diagram, thick black lines indicate a fragment whose mobility shifted upon the addition of the ChvI(D52E) protein, and thick gray lines indicate a fragment that was not shifted. Arrowheads indicate a DNA fragment whose mobility shifted upon the addition of ChvI(D52E). Note that many of the restriction digests are partial digests so that some full-length fragment is still present, serving as an internal positive control for a mobility shift. Asterisks indicate fragments used for footprinting in Fig. 4.

SMc02986, SMb21285, and SMc00159) clearly appear to belong to operons. Although the other genes in these operons appear to be coregulated to some extent, their average SLRs or percentages of pairwise comparisons with significant changes in expression did not meet the cutoff values that we selected for inclusion in Table 2 (not shown). Similarly, we cannot rule out the possibility that more than 3 of the 19 intergenic regions tested in the gel shift assays are direct targets of ChvI binding in vivo. For example, we were surprised that we did not detect direct ChvI binding to exo gene upstream regions in our gel shift assays. However, previous deletion analyses of the exoY upstream region indicated that its transcriptional regulation is complex, involving distal and proximal promoter regions and perhaps additional regulatory sites (12, 42). Moreover, it is possible that our in vitro conditions for ChvI binding in the gel shift assay are more stringent than conditions in vivo. One group of genes conspicuously absent from Table 2 is genes involved in chemotaxis and flagellar motility, which were

among the most strongly downregulated genes in previous microarray experiments with exoR95::Tn5 and exoS96::Tn5 (47, 53, 55). Since the level of expression of these genes was strongly decreased in the chvI(D52E)/chvI⫹ strain, their absence from Table 2 is due to their failure to show increased expression levels in the chvI(K214T) mutant compared to wildtype levels. One possible explanation is that flagellar motility genes may be expressed at maximum levels in wild-type S. meliloti, so regulatory mechanisms to increase their expression in a chvI partial-loss-of-function mutant may simply not exist. An alternative explanation is that the downregulation of motility genes in exoR95::Tn5, exoS96::Tn5, and chvI(D52E)/ chvI⫹ may be an indirect physiological consequence of elevated ExoS/ChvI activity. A ChvI-dependent regulator of motility is not evident in Table 2. Although MucR has been shown to inhibit the transcription of the rem motility regulator by binding to sequences upstream of rem (1), the MucR family transcriptional regulator SMa1705 (Table 2) lacks the C2H2

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FIG. 4. ChvI protects a region upstream of SMc01580 from cleavage during DNase I footprint analysis. (A) The small NruI fragment from pRF1211 that showed a mobility shift in Fig. 3C was radiolabeled on the lower strand at its 3⬘ end and subjected to DNase I footprint analysis with purified ChvI(D52E) protein (100 ng). The left and right panels have the identical samples loaded, but those in the left panel were run longer than the samples in the right panel. Thus, much of the same protected sequence at the bottom of the left panel can also be seen at the top of the right panel. The black line to the right of each autoradiograph indicates the region protected by ChvI, and asterisks indicate hypersensitive cleavage sites. The gray line indicates a vector sequence from pRF1211. Lanes 1 and 2, Maxam-Gilbert sequencing reactions for G or G⫹A; lanes 3 and 6, no ChvI added; lanes 4 and 5, ChvI(D52E) added; lane 7, no DNase I added. (B) The small SalI fragment from pRF1218 that showed a mobility shift in Fig. 3C was radiolabeled on the upper strand at its 3⬘ end and subjected to DNase I footprint analysis with purified ChvI(D52E) or wild-type (WT) ChvI protein. The black line indicates the SMc01580 upstream region protected by ChvI, and the gray line indicates the vector sequence from pRF1218. Lanes 1, 2, and 10, Maxam-Gilbert sequencing reactions for G or G⫹A; lanes 3, 6, and 9, no ChvI added; lanes 4 and 5, ChvI(D52E) added; lanes 7 and 8, ChvI (wild type) added. (C) Sequence of the upstream region of SMc01580 used in the footprint analysis shown in A. Lines indicate the protected regions from A and B, and asterisks indicate hypersensitive cleavage sites. Numbering is with respect to the start codon of SMc01580.

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FIG. 5. Motif found in the upstream region of each ChvI-dependent gene or operon. The footprinted region upstream of SMc01580, the mobility-shifted regions upstream of ropB1 and SMb21440, and the remaining 44 intergenic regions (ⱖ50 bp long) corresponding to each gene or operon in Table 2 were analyzed using the MEME algorithm. The sequence logo represents a motif that occurs at least once in each input sequence; the log likelihood ratio of this motif is 349, and the E value is 8.0e⫺006. The E value is an estimate of the number of 11-bp-long motifs with a log likelihood ratio of 349 or higher that would be expected to occur once in each input sequence of a similarly sized set of 47 random sequences. SSC, small-sample correction.

zinc finger DNA binding motif found in MucR (3). Also, sinI (Table 2) has been shown to regulate flagellar motility in an expR⫹ strain background, but no link has been found between sinI and motility in an expR mutant strain such as Rm1021 (26, 28). Through in silico analyses, we identified a possible ChvI binding motif (Fig. 5) that is present at least once in all of the sequences upstream of the ChvI-regulated genes listed in Table 2. This motif is found sometimes on the plus strand and sometimes on the minus strand, and the distances between the motif and the start codon varied from gene to gene. Whether the motifs are found at a uniform distance from the transcription start sites awaits the determination of the transcriptional start for each of these genes. For ropB1, the transcriptional start site was previously identified (34). The SspI-Sau3AI region that contains the motif and binds to ChvI in gel shift assays (Fig. 3A) spans from 16 to 124 bp upstream of the previously reported ropB1 transcriptional start site. ExoS/ChvI is a signaling pathway of central importance in S. meliloti, with roles in symbiosis, free-living viability, succinoglycan production, biofilm formation, motility, and nutrient utilization (53, 55). By identifying new ExoS/ChvI transcriptional target genes, including three direct transcriptional target genes, and a DNA sequence directly bound by ChvI, our study provides an important foundation for elucidating how ExoS/ ChvI regulates its many downstream functions. ACKNOWLEDGMENTS We are grateful to M. Barnett and C. Toman for their generosity and guidance with Affymetrix GeneChip microarrays and analysis, to N. Nikolaidis and D. Wang for assistance with bioinformatics searches, and to M. Benam for construction of the SMc01855::uidA transcriptional fusion plasmid. We thank M. Barnett, J. Griffitts, and C. Haney for critical reading of the manuscript. This work was supported by NSF award IOS-0818981 (to E.J.C.), a California State University Program in Education and Research in Biotechnology faculty-student seed grant (to E.J.C.), postdoctoral fellowship PF0507301MBC from the American Cancer Society (to E.J.C.), and NIH award R01-GM30692 (to S.R.L.).

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