streptomyces scabies 87-22 possesses a functional tomatinase · pathogenesis and may be involved in...

JOURNAL OF BACTERIOLOGY, Dec. 2008, p. 7684–7692 Vol. 190, No. 230021-9193/08/$08.00�0 doi:10.1128/JB.01010-08Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Streptomyces scabies 87-22 Possesses a Functional Tomatinase�

Ryan F. Seipke and Rosemary Loria*Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, New York 14853

Received 22 July 2008/Accepted 22 September 2008

The actinomycete Streptomyces scabies 87-22 is the causal agent of common scab, an economically importantdisease of potato and taproot crops. Sequencing of the S. scabies 87-22 genome revealed the presence of a genewith high homology to the gene encoding the �-tomatine-detoxifying enzyme tomatinase found in fungal tomatopathogens. The tomA gene from S. scabies 87-22 was cotranscribed with a putative family 1 glycosyl hydrolasegene, and purified TomA protein was active only on �-tomatine and not potato glycoalkaloids or xylans.Tomatinase-null mutants were more sensitive to �-tomatine than the wild-type strain in a disk diffusion assay.Interestingly, tomatine affected only aerial mycelium and not vegetative mycelium, suggesting that the target(s)of �-tomatine is not present during vegetative growth. Severities of disease for tomato seedlings affected by S.scabies 87-22 wild-type and �tomA1 strains were indistinguishable, suggesting that tomatinase is not importantin pathogenicity on tomato plants. However, conservation of tomA on a pathogenicity island in S. acidiscabiesand S. turgidiscabies suggests a role in plant-microbe interaction.

Streptomycetes are mainly benign saprophytic soil bacteriathat produce nearly two-thirds of the world’s naturally occur-ring antibiotics (5). However, in the genus Streptomyces, thereare a few pathogens, some of which cause common scab ofpotatoes and other taproot crops (38). These pathogens aregeneral necrotic pathogens and aggressively colonize rootstructures and possess multiple virulence factors (36, 37). Pro-duction of the nonribosomally synthesized dipeptide phyto-toxin thaxtomin is required for pathogenicity (20, 33). Thaxto-min inhibits cellulose synthesis on actively growing plant tissue(49). The biosynthesis of thaxtomin has been characterized (19,20, 29), and transcription of the biosynthetic genes is activatedby the AraC family regulator TxtR, which binds cellobiose tofacilitate transcription (25). Scab-causing streptomycetes alsocontain a novel necrogenic protein, Nec1, which is important inpathogenesis and may be involved in the suppression of plantdefenses (10, 24). Recently, sequencing of a self-mobilizablepathogenicity island in S. turgidiscabies revealed that a putativetomatinase gene was conserved among three phylogeneticallydistinct scab-causing streptomycetes, S. turgidiscabies Car8, S.acidiscabies, and S. scabies (also known as S. scabiei) 87-22(28).

Genes encoding tomatinase enzymes are typically found infungal pathogens of tomato plants, such as Septoria lycopersiciand Fusarium oxysporum f. sp. lycopersici, which cause diseaseon tomato plants. Tomatinase belongs to a class of enzymescalled saponinases; such enzymes function as glycosyl hydro-lases that detoxify phytoanticipins, which are preformed anti-microbial molecules produced by plants (52). Tomatinase de-toxifies the steroidal tomato phytoanticipin �-tomatine byhydrolysis of one or more sugar residues (43) that are essentialfor �-tomatine activity (30). The toxicity of �-tomatine is typ-ically mediated through complexing with sterols in eukaryoticmembranes to ultimately cause membrane pores and cell lysis

(30, 31). In addition to nullification of an antimicrobial com-pound, tomatinases are able to indirectly suppress the inducedplant defense response; this presumably results from plantrecognition of by-products from �-tomatine hydrolysis (8, 22).Despite the obvious advantage for a tomato pathogen to havea tomatinase in its arsenal, tomatinase-null mutants are notimpaired in their ability to cause disease on tomato plants (42,46). However, a tomatinase enzyme in Septoria lycopersici wasshown to be important for infection of tobacco (8), and theavenacin-detoxifying enzyme from Gaeumannomyces graminisis required for pathogenicity in oats and contributes to hostrange (9).

Recently, a bacterial tomatinase gene in the tomato wiltpathogen Clavibacter michiganensis subsp. michiganensis wascharacterized; however, tomatinase mutants were not compro-mised in virulence (27). Interestingly, the closely related sub-species C. michiganensis subsp. sepedonicus, a potato pathogen,lacks a tomatinase gene (4). The presence of a saponinase inthe actinomycetes C. michiganensis subsp. michiganensis, S.scabies 87-22, S. acidiscabies, and S. turgidiscabies but not inother bacterial plant pathogens is curious. The location of thetomatinase gene on pathogenicity islands suggests a role inpathogenicity (17, 28). The objectives of this study were to (i)characterize the substrate specificity of the saponinase fromthe broad-host-range pathogen S. scabies 87-22, (ii) describestreptomycete growth in the presence of saponins and assesswhether the saponinase mitigated any negative effects, and (iii)explore the role of the saponinase in virulence of S. scabies87-22.

MATERIALS AND METHODS

Bacterial strains and culturing conditions. Escherichia coli strains were cul-tured as previously described (32, 47). Streptomyces strains were cultured at 28°Cusing International Streptomyces Project 4 agar media, mannitol-soya flour agar,or tryptic soy broth (TSB) medium. All liquid cultures were shaken at �250 rpm.Media were supplemented with antibiotics at the following concentrations: 100�g/ml apramycin, 50 �g/ml kanamycin, 100 �g/ml ampicillin, 50 �g/ml strepto-mycin, 25 �g/ml nalidixic acid, and 10 �g/ml thiostrepton. All Streptomycesstrains were generated by cross-genus conjugation from the nonmethylating E.

* Corresponding author. Mailing address: Cornell University, 334Plant Science Building, Ithaca, NY 14853. Phone: (607) 255-7831. Fax:(607) 255-4471. E-mail: [email protected].

� Published ahead of print on 3 October 2008.

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coli strain ET12567/pUZ8002 as previously described (32). Strains and plasmidsare described in Table 1.

Cloning and plasmid construction. Standard molecular biology procedureswere used for all DNA manipulations and plasmid construction in this study (32,47). Commercial enzymes were purchased from New England Biolabs (Beverly,MA). Unless otherwise noted, insert DNA for cloning was generated by PCRusing Phusion Taq polymerase. Integrated DNA Technologies (Coralville, IA)synthesized all oligonucleotides used in PCRs. PCR products were cloned intopCR2.1TOPO (Invitrogen) and sequenced (Biotechnology Resource Center,Cornell University). Vector and pCR2.1TOPO insert-containing plasmids weredigested with an appropriate restriction enzyme. Gel-purified insert and vectorfragments were ligated together using T4 DNA ligase and transformed into E.coli DH5� cells. Clones were verified by restriction digestion.

Overexpression and purification of TomA-His6. The tomA gene without itsputative secretion signal (15) from S. scabies 87-22 was PCR amplified andcloned into pET30a (Novagen) to produce pRFSRL13 and moved into the E.coli expression strain BL21(�DE3). An overnight culture of BL21(�DE3)/pRFSRL13 (5 ml) was used for inoculation of 500 ml of LB containing kanamycin.Cells were grown to an optical density at 600 nm (OD600) of �0.600, at whichpoint TomA-His6 protein production was induced with 1 mM IPTG (isopropyl-D-thiogalactopyranoside). After 3 h of induction, cells were harvested by centrif-ugation. The cell pellet was resuspended in 50 ml 10 mM Tris-Cl (pH 8.5) andmechanically disrupted by sonication (10-min duration, 20-s pulse, and 20-s reston ice). TomA-His6 was purified from lysate by Ni-nitrilotriacetic acid (Qiagen)affinity chromatography according to the manufacturer’s instructions with a 40mM imidazole wash step. Eluted fractions were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). TomA-His6 was puri-fied to �90% purity and quantified using the Bradford method with bovineserum albumin as a standard. Prior to activity assays, a buffer exchange usingMicrocon YM-30 columns (Millipore) was performed with 10 mM Tris-Cl (pH8.5) to ensure that protein function would not be inhibited by the high concen-tration of imidazole present in the elution buffer.

TLC analysis. TomA-His6 reaction products were examined using the methodused in reference 46 with the following modifications. TomA-His6 protein (6 �g)or crude lysate from BL21(�DE3)/pET30a (100 �g) was incubated with 1 mM�-tomatine and incubated at 42°C for 2 h. Following NH4OH precipitation ofreaction products, pellets were dissolved in 15 �l of 100% methanol and 10 �l ofthin-layer chromatography (TLC) solvent and spotted onto a silica 60 TLC plate(Whatman). Metabolites were identified by cochromatography of standards. Thechemicals �-tomatine and tomatidine were purchased from LTD chemicals andMP Biomedicals, respectively.

Enzyme activity assays. Enzyme activity was measured with PAHBAH (p-hydroxybenzoic acid hydrazide) to determine the amount of reducing sugarpresent (35). Reaction mixtures containing 0.5 mM to 4.0 mM �-tomatine, 1 mM�-solanine or �-chaconine, or 0.5% xylans from birchwood or oat spelt (Sigma)were prepared essentially as described for the TLC analysis; however, 5 �g ofTomA-His6 was used instead of 6 �g. One-hundred micrograms of crude lysatefrom BL21(�DE3)/pET30a served as a negative control. Reactions were stoppedby the addition of 800 �l of PAHBAH reagent and processed as describedpreviously (35). The kinetic constant (Km) was estimated by the Lineweaver-Burkmethod, using glucose as a standard.

Generation of mutant strains. S. scabies �tomA strains were created using thePCR-targeting Redirect technology (18). A disruption cassette consisting of anoriT and the aac(3)IV apramycin resistance gene from pIJ773 was generated byPCR amplification with primers that contained 39 nucleotides (nt) of homologythat included the start or stop codons of tomA and 36 nt downstream or upstreamof the open reading frame. The resulting PCR product was gel purified andelectroporated into E. coli BW25113 containing the � Red gene-carrying plas-mid, pKD46 and tomA bacterial artificial chromosome (BAC) 14h02. Transfor-mants were screened for the presence of mutagenized BAC by colony PCR.Because of antibiotic incompatibilities in downstream procedures, the chloram-phenicol resistance gene present on the BAC backbone was replaced with thestreptomycin resistance gene aadA from pIJ779 by using � Red recombination ina manner similar to that described above. Mutagenized BAC DNA was trans-

TABLE 1. Bacterial strains and plasmids used in this study

Strain or plasmid Genotype or commentsa Source orreference

S. scabies strains87-22 Wild type This study�tomA1 87-22 tomA-null mutant (isolate 1) This study�tomA2 87-22 tomA-null mutant (isolate 2) This study�tomA1 attB �C31::pAU34-5 tomA1-null mutant bearing empty complementation vector This study�tomA1 attB �C31::pRFSRL20 Complemented tomA1-null mutant This study

E. coli strainsBW25113 Host for Redirect PCR targeting system 18ET12567 Nonmethylating host for transfer of DNA into Streptomyces spp. (dam, dcm, and

hsdS)41

DH5� F general cloning host Gibco-BRLTOP10 Host for pCR2.1TOPO cloning system Invitrogen

Plasmids This studypBACher BAC backbone for S. scabies 87-22 BACs; Camr Invitrogen14h01 pBACher derivative containing tomA locus; Camr This studypCR2.1TOPO Cloning vector for PCR products; Kanr Ampr InvitrogenpET30a Protein expression vector; IPTG-inducible T7 promoter system; Kanr NovagenpIJ773 PCR template for aac3(IV) plus oriT cassette used in Redirect PCR targeting system 18pIJ779 PCR template for aadA cassette used in Redirect PCR targeting system 18pUZ8002 Encodes conjugation machinery for mobilization of plasmids from E. coli to

Streptomyces; Kanr41

pKD46 Encodes lambda Red recombination machinery induced by arabinose; Ampr 12pAU34-5 pSET152 derivative, integrates into the �C31 attB site in Streptomyces; Aprr Tspr 7pHZ1272 PCR template for pIJ101 origin of replication used to construct pRFSRL36 Zixin DengpRFSRL13 pET30a derivative with �1.6-kb tomA gene cloned into NdeI-HindIII sites; Kanr This studypRFSRL20 pAU3-45 derivative with a �3.4-kb fragment containing the tomA locus cloned into

the XbaI siteThis study

pRFSRL36 pRFSRL20 derivative with a �2.2-kb fragment containing the pIJ101 origin ofreplication cloned into the SphI-HindIII sites

This study

a Amp, ampicillin; Apr, apramycin; Cam, chloramphenicol; Kan, kanamycin; Tsp, thiostrepton.

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ferred to S. scabies via conjugation. Transconjugants were selected for apramycinresistance and streptomycin sensitivity. Deletion of tomA was confirmed bycolony PCR and Southern blot hybridization.

Complementation of the tomA1-null mutant. To complement the �tomA1mutant, a 3.4-kb fragment containing the tomA gene and �1,800 bp of upstreamDNA was PCR amplified and cloned into pAU34-5 at the XbaI site. The result-ing plasmid, pRFSRL20, was moved to the �tomA1 strain by conjugation.Transconjugants were selected for thiostrepton resistance.

�-Tomatine bioassay. A disk diffusion assay with �-tomatine was used toassess saponin sensitivity. Mannitol-soya flour agar plates (pH 6.8) were seededwith a lawn of bacteria by streaking 100 �l of spores. Sterilized paper disks witha diameter of 6 mm were securely placed on top of the agar. �-Tomatine (0.3�mol) dissolved in 50 mM Na citrate (pH 4.0) was applied to the center of threedisks. Sodium citrate buffer without �-tomatine was used as a negative control.Plates were incubated at 28°C for approximately 3 days before measuring zonesof inhibition. A zone of inhibition was defined as the diameter over which aerialmycelium was absent.

To assess �-tomatine sensitivity in liquid culture, we generated a growth curvein the presence or absence of �-tomatine. Five-hundred microliters of overnightculture of wild-type or �tom1A S. scabies was used for inoculation of 25 ml ofTSB (pH 7.0) in a 250-ml flask. Cultures were supplemented with �-tomatine toa final concentration of 0.1 mM or an equivalent volume of Na citrate bufferduring early exponential growth phase. One milliliter of culture was harvestedevery hour, and a spectrophotometer was used to read the absorbance at 600 nmof three biological replicates for each treatment.

Southern blot hybridization. Genomic DNA was isolated from overnight TSB-grown cultures by using the MasterPure kit for gram-positive bacteria (Epicen-tre) according to the manufacturer’s instructions. Five micrograms of genomicDNA was digested with KpnI and run on a 0.8% agarose gel. The DNA wasdenatured with 0.5 M NaOH and 1.5 M NaCl and transferred to a nylonmembrane (Whatman) by capillary transfer with 20 SSC (1 SSC is 0.15 MNaCl plus 0.015 M sodium citrate). DNA was UV cross-linked to the membraneby applying 120,000 �J/cm2 for 4 min. The membrane was probed with a gene-specific probe for tomA or aac(3)IV that was labeled with dioxigenin-11-dUTP(Roche). Hybridization was performed in a rotary oven at 42°C. Stripping con-ditions were as follows: 2 SSC at room temperature for 10 min and 0.1 SSCat room temperature for 15 min. Processing of the membrane from this pointforward was performed according to the manufacturer’s instructions (Roche).

Cotranscription and 5� transcript mapping. For cotranscription and transcriptmapping experiments, wild-type S. scabies was grown overnight in TSB liquidmedium. RNA was extracted as previous described (25). Five micrograms ofDNase-treated RNA was reverse transcribed using the Superscript III first-strand synthesis system and 250 ng of random hexamer primers (Invitrogen).Control reactions (non-RT) in which enzyme was omitted were also performed.Cotranscription analysis was performed by PCR using cDNA as the template andfour primers (P1 [CACCACCTCAACCTTTC], P2 [CGGTCAAGAGCCCTATGG], P3 [CAGCCCATCGACCTGCT], and P4 [TGCGTCTGCTGATCCA]).PCR products were then gel purified and sequenced to confirm their identities.

To map the transcription start site, we used a 5� rapid amplification of cDNAends (5� RACE) system (version 2.0; Invitrogen). Due to the apparent lowabundance of SCAB77311-tomA transcript present in TSB-grown cultures, wecloned the multicopy origin of replication (pIJ101) from pHZ1272 (32) intopRFSRL20 to increase the number of SCAB77311-tomA transcripts and tofacilitate mapping of the transcriptional start site. This plasmid pRFSRL36 wasintroduced into S. scabies by conjugation, and RNA was isolated from theresulting strain and was processed exactly as described by the manufacturer for5� RACE of high-G�C-content DNA. Sequences of gene-specific primers wereas follows: for GSP1, CTCCGCGTACTTCTCGAA; for GSP2, GGTGGAGCGAGGCCATCAG; and for GSP3, TGAACGTGTCCCAGAT. The final PCRproduct was gel purified, cloned into pCR2.1TOPO, and sequenced using M13fand M13r primers (Invitrogen). The transcriptional start site was determined tobe the nucleotide immediately adjacent to the string of poly(A)s that resultedfrom deoxyribosyladenine tailing of cDNA.

Quantitative RT-PCR. S. scabies wild-type and �tomA1 strains were grownovernight in TSB liquid medium, and then 200 �l of each was subcultured into10 ml of fresh TSB medium. Cultures were grown to an OD600 of �0.700, atwhich point either 0.1 mM �-tomatine or an equivalent amount of empty buffer(50 mM Na citrate, pH 4.0) was added. Culture samples were harvested in20-min intervals until 80 min was reached. RNA was extracted and quantitativereverse transcription-PCR (RT-PCR) was performed exactly as previously de-scribed (25). Induction was defined as a �2-fold change in transcript level.

In planta bioassays. Excised potato tuber (cv. Russet Burbank) assays wereperformed as described previously (39). Virulence assays with tomato (cv. Pto)

were performed essentially as described previously (25), in Magenta boxes onMurahige and Skoog agar medium with 2% sucrose. Overnight cultures of S.scabies wild-type and �tomA1 strains were grown in TSB liquid medium. Onemilliliter of culture was pelleted in a microcentrifuge, washed with 1 ml of sterilewater, and resuspended in 1 ml of water. The resuspended cells were then usedfor inoculation of an entire Magenta box (containing �4 plants) by swirling theliquid across the agar surface. Inoculations were performed �2 weeks after seedgermination, and infection was allowed to proceed for �4 weeks. Infection ofhydroponically grown radish seedlings was performed with 10 104 spores asdescribed previously (24). Plants were inoculated 5 days after germination andinspected for disease 3 weeks after inoculation.

RESULTS

The deduced amino acid sequence of the tomA gene productis homologous with known �-tomatine-detoxifying enzymes.The tomA gene product encodes a 545-amino-acid (aa) protein(55 kDa) that contains a predicted secretion signal at its Nterminus (aa 1 to 27) (15). The TomA protein has two do-mains, an N-terminal catalytic domain (aa 28 to 356) thatbelongs to glycosyl hydrolase family 10 and a C-terminal do-main (aa 357 to 507) with weak similarity to the E-set super-family that has been found in other bacterial hydrolases butdoes not have an ascribed function.

A CLUSTAL W alignment of the predicted amino acidsequence from the S. scabies TomA protein and from closelyrelated proteins showed that TomA is homologous to known�-tomatine-detoxifying enzymes as well as bacterial xylanases(Fig. 1). It is most closely related to the well-characterizedtomatinase from the tomato fungal pathogen F. oxysporum f.sp. lycopersici (60% identical) (46) and to the tomatinase fromthe bacterial wilt pathogen C. michiganensis subsp. michiganen-sis (58% identical) (27). The TomA protein also contains Gluresidues at positions 158 and 266, which align with the acid/base catalysis and nucleophile residues, respectively, from thefamily 10 glycosyl hydrolase Cex of Cellulomonas fimi (40).TomA protein catalyzes hydrolysis of an O-glycosyl compound,a unique reaction among family 10 glycosyl hydrolases, whichcharacteristically possess either xylanase (EC 3.2.1.8) or endo-1,3-�-xylanase (EC 3.2.1.32) activity (21). The range of cata-lytic activity observed in glycosyl hydrolase family 10 is inagreement with the low conservation among substrate bindingsites in this family (11).

Heterologous overexpression and purification of TomA in E.coli. Our initial attempts to purify the TomA protein weremade in its natural producer, S. scabies, using pRFSRL12 withtomA transcription driven by ermEp* (6). Despite the use ofthis strong promoter, we were able to obtain only a smallamount of purified protein from culture supernatants. Wetherefore selected an E. coli expression system for proteinexpression. A polyhistidine tag (His6) was attached to the Cterminus of the TomA protein that lacked the secretion signal.TomA-His6 was purified from E. coli extracts by Ni-nitrilotri-acetic acid affinity chromatography. With this purification tech-nique, we were able to achieve �90% purification of TomA-His6 as analyzed by 12% SDS-PAGE (Fig. 2A).

TLC of reactions containing TomA-His6 and �-tomatineshowed that the recombinant enzyme was able to catalyze theconversion of �-tomatine to tomatidine, releasing lycotetraose,whereas vector control lysate could not (Fig. 2B and C). Thetomatinase enzymes from F. oxysporum f. sp. lycopersici and C.michiganensis subsp. michiganensis also catalyze this reaction

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(16, 27, 34); however, the tomatinase from Septoria lycopersicihydrolysizes only the terminal �-1,2-linked D-glucose (1, 14).Since TomA-His6 catalyzes the release of the sugar moietyfrom tomatine, we were able to use a reducing-sugar assay inorder to measure enzyme activity (35). We determined a Km of650 �M for TomA-His6, which is comparable to the Km of 1.1mM reported for TomA of F. oxysporum f. sp. lycopersici (34),considering the error associated with the Lineweaver-Burk pa-rameter estimation technique (13). Since S. scabies is a generalnecrotic pathogen with a broad host range, we also analyzedwhether TomA-His6 had activity against the potato glycoalka-loids �-chaconine and �-solanine. Incubation of TomA-His6

with these substrates did not yield significant activity (data notshown), suggesting that the tomatinase from S. scabies may notbe important in pathogenesis in potatoes. In addition, becauseTomA displayed very high homologies to xylanases, we assayedTomA-His6 for xylanase activity on xylans from oats or birch-wood. Significant xylanase activity was not detected with eitherxylan (data not shown).

Analysis of the genomic region surrounding tomA in S. sca-

bies revealed the presence of a gene, SCAB77311, encoding aputative family 1 glycosyl hydrolase. This gene is located up-stream of tomA and is predicted to be transcribed in the samedirection as tomA, which led us to investigate whether the twogenes are cotranscribed in an operon. We isolated RNA fromlog-phase cells grown in TSB and performed RT-PCR with twopairs of oligonucleotide primers that spanned the intergenicregion (Fig. 3A). PCR products were obtained for both sets ofprimers and were sequenced to verify their identities (Fig. 3B);NRT controls confirmed that the PCR products were gener-ated from cDNA and not from genomic DNA, which cancontaminate RNA preparations. These data suggest that tomAand SCAB77311 are cotranscribed, despite the relatively large(164-nt) intergenic region.

Since tomatinase gene expression is induced by �-tomatinein F. oxysporum f. sp. lycopersici and Septoria lycopersici, weanalyzed whether or not the same was true for S. scabies. Weperformed quantitative RT-PCR of RNA isolated from expo-nentially growing cells in TSB liquid medium. Cells were sup-plemented with �-tomatine and transcript abundance was as-

FIG. 1. Comparison of the amino acids of the predicted catalytic domain from the S. scabies 87-22 TomA protein with closely related proteins.Ssc, tomatinase from S. scabies 87-22 (coordinates 8542762 to 8544363 at http://www.sanger.ac.uk/Projects/S_scabies/); Cmm, tomatinase from C.michiganensis subsp. michiganensis (GenBank accession no. AAP57293); Fol, tomatinase from F. oxysporum f. sp. lycopersici (CAA10112); Sco,xylanase from S. coelicolor A(3)2 (CAB61191); Tfu, xylanase from Thermobifida fusca YX (YP_290847); Tma, xylanase from Thermotoga maritima(AAP97078); Kra, xylanase from Kineococcus radiotolerans (EAM76898); Cfi, xylanase from Cellulomonas fimi (Q59277). Residues identical forall proteins are boxed in black, and residues identical in four or more proteins are boxed in gray. Asterisks mark catalytic residues Glu 158 andGlu 266.



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sessed in 20-min intervals until 80 min after supplementation.The addition of �-tomatine did not increase transcript levels ofthe SCAB77311-tomA locus (data not shown), suggesting thattranscription of tomA is not induced by its substrate.

Mapping of the tomA transcriptional start site by 5� RACE.Our initial attempts at mapping the SCAB77311-tomA tran-scriptional start site failed because of the presence of several

nonspecific PCR products, presumably resulting from low tran-script abundance for the SCAB77311-tomA locus. Indeed, ex-pression analysis using quantitative RT-PCR suggested thatthe tomA gene is transcribed from a weak promoter (data notshown). In order to facilitate mapping of transcriptional startsite by 5� RACE, we cloned the high-copy-number origin ofreplication from pIJ101 into pRFSRL20, and the resulting

FIG. 2. Purification of S. scabies 87-22 TomA-His6 protein and TLC analysis of �-tomatine reaction products. (A) Protein extracts wereanalyzed by 12% SDS-PAGE and were stained with SimplyBlue SafeStain. Lanes: 1, total soluble protein from pET30a vector control; 2, totalsoluble protein from induced TomA-His6 cultures; 3, TomA-His6 protein purified by nickel affinity chromatography. (B) TLC of TomA-His6reaction products. Lanes: 1, TomA-His6 (6 �g) plus �-tomatine (0.2 �mol); 2, vector control lysate (100 �g) plus �-tomatine (0.2 �mol); 3,tomatidine standard (0.2 �mol); 4, �-tomatine standard (0.2 �mol). (C) Structure of �-tomatine from tomato. The arrow indicates the point ofhydrolysis of lycotetraose by tomatinase enzymes from S. scabies 87-22, C. michiganensis subsp. michiganensis, and F. oxysporum f. sp. lycopersici.(Adapted from reference 46.)



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plasmid, pRFSRL36, was conjugated into wild-type S. scabies.RNA was isolated from log-phase cells grown in TSB and wassubjected to 5� RACE analysis. Using this technique, we wereable to successfully map the transcriptional start site for theSCAB77311-tomA operon (Fig. 4). Transcription is initiatedfrom a cytosine nucleotide located 23 bp upstream of theputative ATG start codon for SCAB77311. Sequence analysisidentified 10 and 35 promoter regions that were identicalto Streptomyces promoters that do not display typical charac-teristics of promoters recognized by E. coli 70-like sigma fac-tors (51). The 10 region was identical to the 10 region ofthe hydroxystreptomycin phosphotransferase gene, sphP, fromS. glaucescens (51, 53) and the 35 region was identical to thatof the bialaphos resistance gene, bar, from S. coelicolor A3(2)(2, 51). The distance between 10 and 35 elements is 20 nt,longer than the typical 17- or 18-nt spacer of streptomycetepromoters.

�-Tomatine inhibits aerial but not vegetative growth of S.scabies. A tomatinase-null mutant strain was created using the� Red-based Redirect system for gene deletions in Streptomy-ces spp. (18). Two mutant strains (�tomA1 and �tomA2) wereisolated and subsequently verified by Southern blot hybridiza-tion (Fig. 5). S. scabies wild-type and �tomA strains were an-alyzed for sensitivity to �-tomatine in a disk diffusion assay.The �tomA1 and �tomA2 mutant strains behaved similarlyin this assay; therefore we present data for only the �tomA1strain.

The growths of S. scabies wild-type and �tomA1 strains wereunaffected by the addition of buffer alone. However, the addi-tion of 0.3 �mol of �-tomatine inhibited the formation of thefluffy white aerial mycelium that is characteristic of streptomy-cetes (Fig. 6A and B). The size of the zone of inhibitionincreased in a dose-dependent manner (data not shown); how-ever, for experimental ease, we chose to work with 0.3 �mol of�-tomatine only, which is within the physiologically relevantrange for the level of �-tomatine that is produced by tomatoplants (3). Inhibition of aerial mycelium formation was moresevere in the �tomA1 strain, with nearly a 50% increase in thediameter of inhibited growth (Table 2), suggesting that TomAdetoxifies �-tomatine in vivo. To ensure that the observedphenotype was due solely to the deletion of tomA, we cloned a�3.4-kb fragment containing the tomA gene into the integra-tive vector pAU3-45 (7) and moved it to the �tomA1 strain byconjugation. As expected, the complemented strain returnedto wild-type levels of inhibited aerial growth, whereas the pres-ence of the vector alone had no effect (Fig. 6C and D andTable 2).

Next, we analyzed vegetatively growing cells of S. scabieswild-type and �tomA1 strains in liquid TSB medium for inhi-bition by �-tomatine. Cultures were grown to an OD600 of�0.200, and then either buffer alone or �-tomatine (final con-

FIG. 3. The tomA gene is cotranscribed with SCAB77311, a puta-tive family 1 glycosyl hydrolase. (A) Schematic illustrating the S. sca-bies 87-22 SCAB77311-tomA locus. P1, P2, P3, and P4 refer to oligo-nucleotide primers used to demonstrate cotranscription. (B) Results ofRT-PCR with oligonucleotide primers P1/P2 and P3/P4. Lanes: 1,RT�; 2, non-RT control; 3, RT�; 4, non-RT control. PCR productswere sequenced to confirm their identities.

FIG. 4. Mapping of the SCAB77311-tomA transcriptional start siteby 5� RACE. (A) A 2% agarose gel showing the PCR product obtainedby 5� RACE. (B) Nucleotide schematic showing the transcriptionalstart site determined by sequencing of the PCR product shown in panelA. A double underline indicates the 35 sequence, and a single un-derline indicates the 10 sequence; the bold nucleotide with an arrowindicates the initiating nucleotide and direction of transcription, andthe bold “atg” indicates the putative start codon of the SCAB77311gene.

FIG. 5. Deletion of the S. scabies 87-22 tomA gene. (A) Southernblot hybridization using a PCR-generated dioxigenin-11-dUTP-labeledfragment internal to tomA. The positions of the DNA marker areindicated at the left in kb. Lanes: 1, S. scabies wild type; 2, �tomA1mutant; 3, �tomA2 mutant. (B) The membrane from panel A wasstripped and probed with a dioxigenin-11-dUTP-labeled internal frag-ment of the apramycin resistance gene aac(3)IV.



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centration, 0.1 mM) was added. The addition of 0.1 mM �-to-matine is equivalent to the addition of 0.3 �mol of �-tomatinein a disk diffusion assay. Samples were harvested at 1-h inter-vals and were analyzed spectrophotometrically. There was noobservable change in growth of S. scabies wild-type or �tomA1strains after the addition of �-tomatine compared to that ofcells treated with buffer alone (Fig. 7).

tomA is not required for virulence on excised potato tissueor tomato seedlings. To analyze the importance of TomA inplant pathogenicity, we first inoculated excised potato tuberdisks with plugs of agar containing mycelia of wild-type or�tomA1 S. scabies. Potato tuber slices were heavily necrotizedby both wild-type and �tomA1 strains 7 days postinoculation,suggesting that TomA is not important in pathogenicity onpotatoes.

Next, we infected tomato seedlings in vitro. Plants weregrown in Magenta boxes on Murahige and Skoog agar con-taining 2% sucrose. Seedlings were inoculated 1 week aftergermination with mycelia grown overnight in TSB. Necrosis onprimary and lateral roots was apparent on S. scabies wild-type

strain- and �tomA1 strain-inoculated plants as early as 1 weekpostinoculation; roots of mock-inoculated seedlings were notnecrotic. Four weeks postinoculation, roots of wild-type strain-and �tomA1 strain-inoculated plants were severely stunted,and lateral root tips were necrotized and swollen compared tothose of control plants (data not shown). These data suggestthat the tomatinase protein in S. scabies is not important forpathogenicity on tomato plants. We also inoculated hydropon-ically grown radish (cv. Burpee White) seedlings and did notobserve a difference in pathogenicity for S. scabies wild-typeand �tomA1 strains.

DISCUSSION

We have shown that the saponin-detoxifying enzyme presentin S. scabies is a tomatinase that hydrolyzes �-tomatine, releas-ing lycotetraose. We determined a Km of 650 �M for thisenzyme, which is comparable to the Km of 1.1 mM reported forthe tomatinase enzyme from F. oxysporum f. sp. lycopersici (34)when the error associated with the Lineweaver-Burk parame-ter estimation technique is considered (13). Our substrate uti-lization analysis showed that TomA from S. scabies does nothave xylanase activity, nor is it active on �-chaconine or �-so-lanine. Taken together, these data suggest that the tomatinasefrom S. scabies has a high substrate specificity, which is inagreement with the case for tomatinases from other phyto-pathogens (34, 42).

Our genetic analysis showed that the tomA gene is cotrans-cribed with SCAB77311, which encodes a putative family 1glycosyl hydrolase. The closest homolog of this protein is BlgA(73% identity) from C. michiganensis subsp. michiganensis; theblgA gene is located just downstream of the tomA homologuein this organism (27). InteroPro scanning of the deducedamino acid sequence for the SCAB77311 protein showed highhomology with family 1 glycosyl hydrolase proteins (InterProidentification no. IPR001360). These proteins hydrolyze theglycosidic bond between two carbohydrates or between a car-bohydrate and a noncarbohydrate moiety. The presence of afamily 1 glycosyl hydrolase near the tomA gene in both S.

FIG. 6. TomA mitigates the effect of �-tomatine on aerial growthof S. scabies 87-22. (A) S. scabies wild type. (B) �tomA1 mutant.(C) �tomA1 attB �C31::pAU34-5. (D) �tomA1 attB �C31::pRF-SRL20. One spot of 50 mM Na citrate control buffer (upper leftcorner) and three replicate spots of 0.3 �mol of �-tomatine werespotted on each plate.

TABLE 2. Inhibition of aerial growth of S. scabies by �-tomatinea

S. scabies strainDiam of

inhibition(mm)b

Inhibitionincrease over

wild-typevalue (%)c

Wild-type 87-22 11.7 � 1.2�tomA1 17.2 � 0.3 47.0�tomA1 attB �C31::pAU34-5 21.0 � 1.0 80.0�tomA1 attB �C31::pRFSRL20 11.3 � 0.6 3.4

a A total of 0.3 �mol �-tomatine was used in this assay.b Zone of inhibition � standard deviation for three replicates.c Increase of inhibition is expressed as a percentage of the wild-type value.

FIG. 7. The growth of S. scabies 87-22 vegetative mycelium is notaffected by �-tomatine. Vegetatively growing cells were supplementedat �3.5 h (arrow) with 0.1 mM �-tomatine or 50 mM Na citrate bufferand analyzed spectrophotometrically. Diamonds, wild type plus Nacitrate; squares, wild type plus 0.1 mM �-tomatine; triangles, �tomA1plus Na citrate; asterisks, �tomA1 plus 0.1 mM �-tomatine.



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scabies and C. michiganensis subsp. michiganensis suggests thatthe SCAB77311 protein and BlgA might perform the samefunction and might possibly be involved in hydrolyzing thelycotetraose released during �-tomatine hydrolysis. It is attrac-tive to speculate that the family 1 glycosyl hydrolase encodedby SCAB77311 acts either to hydrolyze lycotetraose into fourmonosaccharides or two disaccharides, the latter of whichwould require another protein for hydrolysis to four monosac-charides before entry into glycolysis.

Mapping the transcriptional start site by 5� RACE identifieda promoter sequence that does not display characteristics typ-ical of promoters recognized by E. coli 70-like sigma factorsbut is identical to streptomycete promoters involved in antibi-otic resistance (2, 51, 53). Our quantitative RT-PCR experi-ments analyzing �-tomatine-induced transcription of tomAsuggest that this gene is not induced by the presence of itssubstrate. This contrasts with tomatinase transcription in Sep-toria lycopersici and F. oxysporum f. sp. lycopersici, as well as C.michiganensis subsp. michiganensis, in which tomA gene ex-pression is induced by the presence of �-tomatine (27, 46, 48).The mechanism by which �-tomatine induces transcription inunknown.

Our �-tomatine bioassays showed that S. scabies is sensitiveto �-tomatine but that only aerial growth was inhibited, whilevegetative mycelium was unaffected. This was an unexpectedresult, because in both Septoria lycopersici and F. oxysporum f.sp. lycopersici as well as in C. michiganensis subsp. michiganen-sis, total inhibition of growth was observed upon treatmentwith �-tomatine (27, 46, 48). It is curious why S. scabies and C.michiganensis subsp. michiganensis are sensitive to �-tomatinein the first place, because �-tomatine complexes with sterols toform membrane pores (30, 31) and analysis of the genomic se-quences of both of these organisms did not reveal the presence ofa sterol biosynthetic pathway (http://www.sanger.ac.uk/Projects/S_scabies/ and http://gib.genes.nig.ac.jp/single/index.php?spid�Cmic_NCPPB382). However, growth is clearly adversely affectedin both S. scabies and C. michiganensis subsp. michiganensis, so�-tomatine must interact with a target that is critical for nor-mal growth by these bacteria. Interestingly, analysis of the S.scabies genome sequence revealed the presence of a putativehopanoid biosynthetic gene cluster. Hopanoids are function-ally analogous to sterols and are structurally similar (26) andcould potentially serve as a target for �-tomatine in S. scabies.However, analysis of the C. michiganensis subsp. michiganensisgenome sequence did not reveal an obvious hopanoid biosyn-thetic gene cluster. It is interesting that in F. oxysporum f. sp.lycopersici, the observed sensitivity to �-tomatine is a result of�-tomatine-induced programmed cell death in the fungusrather than an effect on membranes (23). Furthermore, the�-tomatine aglycone tomatidine inhibits sterol biosynthesis inSaccharomyces cerevisiae (50), indicating that the biologicalproperties of saponins and their degradation products arecomplex.

Uncompromised virulence of the �tomA1 mutant in a po-tato tuber disk assay is consistent with our in vitro data showingthat TomA from S. scabies does not act on the importantglycoalkaloids in potatoes. Infection of tomato plants with to-matinase-null mutants from C. michiganensis subsp. michi-ganensis, F. oxysporum f. sp. lycopersici, and Septoria lycopersicidid not yield observable differences in virulence (27, 42, 46).

However, recent studies claim that the F. oxysporum f. sp.lycopersici tomatinase is important during the early stages oftomato infection (44), and curiously, tomatinase was a re-quired virulence factor in Septoria lycopersici for infection ofNicotiana benthamiana, a species that presumably does notproduce �-tomatine (8). Since tomatinase is not required forfull virulence of C. michiganensis subsp. michiganensis, F. oxy-sporum f. sp. lycopersici, and Septoria lycopersici on tomatoplants, it is not surprising that we did not observe a decrease invirulence on potato tuber tissue or tomato plants inoculatedwith the S. scabies �tomA1 mutant. The inoculation of rootsrather than leaves is consistent with the manner in which S.scabies infects plants in the field. However, since tomato rootslikely contain lower levels of �-tomatine than leaves (43, 45), itis possible that the inoculation of leaves could have revealed avirulence phenotype for �tomA1. It may be that the TomAprotein facilitates reproduction on �-tomatine-containing tis-sue, since �-tomatine inhibits aerial growth and thereby sporu-lation of S. scabies. Data presented here do not rule out thepossibility that tomatinase has a role in suppression of inducedplant defenses in some hosts, just as it does in F. oxysporum f.sp. lycopersici and Septoria lycopersici (8, 22). Expression anal-ysis of genes involved in induced defense responses of wild-type strain- and �tomA1 strain-inoculated plants will evaluatethis hypothesis.

ACKNOWLEDGMENTS

We thank D. R. D. Bignell for performing quantitative RT-PCRexperiments and for providing suggestions to improve the manuscript.

This project was supported by the National Research Initiative ofthe United States Department of Agriculture (USDA) CooperativeState Research, Education, and Extension Service, grant no. 2005-35319-15289. Financial support was also provided to R.F.S. throughthe USDA Multidisciplinary Graduate Education Traineeship Pro-gram.

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