GxySBA ABC Transporter of Agrobacterium tumefaciens and Its Rolein Sugar Utilization and vir Gene Expression

Jinlei Zhao, Andrew N. Binns

Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA

Monosaccharides available in the extracellular milieu of Agrobacterium tumefaciens can be transported into the cytoplasm, orvia the periplasmic sugar binding protein, ChvE, play a critical role in controlling virulence gene expression. The ChvE-MmsABABC transporter is involved in the utilization of a wide range of monosaccharide substrates but redundant transporters arelikely given the ability of a chvE-mmsAB deletion strain to grow, albeit more slowly, in the presence of particular monosaccha-rides. In this study, a putative ABC transporter encoded by the gxySBA operon is identified and shown to be involved in the utili-zation of glucose, xylose, fucose, and arabinose, which are also substrates for the ChvE-MmsAB ABC transporter. Significantly,GxySBA is also shown to be the first characterized glucosamine ABC transporter. The divergently transcribed gene gxyR encodesa repressor of the gxySBA operon, the function of which can be relieved by a subset of the transported sugars, including glucose,xylose, and glucosamine, and this substrate-induced expression can be repressed by glycerol. Furthermore, deletion of the trans-porter can increase the sensitivity of the virulence gene expression system to certain sugars that regulate it. Collectively, the re-sults reveal a remarkably diverse set of substrates for the GxySBA transporter and its contribution to the repression of sugar sen-sitivity by the virulence-controlling system, thereby facilitating the capacity of the bacterium to distinguish between the soil andplant environments.

Agrobacterium tumefaciens is a Gram-negative soil bacteriumthat, upon infection of a wound site, is capable of initiating the

development of crown gall tumors in dicotyledonous plants bytransferring transfer DNA (T-DNA) from the tumor-inducing(Ti) plasmid into host cells (1). Expression of the Ti plasmid vir-ulence genes is activated by a two-component system, VirA/VirG,in response to plant-derived signals, including phenolics, mono-saccharides, and acidic pH (2). The monosaccharide signals bind aperiplasmic sugar binding protein, ChvE, and the proposed inter-action between sugar-bound ChvE and the periplasmic domain ofVirA results in increased sensitivity for the phenolic signals recog-nized by a cytoplasmic domain within VirA (3–7).

When free living as a saprophytic bacterium, A. tumefaciens hasto compete for a variety of nutrient sources. Accordingly, it hasevolved a significant number of high-affinity transporters for theacquisition of nutrients. Of these, there are a large number ofATP-binding cassette (ABC) transporters, one of the largestclasses of transporters which exist universally from bacteria tohumans (8, 9). Sequence comparisons indicate that 57 ABC trans-porters are found in Escherichia coli (10), whereas A. tumefaciensstrain C58 has 153 ABC transporters plus additional “orphan”subunits in a genome size slightly larger than that of E. coli (11).Despite the proposed importance of ABC transporters for nutri-ent transport by Agrobacterium, few of these have yet been char-acterized.

One exception to this is the ChvE-MmsAB ABC transporter (4,12) which has been shown to be important in the utilization of awide variety of monosaccharides, including arabinose, fucose, ga-lactose, glucose, and xylose. Earlier studies also demonstrated thatdeletion of the MmsA or MmsB transporter component resultedin strains that were significantly more sensitive to several vir-in-ducing sugars that interact with ChvE (7). These data are consis-tent with the hypotheses that the regulatory system has evolved torespond to the relatively high sugar concentrations found in theplant compared to those found in the soil. They also suggest that

this is accomplished by the activity of the transporter which keepsthe concentration of the sugar in the periplasm sufficiently lowthat it cannot function in the vir induction. However, strains car-rying deletions in this system remain capable of growing in thepresence of the sugars, albeit at a lower rate (12), suggesting thatthere is at least one other transport system for these sugars. More-over, preliminary studies indicated that deletion of mmsB resultedin only a modest reduction in sensitivity to glucose (or xylose) interms of vir gene induction. Therefore, examination of other sugartransporters that could be involved in controlling these pheno-types was undertaken. In Agrobacterium radiobacter, two high-affinity glucose binding proteins (GBP1 and GBP2) have beenpurified, and the binding specificity was determined (13). GBP1 ishomologous to ChvE and was shown to bind glucose and galac-tose with high affinity, while GBP2 bound glucose and xylose withhigh affinity.

In this article, we used bioinformatics to identify the gene en-coding a protein homologous to GBP2 in A. tumefaciens. Se-quence analysis and genetic characterization revealed that thisgene is located in an operon that also includes two downstreamgenes encoding a putative ABC transporter. Growth assays dem-onstrate that this operon is involved in utilization of multiplemonosaccharides, including glucose, xylose, fucose, arabinose,and glucosamine, and the genes have been named gxySBA (glu-cose and xylose transporter SBA). An adjacent, divergently tran-

Received 18 March 2014 Accepted 17 June 2014

Published ahead of print 23 June 2014

Address correspondence to Andrew N. Binns, abinns@sas.upenn.edu.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.01648-14.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JB.01648-14

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scribed gene, gxyR, encodes a repressor of the gxySBA operon, thedeletion of which leads to overexpression of GxySBA. Intrigu-ingly, we find that glycerol suppresses substrate-induced expres-sion of gxySBA. Furthermore, we show that the abundance of thisABC transporter affects the sensitivity of Agrobacterium to glu-cose-mediated induction of vir gene expression. This is consistentwith the hypothesis (7) that control of sugar concentration in theperiplasmic space contributes to a system that can distinguish be-tween plant tissues with high concentrations of inducing sugarsand the soil environment that would have significantly lower lev-els of free sugars.

MATERIALS AND METHODSBacteria, media, and growth conditions. Escherichia coli strains weregrown in Luria-Bertani (LB) medium (14) with appropriate antibiotics at37°C. Agrobacterium tumefaciens strains (Table 1) were routinely main-tained on LB medium at 25°C, except where noted. The defined AB min-imal medium (15) was used for growth assays. For vir gene induction, ABinduction medium (ABI) (16), was used except 7.8 g/liter MES (morpho-lineethanesulfonic acid) (pH 5.5) was used, acetosyringone (AS) was in-cluded at 10 �M, and thiamine and Casamino Acids were omitted. Anti-biotics were used at the following concentrations. For A. tumefaciens,carbenicillin was used at 30 or 100 �g/ml, spectinomycin at 25 or 50�g/ml, and kanamycin at 10 or 50 �g/ml (in liquid or solid medium,respectively). For E. coli, carbenicillin was used at 100 �g/ml and kana-mycin at 50 �g/ml in liquid or solid medium.

Genetic modification of bacterial strains and construction of plas-mids. We used marker-exchange eviction mutagenesis (17) as describedearlier (12) to create strains carrying nonpolar deletions of gxySBA in thechromosome of strain A348 (18) or in the chromosome of AB520 (anmmsB deletion strain derived from A348) (12). Primer Datu35746.P2 (seeTable S1 in the supplemental material) was used with Datu35746.P1 toamplify a 600-bp fragment containing the downstream gxySBA flankingsequence. PCR to amplify the upstream gxySBA flanking sequence usedprimers Datu35746.P3 and Datu35746.P4 to create a 593-bp product. The5= ends of primers Datu35746.P2 and Datu35746.P3 had been designed toinclude complementary sequence so that the products from the first twoPCRs could function as self-annealing templates in a third PCR withprimers Datu35746.P1 and Datu35746.P4 to generate a 1.2-kb fragmentcarrying gxySBA flanking sequences. This 1.2-kb PCR fragment was di-gested with HindIII and EcoRI and cloned into pK18mobsacB (17). Theresulting construct was electroporated into strain A348 or AB520. Thedeletion of the gxySBA genes in these strains was obtained and con-firmed using the methods described in reference 12. The primers used forconfirmation are outside the amplified gxySBA flanking sequences:Con35746.P1 (downstream) and Con35746.P2 (upstream). The gxySBAdeletion strain and mmsB gxySBA double deletion strain were namedAB650 and AB662, respectively.

The same methods and confirmation strategies were used to makeA348 derivative strains with in-frame deletions of gxyS, gxyA, and gxyRindividually and AB655 (see below) derivative strains with deletions ofgxyR and gxyA individually (Table 1), using primers in Table S1 in thesupplemental material. All strains were confirmed by PCR.

TABLE 1 Strains and plasmids used in this study

Strain or plasmid CharacteristicsReference orsource

StrainsE. coli XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac[F= proAB lacIqZ�M15 Tn10 (Tcr)] Stratagene

A. tumefaciensA348 C58 background carrying pTiA6 18AB325 AB685 with gxyR deletion This studyAB520 A348 mmsB deletion 12AB529 A348 mmsB and gxyR deletion This studyAB650 A348 gxySBA deletion This studyAB652 A348 gxyS deletion This studyAB655 A348 but with RGS-6�His-tagged gxyS This studyAB656 AB655 with gxyR deletion This studyAB662 A348 mmsB and gxySBA deletion This studyAB685 AB655 with mmsB deletion This studyAB686 A348 mmsB and gxyA deletion This studyAB689 A348 with gxyR deletion This study

PlasmidspBBR1MCS-4 Broad-host-range plasmid cloning vector; Carbr 19pK18mobsacB Suicide plasmid in Xanthomonas campestris pv. campestris; Kmr 17pSW209� Reporter plasmid carrying a PvirB::lacZ fusion; Specr 21pJZ31 pK18mobsacB derivative containing flanking sequences of gxySBA as a HindIII/EcoRI fragment; used for gxySBA deletion This studypJZ32 pK18mobsacB derivative containing flanking sequences of gxyS as HindIII/EcoRI fragment; used for gxyS deletion This studypJZ33 pK18mobsacB derivative containing flanking sequences of gxyA as HindIII/EcoRI fragment; used for gxyA deletion This studypJZ34 pK18mobsacB derivative containing flanking sequences of gxyR as BamHI/SalI fragment; used for gxyR deletion This studypJZ35 pK18mobsacB derivative containing RGS-6�His-tagged gxyS and its flanking sequences as a BamHI/SalI fragment; used

for RGS-6�His-tagged gxyS recombination into chromosomeThis study

pJZ36 gxySBA with the promoter of gxySBA (PgxySBA) in pBBR1MCS-4 This studypJZ37 gxyA with the promoter of PgxySBA in pBBR1MCS-4 This studypJZ38 gxyA·RGS-6�His with the promoter of PgxySBA in pBBR1MCS-4 This studypJZ39 gxyAK47A·RGS-6�His with the promoter of PgxySBA in pBBR1MCS-4 This study

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To monitor the expression of GxyS, an RGS-6�His tag was fused tothe C terminus of gxyS, and this fusion was used to replace gxyS at itslocation in the chromosome. Two primer pairs, 3576RGS.P1/3576RGS.P2 and 3576RGS.P3/3576RGS.P4, were used to do two initialPCRs. Subsequently, the first two PCR products were used as templates toamplify a fragment carrying the RGS-6�His-tagged gxyS gene along withadditional upstream and downstream sequences. This third PCR usedprimers 3576RGS.P1 and 3576RGS.P4, and the resultant 2.2-kb fragmentwas digested with BamHI and SalI and cloned into pK18mobsacB. Theplasmid was introduced into gxyS deletion strain AB652. The resultingstrain, containing C-terminal RGS-6�His-tagged GxyS, was namedAB655 (Table 1). Strain AB685 carrying a deletion of mmsB as well as theRGS-6�His-tagged gxyS was derived from AB655 using the methods de-scribed in reference 12.

For complementation of the gxySBA deletion, primers 35746com.P1and 35746com.P2 were used to amplify a fragment containing the entiregxySBA region, including a 221-bp sequence upstream of the gxyS startcodon, which contains the promoter of gxySBA. This PCR product wasdigested with SalI and XbaI and cloned into pBBR1MCS-4 (19), yieldingpJZ35.

For complementation of the gxyA deletion, primers 35746com.P1 and3574com.P11 were used to amplify the promoter region of gxySBA. Prim-ers 35746com.P2 and 3574com.P22 were used to amplify gxyA togetherwith a 60-bp upstream region containing its ribosome binding site (RBS).The two PCR products were mixed and used as the template to conduct athird PCR with primers 35746com.P1 and 35746com.P2. The 1.1-kb PCRfragment was digested with SalI and XbaI and cloned into pBBR1MCS-4to make the plasmid pJZ36 (Table 1), which has gxyA driven by thegxySBA promoter.

To monitor the expression of gxyA, we added a RGS-6�His tag to theC terminus of gxyA. Primer com3574RGSHIS.P2, which has an addedsequence encoding RGS plus 6 His amino acids, was used with primercom35746.P1 to amplify the gxyA with the promoter of the gxySBAoperon using pJZ36 as the template. The PCR fragment was cloned intopBBR1MCS-4 to make the RGS-6�His-tagged gxyA complementationplasmid, pTZ37 (Table 1).

We used overlap-extension PCR to create a mutation (K47A) in theGxyA Walker A ATP binding site. Primers com35746.P1 and3574K47A.P111 were used to amplify the promoter and the first 47codons of gxyA. Primers Com3574RGSHIS.P22 and 3574K47A.P222were used to amplify the remainder of gxyA. These two PCR productswere mixed and used as the template for the third PCR with primerscom35746.P1 and Com3574RGSHIS.P22. The third PCR product wasdigested by XhoI and XbaI and cloned into pBBR1MCS-4 to create pJZ38(Table 1).

Growth assays. The growth assays were carried out as described pre-viously (12), except 3 mM concentrations of sugars were used here.Briefly, overnight cultures of A. tumefaciens in LB medium were centri-fuged, and the pellet was resuspended to an optical density at 600 nm(OD600) of 0.1 in AB minimal medium with 10 mM glycerol. After ap-proximately 20 h of growth, the cultures were pelleted, washed with ABmedium (without any carbon source), and inoculated into AB mediumwith 3 mM various carbon sources at an OD600 of 0.06 and incubated at30°C. Bacterial growth was measured at 600 nm with a spectrophotometer(Beckman DU 520).

Immunoblot analysis for the expression of GxyS and GxyA. Immu-noblot analysis was performed as previously described (12). In brief, cul-tures grown in AB or ABI medium were spun down and resuspended inSDS-loading buffer (20) with an OD600 of 5. Samples were boiled for 5min, and 5 �l was subjected to SDS-PAGE and subsequently transferredto polyvinylidene difluoride (PVDF) membrane (Millipore). For immu-noblot analysis, RGS-His mouse monoclonal antibody (Qiagen) was usedto detect RGS-6�His-tagged GxyA and GxyS. The immunoblots weredeveloped with an ECL Plus enhanced chemiluminescence kit (Amer-sham) per the manufacturer instructions.

�-Galactosidase assay. �-Galactosidase assays were performed as de-scribed previously (7). Briefly, strains carrying a PvirB::lacZ reporter con-struct on plasmid pSW209� (21) were first grown in LB medium plusappropriate antibiotics overnight at 25°C and inoculated into ABI me-dium containing 0.25% glycerol plus the indicated concentrations of var-ious sugars and 10 �M AS. After a 24-h induction, strains were assayed for�-galactosidase by the method of Miller (14). The sensitivity of strains todifferent sugars was examined via dose-response curves with the 50%effective concentration (EC50) defined as the sugar concentration yielding50% of the maximal activity in the �-galactosidase assay (7).

Tobacco tumor assay. Tumor formation on Nicotiana tabacum cv.H425 leaf explants was performed as described previously (7).

RESULTSSequence analysis identifies a second potential glucose ABCtransporter. Since glucose, xylose, and some other sugars werepreviously shown to serve as carbon sources in a chvE-mmsABdeletion strain (12), we hypothesized that there must be anothertransport system(s) for these substrates. A glucose binding protein(GBP2) which, in addition to glucose, also binds D-xylose withhigh affinity was previously identified in Agrobacterium radiobac-ter (13) suggesting it as a candidate protein involved in glucoseand xylose transport. Based on the N-terminal amino acid se-quence of GBP2, a homologue (protein product of gene locusAtu3576) on the linear chromosome of A. tumefaciens strain C58was identified by using BLAST (http://blast.ncbi.nlm.nih.gov/).The products encoded by the downstream gene loci Atu3574 andAtu3575 are predicted to be an ATP-binding protein and a trans-membrane protein, respectively (Fig. 1). Together, the three pro-teins are predicted to constitute a binding-protein-dependentABC transporter. As shown below, this operon is indeed involvedin glucose and xylose utilization and therefore gene loci Atu3576,Atu3575, and Atu3574 were renamed gxyS, gxyB, and gxyA, re-spectively, for glucose/xylose transport. Directly upstream ofgxySBA is a divergently transcribed gene locus Atu3577 (renamedgxyR) predicted to encode an ROK (repressor, open readingframe, and kinase) family transcriptional regulator (22) with a

FIG 1 Genetic map of the gxyR-gxySBA region in A. tumefaciens and otheralphaproteobacteria. Arrows indicate the direction of transcription with thenames of gene loci. The percentage identity compared with correspondinggene product of A. tumefaciens is presented above the arrows representing eachopen reading frame. Atu, Ara, Sme, Rle, and Rsp represent Agrobacteriumtumefaciens C58, Agrobacterium radiobacter K84, Sinorhizobium meliloti 1021,Rhizobium leguminosarum bv. trifolii WSM1325, and Rhodobacter sphaeroides2.4.1, respectively. For accession numbers for all gene loci, see Table S2 in thesupplemental material.

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helix-turn-helix (HTH) DNA binding domain in the N-terminaldomain (http://pfam.sanger.ac.uk/).

Analysis using SignalP (http://www.cbs.dtu.dk/services/SignalP/) (23) predicted that GxyS has a secretion signal (the N-terminal23 amino acids) and that it should localize to the periplasm. Al-though GxyS shares low identity (32%) with ChvE, the key aminoacids in the sugar-binding site (R17/D91/R92/D143/N145/D242/K262) of ChvE (7) are conserved in GxyS, except S15, which is E inGxyS (see Fig. S1 in the supplemental material). Further analysisusing IMG (http://img.jgi.doe.gov/) indicates that the operongxySBA, together with gxyR, is highly conserved and exists widelyin Agrobacterium radiobacter K84, Sinorhizobium meliloti 1021,

Rhizobium leguminosarum bv. trifolii WSM1325 and even Rhodo-bacter sphaeroides 2.4.1. (Fig. 1).

GxySBA is involved in utilization of diverse sugars. The func-tion of gxySBA was tested by examining the growth of strainscarrying a deletion of the entire operon in the presence of variouscarbon sources. The gxySBA deletion strain AB650 grew normallyin minimal medium with glycerol as the sole carbon source (Fig.2A) but exhibited a growth defect in minimal medium with glu-cose and xylose (Fig. 2A). This is consistent with the glucose andxylose binding activity of GBP2 in A. radiobacter (13). In light ofthe chvE-mmsAB operon’s involvement in glucose and xylose uti-lization (12), we proposed that a double deletion strain (�mmsB

FIG 2 Growth of A. tumefaciens mutant strains (A) and complemented strains (B) in minimal medium containing 3 mM various sugars as the sole carbon source.At intervals, the optical density at 600 nm of the cultures was checked. The data are the means of three samples, and the error bars indicate standard deviations.The strains used are wild-type strain A348, mmsB deletion strain AB520, gxySBA deletion strain AB650, and mmsB gxySBA double deletion strain AB662.

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�gxySBA) would have a more severe glucose/xylose-dependentgrowth defect. Strain AB662 carrying both deletions grew nor-mally in minimal medium with glycerol as the sole carbon source(Fig. 2A). However, as predicted, growth of AB662 was muchslower in glucose and xylose, compared with the single mutantsAB520 (�mmsB) or AB650 (�gxySBA), indicating both transport-ers contribute to the utilization of glucose and xylose (Fig. 2A).

We also measured the growth of the mutants in minimal me-dium with arabinose, fucose, galactose, glucosamine, galacturonicacid, or glucuronic acid as the sole carbon source. All the strainsdemonstrated wild-type growth in galacturonic acid and glucu-ronic acid (Fig. 2A), which suggests neither ChvE-MmsAB norGxySBA is involved in sugar-acid transport. The deletion ofgxySBA alone (strain AB650) had no discernible effect on growthin the presence of arabinose or fucose, but the mmsB gxySBA dou-ble deletion strain AB662 grew significantly slower than was ob-served in the mmsB deletion strain AB520, suggesting that arabi-nose and fucose are substrates for both transporters (Fig. 2A). Ingalactose-containing medium, strains AB662 and AB520 grewsimilarly, suggesting that GxySBA is not involved in galactosetransport (Fig. 2A). Interestingly, in glucosamine-containing me-dium, the gxySBA deletion strain AB650 has a severe growth de-fect, indicating that GxySBA may be the principal glucosaminetransporter (Fig. 2A).

To show that the growth defect observed in AB662 was due tothe gxySBA deletion, we performed a complementation assay. Inminimal medium with glucose as the sole carbon source, a plas-mid harboring the gxySBA operon, including the native promoterrestored the growth of the mmsB gxySBA double deletion strainAB662 to the level seen in the mmsB deletion strain AB520 (Fig.2B).

Collectively, these data demonstrate that the gxySBA and chvE-

mmsAB operons are both important in the utilization of glucoseand xylose and can also contribute to arabinose and fucose utili-zation. GxySBA, though, appears to be the predominant facilitatorof glucosamine utilization, while ChvE-MmsAB has the predom-inant role in galactose utilization.

The amino acid K47 is essential for the function of GxyA.Based on its amino acid sequence, GxyA is predicted to be an ATPbinding protein (http://pfam.sanger.ac.uk/), with a typical WalkerA motif GXXGXGKST (G41HNGAGKST49). The highly con-served residue K47 is vital for binding ATP/ADP in other ho-mologs (24) and is expected to be necessary for function of GxyA.To verify this prediction, site-directed mutagenesis was per-formed to change K47 to A (lysine to alanine) in this protein.Plasmids carrying gxyA, gxyA·RGS-6�His, or gxyAK47A·RGS-6�His (see Materials and Methods) were electroporated into themmsB gxyA double deletion strain AB686 and tested for growth inmedium with glucose as the sole carbon source. The plasmid car-rying the gxyAK47A·RGS-6�His mutation could not restoregrowth of AB686, while native gxyA and gxyA·RGS-6�His sub-stantially restored the wild-type phenotype, with the added RGS-6�His tag having a slightly positive effect on GxyA’s functionbased on the growth assay (Fig. 3A). An immunoblot assayshowed that RGS-6�His-tagged gxyA and gxyAK47A had similarexpression levels (Fig. 3B). Thus, K47 is required for the functionof GxyA and this supports the hypothesis that GxyA is an ATPbinding protein.

Expression of the gxySBA operon is induced by a subset ofutilized sugars and regulated by the repressor GxyR. Expressionof an ABC transporter operon is often induced by substrates of thesystem (25). Expression of the gxySBA transporter in response todifferent sugars was examined by monitoring GxyS. For these ex-periments, a sequence encoding an RGS-6�His tag was fused tothe 3= terminus of gxyS, resulting in a C-terminal tag on GxyS, andthe resultant construct was used to replace the wild-type gxySsequences at its native location on the chromosome of A348, re-sulting in strain AB655. Growth assays in glycerol, xylose, glucose,and glucosamine showed that this strain exhibited wild-typegrowth, and thus the RGS-6�His tagged version of GxyS is fullyfunctional (see Fig. S2 in the supplemental material). Consistentwith growth data described above, GxyS was found to be inducedby its substrates glucose, xylose, and glucosamine (strain AB655)(Fig. 4). Interestingly, GxyS accumulation was also moderatelyinduced by glucuronic acid (Fig. 4) and much less so, but detect-ably after long exposure, by arabinose (Fig. 4) (data not shown).These results are consistent with those of Mauchline et al. (25)

FIG 3 Effect of the K47A mutation of GxyA on the growth of strain AB686(�mmsB �gxyA). (A) Growth curves of mmsB gxyA double deletion strainAB686 carrying gxyA, gxyA with the RGS-6�His tag, the RGS-6�His-taggedgxyAK47A mutant, or the vector control. mmsB deletion strain AB520 withvector served as a control. The strains were grown in minimal medium con-taining 3 mM glucose as the sole carbon source. Data are the averages oftriplicate values with standard deviations. (B) Expression level of GxyA in thegxyA deletion strain AB686 carrying the vector control (lane 1), gxyA (lane 2),gxyA with the RGS-6�His tag (lane 3), or the RGS-6�His-tagged gxyAK47A

mutant (lane 4). The strains were grown in minimal medium containing 3 mMglycerol as the sole carbon source.

FIG 4 Expression of GxyS in strains grown at 30°C in minimal medium with3 mM glycerol, arabinose, fucose, galactose, glucose, xylose, galacturonic acid(gal. acid), glucuronic acid (glu. acid), or glucosamine as the sole carbonsource. “WT” represents AB655, which is similar to wild-type strain A348,except gxyS is tagged with RGS-6�His. “�gxyR” represents AB656, which hasa gxyR deletion and is derived from AB655.

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showing that transcription of SMb20902 (homologous to gxyS) inSinorhizobium meliloti 1021 was induced by glucose.

Directly upstream of gxySBA is the divergently transcribedgene locus Atu3577, the protein product of which is homologousto the ROK family transcriptional regulators, including the signa-ture HTH DNA binding domain in its N terminus (22). Based onthis predicted function and its proximity to the gxySBA operon,we hypothesized that this gene locus encodes the regulator forgxySBA, and therefore we named it gxyR. A gxyR deletion strain(AB656) was derived from the wild-type strain, A348, carryingRGS-6�His-tagged gxyS (AB655) to study the role of GxyR ingxySBA operon expression. In the wild-type strain, only glucose,xylose, and glucosamine strongly induced expression of GxyS,with weak induction by glucuronic acid and no induction by theother sugars (Fig. 4). Deletion of gxyR resulted in expression ofGxyS in the presence of all of the tested carbon sources (Fig. 4).Overall, these data suggest that GxyR is a repressor of gxySBAexpression, and at least some of the sugar substrates induce ex-pression by relieving GxyR’s repressive function.

gxyR deletion leads to faster growth in GxySBA’s substratesugars. Experiments in Fig. 2 showed that in comparison to themmsB deletion strain the mmsB gxySBA deletion strain exhibitsvirtually no growth in fucose and very slow growth in arabinose,indicating both of these substrates can be transported by GxySBA.However, these two sugars had only very little (if any) capacity toinduce expression of GxySBA. If the GxySBA proteins are in factimportant for arabinose and fucose utilization, we would predictthat the deletion of gxyR would result in strains that could betterutilize these substrates. The mmsB deletion strain AB685 andmmsB gxyR double deletion strain AB325, both of which carry anRGS-6�His-tagged gxyS gene, were used to test this hypothesis.As predicted, the mmsB gxyR deletion strain shortened the lagphase in all sugars tested, except glycerol (Fig. 5), which is likelydue to the fact that the transporter is already expressed upontransfer into the growth medium (see Fig. S3 in the supplemental

material). Interestingly, this constitutive expression of the gxySBAoperon resulted in a higher growth rate in fucose but not the othersugars tested (Fig. 5).

gxySBA and vir gene expression. Our previous work showedthat the deletion of the ABC transporter mmsAB leads to strainsthat are substantially more sensitive to certain sugar substrates(arabinose and galactose) in terms of vir gene expression as mea-sured using a PvirB::lacZ reporter construct (7). However, themmsB deletion resulted in only a modest increase in sensitivity toglucose as the vir-inducing sugar (Fig. 6): for the mmsB deletionstrain AB520, the EC50 for glucose is 0.4 mM, contrasting with the1.4 mM EC50 of the wild-type strain. The gxySBA deletion strainAB650 —which exhibited a growth response similar to that ofAB520 when cultured in glucose (Fig. 2A)— had an EC50 for glu-

FIG 5 Growth of the mmsB deletion strain AB685 and mmsB gxyR double deletion strain AB325 in minimal medium containing 3 mM various sugars as the solecarbon source. At intervals, the optical density at 600 nm of the cultures was checked. Data are the means of three samples with standard deviations.

FIG 6 Effects of mmsB gxySBA deletions on glucose dose response for virinduction. Wild-type strain A348, mmsB deletion strain AB520, gxySBA dele-tion strain AB650, and the mmsB gxySBA double deletion strain AB662 carry-ing plasmid pSW209� were grown in AB induction medium containing 10�M AS and various concentrations of glucose. All samples were assayed intriplicate, and results are plotted as means with error bars indicating the stan-dard deviation.

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cose-mediated vir gene induction that was virtually identical tothat of the wild type (Fig. 6). Given the profound effect of themmsB gxySBA double deletion on growth in glucose (Fig. 2A), wetested it in the vir gene expression assay and found vir gene expres-sion in the mmsB gxySBA double deletion strain AB662 is muchmore sensitive to glucose, with an EC50 of 0.01 mM (Fig. 6). Sim-ilar results were observed when xylose was tested in these samestrains, whereas all of the strains exhibited the wild-type sensitivityto glucuronic acid, which is not a substrate of either transporter(see Fig. S4 in the supplemental material). In all of these experi-ments, some activity of the vir promoter was observed in AB662 inthe absence of the inducing sugar, possibly indicating that someinducing sugars are present—possibly produced by the bacteria—even in the absence of an exogenously supplied source.

The presence of glycerol in ABI induction medium inhibitsthe expression of GxyS. Some of the results presented above (Fig.2A and 6) seemed contradictory. Single deletions of mmsB orgxySBA both resulted in strains exhibiting slower growth in me-dium containing glucose as a sole carbon source (Fig. 2A). Thissuggested both deletion mutants have a reduced capacity to trans-port glucose. When the same strains were tested for the effect ofglucose on vir gene expression, the single deletion of mmsBshowed a modest increase in sensitivity to glucose compared towild type, whereas the gxySBA deletion responded like the wild-type strain (Fig. 6). In earlier studies (7), we suggested that dele-tion of the mmsB transport protein might result in an increase inthe sugar concentration in the periplasmic space, thereby increas-ing the sensitivity of the vir-inducing system to glucose. However,the deletion of gxySBA had the same effect as the deletion of mmsBon glucose utilization for growth—which would be predicted toresult in a similar effect on periplasmic glucose concentration.One possible cause of this result could be that under vir-inducingconditions expression of gxySBA is suppressed, resulting in lowlevels of GxySBA. Given that different conditions were used forgrowth assays and the vir gene expression assays, we examined theGxyS expression in bacteria grown in the ABI medium used for thelatter. Interestingly, significantly less GxyS was found in mmsBdeletion strain AB685 tested in the ABI medium containing glyc-erol, which was used to supply the carbon source for growth, evenin the presence of 10 mM glucose, which did induce gxySBA ex-pression in the absence of glycerol (Fig. 7). In contrast, the expres-sion of the chvE-mmsAB operon is not suppressed by glycerol (Fig.7). Thus, a �gxySBA strain cultured in glycerol plus inducingsugar has significant levels of the ChvE-MmsAB transporter,whereas a �mmsB strain cultured under the same conditionswould have very low, noninducible levels of the GxySBA trans-porter.

If transporter activity is in fact affecting the sugar concentra-

tion in the periplasmic space, we reasoned that the deletion ofgxyR, which releases the repression of gxySBA expression even inthe presence of glycerol (Fig. 4), would lead to an even lowersensitivity to glucose in terms of vir gene expression. For this pur-pose, gxyR deletion strain AB689 and mmsB gxyR double deletionstrain AB529 were constructed, and vir gene expression was exam-ined. Compared to wild-type strain A348, the gxyR deletion strainAB689 is less sensitive to glucose, and the mmsB gxyR doubledeletion strain AB529 is less sensitive than mmsB deletion strainAB520 (Fig. 8). Given that the gxyR deletion modestly decreasedthe sensitivity of the strain to the vir-inducing effects of glucosecompared to the wild type, we tested these two strains in the stan-dard tobacco leaf explant system. The results showed no consis-tent effect on virulence (see Table S3 in the supplemental mate-rial), suggesting that the available sugar concentrations at thewound site are sufficiently high to overcome the modest decreasein sensitivity.

DISCUSSION

In the soil, A. tumefaciens faces significant competition for nutri-ents of various abundance and, accordingly, has evolved to adaptto these conditions. Included among these adaptations are multi-ple high-binding-affinity ABC transporters that are used to trans-port nutrients into the cell (11). ChvE-MmsAB is one such trans-porter and was shown to be important in the utilization ofmultiple monosaccharides (12), and it has added relevance forAgrobacterium because the periplasmic sugar binding proteinChvE is also involved in the regulation of the virulence genes ofthis bacterium (3, 4, 6). The relationship between these two activ-ities was revealed in studies that showed an increase in sensitivityto the vir-inducing sugars when mmsB was deleted, suggesting thetransporter activity in some manner keeps the concentration ofsugar required for induction to be at a significantly higher levelthan would be expected based on the dissociation constant (Kd) ofChvE for those sugars (7). Because strains lacking the chvE-mmsAB transporter are still capable of growing in all sugars tested(12) and because an mmsB deletion showed only modest effects onthe sugar sensitivity for induction of vir gene expression for cer-tain sugars, including glucose and xylose, we proposed that addi-tional transporters must exist. This led to the current character-

FIG 7 Expression of GxyS and ChvE in normal and modified ABI inductionmedia. AB655 carrying plasmid pSW209� was grown in normal ABI medium(lanes 1 and 2) or modified ABI medium without glycerol (lanes 3 and 4). A 3mM (lanes 1 and 3) or 10 mM (lanes 2 and 4) concentration of glucose wasincluded in the induction medium. Note for GxyS detection, in lanes 3 and 4only 1/20 of the sample was loaded compared to lanes 1 and 2, while the sameamount of sample was loaded for ChvE detection.

FIG 8 Effects of gxyR deletion on sugar dose response for vir induction. Wild-type strain A348, gxyR deletion strain AB689, mmsB deletion strain AB520, andmmsB gxyR deletion strain AB529 carrying plasmid pSW209� were grown inABI induction medium plus different concentrations of glucose. Note that 10�M AS was used. All samples were assayed in triplicate, and results are plottedas means � standard deviations.

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ization of the gxySBA operon and its role in both sugar utilizationand vir gene expression. It encodes a high-affinity transport sys-tem and, consistent with the results of Cornish et al. (13), is im-portant for utilization of glucose and xylose. The ChvE-MmsABand GxySBA transporters have overlapping characteristics interms of substrate specificity but differ considerably in the controlof their expression. Interestingly, to our knowledge, GxySBA is thefirst characterized glucosamine ABC transporter in bacteria. Fi-nally, as is the case with ChvE-MmsAB (7), GxySBA also plays arole in preventing vir gene induction at low glucose concentra-tions.

The redundancy of multiple transporters for one substrate willbenefit agrobacteria and enhance their survival in competitionwith other soil microbes in their natural habitat. The overlappingsugar specificities of the ChvE-MmsAB and GxySBA transportersare another means that can maximize competiveness for A. tume-faciens in the soil, and these specificities can be categorized intotwo groups. In the first group, fucose, glucose, and glucosamineutilization is dependent on both transporters since elimination ofthe two systems eliminates the capacity for such strains to grow onthese carbon sources (Fig. 2A). The second group of sugars, com-prised of arabinose, xylose, and galactose, are apparently trans-ported by at least one of these two transporter systems. However,given that even the double deletion strains grow under these con-ditions (Fig. 2A), there must be additional transport systems forthis second group of sugars, such as major facilitator superfamily(MFS) permeases (26) or other ABC transporters.

The high-affinity sugar binding proteins, such as ChvE andGxyS, play a key role in defining substrate specificity for the ABCtransporters (27). In the sugar-bound ChvE structure, the sugar isstabilized by eight residues in the sugar binding pocket via hydro-gen bonds as well as hydrophobic interactions with two trypto-phan residues (7). Although GxyS shares low similarity with ChvE(identity, 30%), the two tryptophan residues (W18 and W183 inChvE) and other residues in the ChvE sugar binding pocket areconserved, except one (S15 in ChvE) in the GxyS sequence (seeFig. S1 in the supplemental material). Residue 15 may be the sitethat critically differentiates the substrate binding specificity, butresolution of this will require structural analysis. Besides the func-tion in binding sugars, ChvE also separately interacts with theperiplasmic domain of the membrane sensor protein VirA andABC transporter membrane component MmsB. Previous geneticand structural studies of ChvE identified a surface involved ininteraction with VirA (4, 7). Sequence alignment indicates thatamong the 11 residues in the ChvE-VirA interaction surface, only2 residues (E53 and T180) are conserved in GxyS (see Fig. S1). Fiveof these ChvE residues are also likely to be involved in interactionwith the membrane component of the transport system, as evi-denced by their slow-growth phenotype when tested in definedmedium with glucose as a carbon source (4), and of these, onlyone residue (E53 in ChvE) is conserved in GxyS. This suggests thatalthough ChvE and GxyS have similar sugar binding profiles, theinteraction with their transport partner is, as expected, specific.

An interesting finding related to sugar specificity is thatGxySBA is quite important in the utilization of glucosamine andthat ChvE-MmsAB is involved to a limited extent (Fig. 2A). Glu-cosamine is an abundant amino sugar in soil organic matter, withconcentrations ranging from 717 to 2533 mg kg�1 soil in soilhydrolysates (28), although free levels have not been reported.Although a glucosamine degradation pathway has been character-

ized in A. radiobacter (29) and the gene encoding glucosaminitoldehydrogenase in this pathway was identified (30), until now noglucosamine transporters have been reported in agrobacteria oreven in alphaproteobacteria. In E. coli, Bacillus subtilis, and Co-rynebacterium glutamicum, glucosamine transport is via the phos-phoenolpyruvate-dependent carbohydrate phosphotransferasesystem (PTS) (31–33), while in humans, glucosamine uptake isthrough glucose transporters GLU1, -2, and -4 (34). Thus, theseare the first characterized ABC transporters involved in the utili-zation of glucosamine.

Besides the substrate preferences of ChvE-MmsAB andGxySBA transporters, they also exhibit clear differences in theirexpression patterns. The chvE-mmsAB operon has a significantlevel of constitutive expression (12), whereas the gxySBA operon isstringently regulated (Fig. 4). The chvE-mmsAB operon is regu-lated by repressor GbpR, a LysR family regulator (35), whilegxySBA is regulated by GxyR (Fig. 4), an ROK family regulator(22). Expression of these operons is induced by different carbonsources, with chvE-mmsAB induced by arabinose, fucose, and ga-lactose (12, 35) and gxySBA induced by glucose, xylose, and glu-cosamine (Fig. 4). Taken together with the growth data, theseresults suggest that the ChvE-MmsAB system is an importanttransporter for arabinose, fucose, and galactose, while GxySBA iscritical for glucosamine transport. Given that both transporterscontribute similarly to glucose and xylose utilization (Fig. 2A), thefact that these compounds are inducers only of GxySBA expres-sion suggests this transporter may be preferred for these sub-strates.

A surprising result was that in the presence of glycerol, theinduction of GxyS accumulation by glucose was repressed. Catab-olite repression is a possibility, but this generally is the repressionof utilization of a less efficient carbon source (e.g., glycerol) by amore efficient one (glucose)— exactly the opposite of the casehere (36). Glycerol-mediated repression of glucose metabolismhas been observed in Haloferax volcanii (37) through an as yetuncharacterized mechanism. One possibility accounting for theresults here is that glucose uptake is somehow repressed by glyc-erol. However, earlier work (12) as well as work in this article (Fig.2A) indicates that glucose can be transported not only by GxySBAbut also by the ChvE-MmsAB transporter. ChvE-MmsAB is stillexpressed, although at its basal noninduced level, in the presenceof high concentrations of glycerol and can transport glucose,which should be capable of binding to GxyR and thereby relieverepression even in the presence of glycerol. The basis for this ap-parent contradictory finding is not known. One possibility is thatthe actual molecule capable of binding the repressor GxyR andcausing its release from the promoter is not yet known. For exam-ple, the inducer may be metabolic intermediates derived from thetransported sugar. Precedent for this is seen in A. tumefaciens inthe regulation of the attKLM operon via the activity of the meta-bolic intermediates of the acylhomoserine lactone autoinducer(38). Thus, it is possible that the inducers for GxyR are metabolicintermediates of glucose, xylose, or glucosamine, and the presenceof glycerol may result in a metabolic state that does not allowformation of such an inducer. Further studies will be needed tounravel the role glycerol plays in suppressing GxySBA productionin response to glucose.

In our previous study, we showed that deletion of mmsB leadsto dramatically increased sensitivity of the strain to the vir-induc-ing sugars, which are substrates of ChvE-MmsAB transporter,

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while no such change was observed for sugars which are not suchsubstrates (7). A model was proposed suggesting that transportactivity of ChvE-MmsAB maintains a relatively low concentrationof the sugar in the periplasmic space and high external concentra-tions of the sugar may be required to achieve periplasmic concen-trations that are vir inducing. In this study, we investigated thesensitivity of various strains to glucose, which is transported byChvE-MmsAB and GxySBA but not by other transporters. Thedeletion of gxySBA alone had little effect on vir gene expression,while the mmsB deletion resulted in a modest increase in sensitiv-ity to glucose. We propose that the difference between these tworesults is that under the conditions of the vir gene induction assay,glycerol suppresses accumulation of GxySBA but not ChvE-MmsAB. Thus, in the mmsB deletion mutant there would be lesstransport capacity than would be seen in the gxySBA deletion mu-tant tested under the same conditions. In agreement with thismodel, (i) the double deletion mutant showed a dramatic increasein glucose sensitivity and (ii) deletion of the gxyR gene, whichresults in overexpression of GxySBA under inducing conditions,resulted in decreased sensitivity to the inducing sugar. Assumingthat GxySBA is unlikely to interact with VirA, these data are con-sistent with the hypothesis that the transport systems are main-taining a periplasmic concentration of the sugars below the levelrequired for vir induction, unless high external concentrations ofthat sugar are available. While other mechanisms could be in play,this regulatory interaction between transport activity and vir in-duction could be a selective advantage for the bacteria given thatvir induction at high concentrations of sugar might be indicativeof the host rather than the soil environment. While deletion ofgxyR resulted in strains with modestly decreased sensitivity to glu-cose in vir induction assays, this did not affect their virulence inthe tobacco leaf explant assay system (see Table S3 in the supple-mental material), consistent with the proposal that the leaves havevery high levels of inducing sugars.

In summary, the data presented here demonstrate thatGxySBA is an ABC transporter that has multiple monosaccharidesas substrates and is the crucial means of glucosamine uptake byAgrobacterium, and its expression is controlled by both the GxyRrepressor and, surprisingly, by glycerol. Finally, we have demon-strated that this transporter can affect the sensitivity of the bacte-rium to the vir-inducing sugars, most likely by affecting the con-centration of those sugars in the periplasm. This serves to ensurethat vir induction only occurs in environments, such as the plantwound site, that contain high levels of inducing sugars.

ACKNOWLEDGMENTS

We thank Arlene A. Wise for critically reading an earlier version of themanuscript. We also thank Matthew Verosloff for assistance with thegrowth assays and Cush El for assistance with the plant tumor assays.

This work was funded by National Science Foundation grantIOS11211019.

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