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JOURNAL OF BACTERIOLOGY, Sept. 2007, p. 64156424 0021-9193/07/$08.00 0 doi:10.1128/JB.00398-07 Copyright 2007, American Society for Microbiology.

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Vol. 189, No. 17

The Plant Pathogen Ralstonia solanacearum Needs Aerotaxis for Normal Biolm Formation and Interactions with Its Tomato Host
Jian Yao and Caitilyn Allen*
Department of Plant Pathology, University of WisconsinMadison, Madison, Wisconsin 53706
Received 16 March 2007/Accepted 20 June 2007

Ralstonia solanacearum is a soilborne pathogen that causes bacterial wilt of diverse plant species. To locate and infect host plant roots R. solanacearum needs taxis, the ability to move toward more favorable conditions. However, the specic signals that attract this pathogen were unknown. One candidate is aerotaxis, or energy taxis, which guides bacteria toward optimal intracellular energy levels. The R. solanacearum genome encodes two putative aerotaxis transducers. Cloned R. solanacearum aer1 and aer2 genes restored aerotaxis to an Escherichia coli aer mutant, demonstrating that both genes encode heterologously functional aerotaxis transducers. Site-directed mutants lacking aer1, aer2, or both aer1 and aer2 were signicantly less able to move up an oxygen gradient than the wild-type parent strain; in fact, the aerotaxis of the aer mutants was indistinguishable from that of a completely nonmotile strain. Tomato plants inoculated with either the aer2 or the aer1/aer2 mutant had slightly delayed wilt disease development. Furthermore, the aer1/aer2 double mutant was signicantly impaired in the ability to rapidly localize on tomato roots compared to its wild-type parent. Unexpectedly, all nonaerotactic mutants formed thicker biolms on abiotic surfaces than the wild type. These results indicate that energy taxis contributes signicantly to the ability of R. solanacearum to locate and effectively interact with its host plants. R. solanacearum is a soilborne rod-shaped gram-negative -proteobacterium that causes bacterial wilt disease in many important plants such as potato, tobacco, tomato, banana, and peanut (30). Due to its wide geographic distribution and unusually broad host range (over 50 plant families) the pathogen is responsible for severe crop losses worldwide. R. solanacearum survives long term in water, soil, and latently infected plants and can be transmitted by soil, water, equipment, and infected plant materials (30). The pathogen invades plant roots through wounds or natural openings such as lateral root emergence points. Once the bacteria enter a susceptible host, they colonize the intercellular spaces of the root cortex and vascular parenchyma, eventually entering the xylem vessels and spreading into the upper parts of the plant (59). R. solanacearum virulence is additive and complex. Many factors that contribute to bacterial wilt disease have been identied; these include extracellular polysaccharide, several plant cell wall-degrading enzymes, and some type III-secreted effectors (6, 20, 26, 32, 39). Bacterial motility, including both agellum-driven swimming and pilus-driven twitching, is necessary for full virulence (33, 56). All of these virulence factors are controlled by an elaborate regulatory system that allows the bacterium to adjust to life in different niches. The core of this regulatory network is the Phc (phenotype conversion) system (51), which specically responds to a quorum-sensing molecule, 3-hydroxypalmitic acid methyl ester (15). Many bacteria use a form of motility called taxis to sense specic environmental stimuli and move toward favorable conditions (7, 12), and taxis is directly involved in several plantbacterium interactions (13, 21, 22, 28, 29, 62). The molecular mechanisms underlying taxis have been intensively studied in Escherichia coli (55, 61), and these are largely conserved in eubacteria. Briey, cell membrane-bound receptors (also called methyl-accepting chemotaxis proteins [MCPs] or transducers) sense environmental stimuli and respond with conformational changes. These result in autophosphorylation of a cytoplasmic histidine autokinase, CheA, which associates with MCPs through a coupling protein, CheW. CheA then donates its phosphate group to CheY, a diffusible cytoplasmic response regulator. Phosphorylated CheY alters agellar rotation by binding to the agellar motor, resulting in net movement toward favorable conditions and away from deleterious ones (55, 61). We previously found that R. solanacearum strain K60 is actively attracted to root exudates from the host plant tomato and that this pathogen depends on taxis to locate and colonize plant roots (62). Nontactic cheA and cheW mutants of R. solanacearum have reduced virulence, and they compete poorly with their wild-type parents during host colonization (62). However, since these mutants lack all forms of taxis, we could not discern the role of any particular type of taxis, such as chemotactic response to specic compounds or aerotaxis. Aerotaxis, more accurately known as energy taxis, is a behavioral response that guides bacterial cells toward a location where they achieve an optimal intracellular energy level, as sensed through the electron transport chain (58). This is usually, but not always, optimal oxygen concentration (58). Energy taxis is widespread in motile bacteria and plays a critical ecological role in many bacterial life cycles (1, 58). The molecular mechanisms of bacterial aerotaxis are well understood in E. coli, where two membrane-associated receptor proteins, Aer
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* Corresponding author. Mailing address: Department of Plant Pathology, University of WisconsinMadison, 1630 Linden Dr., Madison, WI 53706. Phone: (608) 262-9578. Fax: (608) 263-2626. E-mail: cza @plantpath.wisc.edu. Present address: DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824. Published ahead of print on 29 June 2007.

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YAO AND ALLEN TABLE 1. Strains and plasmids used in this study
Strain or plasmid Relevant characteristicsa

J. BACTERIOL.

Source or reference

Strains E. coli DH5 BT3388 UU1117 R. solanacearum K60 K60GFP K701 K760 K760GFP K770 K773 K774 K774GFP K60-Q Plasmids pSTBlue-1 pTrc99A pHP45 pUCGM pVO122 pVO155 pSTSM pUC18-mini-Tn7T-oriT pTNS1 pJY-mini-Tn7T-Sm pGH1 pSJYaer1 pSJYaer2 pSJYaer1::Gm pSSJYaer2::Km p13-6 p18-9 pTJYaer1 pTJYaer2 pJYTn7aer1 pJYTn7aer2 pJYTn7aer1/2
a r r

F endA1 relA 80 lacZ M15 hsdR17 supE44 thi-1 recA1 gyrA96 aer tsr tar trg tap aer1 Wild-type race1, biovar 1, isolated from tomato K60 Tn5gfp, constitutively expresses GFP; Tcr; motile and tactic, fully virulent K60 iC::aacC1; Gmr; aagellate and nonmotile K60 cheW::aphA-3, Kmr; nontactic K760 Tn5gfp, constitutively expresses GFP; Kmr Tcr K60 aer1::aacC1; Gmr K60 aer2::nptII; Kmr K60 aer1::aacC1 aer2::nptII; Gmr Kmr K774 Tn5gfp, constitutively expresses GFP; Gmr Kmr Tcr K60 pilQ::nptII; Kmr Cloning vector; Apr Kmr E. coli expression vector; Ptac lacIq; Apr Apr Smr Apr Gmr Apr Smr Apr Kmr pSTBlue-1 1147-2186::aadA; Smr Mini-Tn7 delivery plasmid; Apr Helper plasmid encoding the site-specic TnsABCD Tn7 transposition pathway; Apr 2.0-kb Smr cassette inserted into StuI site of pUC18-mini-Tn7T-oriT; Apr Smr E. coli aer cloned in pTrc99A; Apr PCR-amplied 1.46-kb fragment containing K60 aer1 cloned in pSTblue-1; Apr Kmr PCR-amplied 2.3-kb fragment containing K60 aer2 in pSTblue-1 0.8-kb Gmr cassette inserted into SacII sites of aer1; Apr Kmr Gmr 1.3-kb Kmr cassette inserted into BstEII-BglII site of aer2 and cloned into pSTSM; Kmr Smr pLAFR3 cosmid containing K60 aer1; Tcr pLAFR3 cosmid containing K60 aer2; Tcr K60 aer1 cloned in pTrc99A; Apr K60 aer2 cloned in pTrc99A; Apr 2.3-kb DNA fragment containing K60 aer1 cloned into pJY-mini-Tn7T-Sm ; Apr Smr 3.4-kb DNA fragment containing K60 aer2 cloned into pJY-mini-Tn7T-Sm ; Apr Smr 3.4-kb containing K60 aer-2 cloned into pJYTn7aer1; Apr Smr
r r r

Invitrogen 63 11 34 62 56 62 62 This This This This 38

study study study study

EMD Bioscience Pharmacia 47 53 9 44 This study 14 14 This study 48 This study This study This study This study This This This This This study study study study study

This study This study

Ap , ampicillin resistance; Gm , gentamicin resistance; Km , kanamycyn resistance; Rif , rifampin resistance; Tc , tetracycline resistance; Smr, streptomycin resistance.

and Tsr, monitor changes in cellular energy status and function as aerotaxis transducers (11, 18, 23, 27, 48, 49, 60). Among plant-associated bacteria, Aer homologs in Pseudomonas aeruginosa and P. putida have also been shown to transduce the signal for aerotaxis (31, 43) and a chemoreceptor-like protein in Azospirillum brasilense mediates energy taxis and promotes root colonization (28). Although aerotactic behavior was described in R. solanacearum 40 years ago (35), the role of aerotaxis in the life cycle of this pathogen is still unknown. In the present study we identied two putative aerotaxis receptor proteins, Aer1 and Aer2, in R. solanacearum strain K60 and showed that they function as aerotaxis transducers in both E. coli and R. solanacearum. We determined that R. solanacearum needs aerotaxis for full virulence and for rapid localization on host roots. In addition, we found that aerotaxis

plays a key role in formation of biolms on abiotic surfaces. These results suggest that aerotaxis behavior is important at several points in the life cycle of R. solanacearum and may mediate the transitions between the pathogens different habitats.
MATERIALS AND METHODS Bacterial strains, plasmids, culture media, and growth conditions. The bacterial strains and plasmids used in the present study are listed in Table 1. E. coli cells were grown in Luria-Bertani medium (40) at 37C except for the swarm plate assay described below. All R. solanacearum strains were routinely grown or maintained either in CPG-rich broth or on TZC plates (34). Growth media were supplemented with the antibiotics ampicillin (100 g/ml for E. coli and 250 g/ml for R. solanacearum), gentamicin (15 g/ml), kanamycin (25 g/ml), streptomycin (50 g/ml), and tetracycline (10 g/ml) as necessary. The growth of R. solanacearum wild-type and mutant strains was compared in buffered minimal

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medium (62) with 10 mM glucose, in CPG medium, and in tobacco leaves (Nicotiana tabacum cv. Bottom Special) as previously described (56). Recombinant DNA techniques. The isolation of cosmid, plasmid, and genomic DNA, as well as cloning, PCR, and Southern hybridization, was carried out by using standard protocols (8). E. coli and R. solanacearum were transformed as previously described (3). DNA was sequenced at the University of WisconsinMadison Biotechnology Center. The R. solanacearum strain UW551 (http://vision.biotech.u.edu /mycap/jsp/project/description.jsp? projectID 1) and GMI1000 (http://bioinfo .genopole-toulouse.prd.fr/annotation/iANT/bacteria/ralsto/) genome databases and Jellysh 3.0 software (Field Scientic, Lewisburg, PA) were used to analyze the DNA sequence data. Molecular biology reagents were from either Promega (Madison, WI) or Invitrogen (Carlsbad, CA). Oligonucleotides were from Integrated DNA Technologies (Coralville, IA). Construction of R. solanacearum aer1 and aer2 mutants. A 1,465-bp DNA fragment containing part of the R. solanacearum strain K60 aer1 gene was amplied by PCR using primers aer1-F (5 -ATGCGCGTCAACGAACCC) and aer1-R (5 -ACCAGCGTGGCGTTCTCC) and cloned into vector pSTBlue-1 to create pSJYaer1. The gentamicin resistance cassette (accC1) from pUCGM was inserted into SacII sites in the aer1 fragment to create the pSJYaer1::Gm mutagenesis construct. A 2,309-bp DNA fragment containing a full-length copy of the K60 aer2 gene was PCR amplied by using aer2-F (5 -CGCATTGGAAGA CCGATAG) and aer2-R (5 -ATCTGTACGACGGTGGTGGT) and cloned into pSTBlue-1 to yield pSJYaer2. The kanamycin resistance cassette from pVO155 was inserted into the blunt-ended BglII-BstEII site in aer2; the resulting aer2::Km construct was transferred into the EcoRI site of pSTSM to create the pSSJYaer2::Km mutagenesis construct. The aer1::Gm and aer2::Km constructs were introduced individually into the strain K60 chromosome by double homologous recombination as previously described (3); this resulted in aer1 mutant strain K770 and aer2 mutant strain K773. The aer2::Km construct was introduced into aer1 mutant K770 by the same process to create the aer1 aer2 double mutant strain K774. Fluorescent strain K774GFP was created by using natural transformation (38) to move the Tn5::gfp38 insertion from the chromosome of strain K60GFP (62) into the strain K774 chromosome. This Tn5::gfp insertion, which is in the same locus in all strains, was previously shown to be constitutively expressed and not to affect motility, taxis, virulence, or growth rate (36, 60). Similarly, strain K760GFP was constructed by using natural transformation to move the cheW::Km mutation from cheW mutant strain K760 into the chromosome of uorescent strain K60GFP (38, 62). The correct allelic replacement in each mutant was conrmed by PCR and by Southern hybridization, using as probes DNA fragments from the aer1 and/or aer2 genes with anking regions and the gentamicin and/or kanamycin resistance cassette. Complementation studies. For complementation studies, the R. solanacearum strain K60 cosmid library (3) was screened by colony blotting using the aer1 and aer2 sequences from pSJYaer1 and pSJYaer2 as probes; cosmids p13-6 and p18-9 contained full-length copies of aer1 and aer2, respectively. To study R. solanacearum aer1 and aer2 functions in E. coli, a 1,548-bp DNA fragment containing full-length aer1 and a 1,598-bp DNA fragment containing full-length aer2 without their starting ATG codons were PCR amplied by high-delity PfuUltra DNA polymerase (Stratagene, La Jolla, CA). Cosmid p13-6 was used as a template for primer set aer1-F1 (5 -cacgaattcCGTGTCAA CGAACCCGTTAC) and aer1-R1 (5 -ccaaagcttTCAGCGGAACACGCCG AC), and cosmid p18-9 was the template for primer set aer2-F1 (5 -tctgaattcC GAAACAACCTGCCGGTCAC) and aer2-R2 (5 -tctaagcttacggcTCAAGCGC GGAACAC). The primers contain either EcoRI or HindIII site (marked in italics) at each end to facilitate further manipulation. The resulting PCR products were double digested with EcoRI and HindIII and cloned into the EcoRI and HindIII sites of pTrc99A to create the in-frame expression constructs pTJYaer1 and pTJYaer2; correct constructions were conrmed by sequencing. Both pTJYaer1 and pTJYaer2 were introduced into E. coli aerotaxis mutant strains UU1117 (11) and BT3388 (63) for functional studies. To complement R. solanacearum aer1 and aer2 mutants, a mini-Tn7 delivery system (14) was used to insert intact aer1 or aer2 genes into a unique attTn7 site that is located 25-bp downstream of glmS in the R. solanacearum chromosome. A 2.3-kb fragment containing the full-length copy of K60 aer1 from cosmid p13-6 was subcloned into pJY-mini-Tn7T-Sm to create pJYTn7aer1. Similarly, a 3.4-kb fragment containing full-length K60 aer2 from cosmid p18-9 was subcloned into pJY-miniTn7T-Sm and pJYTn7aer1 to create pJYTn7aer2 and pJYTn7aer1/2. Plasmids pJYTn7aer1, pJYTn7aer2, and pJYTn7aer1/2, together with helper plasmid pTNS1 (14), were electroporated into strains K770, K773, and K774, respectively, to create the corresponding trans-complemented strains. An empty construct, pJY-miniTn7T-Sm , together with pTNS1 was electroporated into K60, K770, K773, and K774 to create negative control strains. The resulting strains were selected as streptomycin resistant (Smr) and ampicillin sensitive (Aps) on TZC plates supplemented

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with appropriate antibiotics. The successful integration of mini-Tn7 transposons and their derivatives was veried by PCR using primer set glmSu (5 -GAATACCGTT ACCGCGACAC) and Tn7R (5 -CAGCATAACTGGACTGATTT). Bacterial behavioral assays. E. coli aerotaxis and chemotaxis were assayed on minimal semisolid agar medium containing 40 mM sodium succinate or 1 mM glycerol, respectively (11). R. solanacearum chemotaxis was assayed as previously described (62). The Aer proteins were expressed from the leaky tac promoter in the pTrc99A plasmid without addition of IPTG (27). A modied chemotaxis chamber method (54) was used to quantify R. solanacearum aerotaxis. Instead of using single chemotaxis chamber wells, we used 96-well nested MultiScreen-MIC Plates (Millipore, Billerica, MA) to measure aerotaxis. R. solanacearum cells were grown to an optical density at 600 nm (OD600) of approximately 0.3 to 0.7 in CPG broth; the cells were collected by centrifugation, washed twice in chemotaxis buffer (62), and resuspended to an OD600 of 0.01. The wells of the bottom plate were lled with 150 l of bacterial suspension, and the wells of the top lter plate, which had 8- m-pore-size lters on the bottom of each well, were lled with 50 l of chemotaxis buffer and left open to the air, thus allowing oxygen to diffuse into the medium in the upper well and create an oxygen gradient between the upper and lower wells. The upper receiving lter plate was carefully placed in contact with the bacterial suspensions in the wells of the bottom plate so that bacteria could navigate across the lter into the upper well in response to the resulting oxygen gradient. After 30 min of incubation at room temperature, the numbers of bacterial cells in the upper and lower wells were determined by dilution plating on TZC plates. The aerotaxis index was calculated as the number of bacterial cells that migrated into the upper lter plate wells divided by the total number of bacteria in both the top and bottom plate wells. For each strain, cell numbers for two individual wells in three separate experiments were averaged to generate the aerotaxis index. Virulence assays. Wilt-susceptible tomato plants (Lycopersicon esculentum Mill. cv. Bonny Best) were used to evaluate the virulence of R. solanacearum mutants by a naturalistic soil-soak assay as previously described (62). Briey, a dilute suspension of bacteria was poured over the soil of unwounded 16-day-old tomato plants to generate a nal concentration of approximately 3 107 CFU/g of potting mix. Plants were maintained at 28C and rated daily using a 0 to 4 disease index as follows: 0 healthy, 1 1 to 25% of leaf area wilted, 2 26 to 50% of leaf area wilted, 3 51 to 75% leaf area wilted, and 4 75% leaf area wilted. Each experiment included 16 plants per treatment, and the assay was repeated four times. Tomato seedling root colonization assays and in situ visualization. Tomato seeds (cv. Bonny Best) were surface sterilized, germinated, and grown as previously described (62) except that the 1% water agar was replaced by half-strength MS basal medium (42) without a carbon source or plant hormones. Seedlings with 5- to 8-cm intact roots were used for further assays. R. solanacearum cell suspensions were prepared as for the aerotaxis assay, above. Two tomato seedlings with intact roots were incubated in 5-ml suspensions of R. solanacearum strain K60GFP, K774GFP, or K760GFP in Falcon six-well plates (BD Biosciences, San Jose, CA) at room temperature. After 30 min, seedling roots were rinsed with sterile water and blotted lightly on absorbent paper. Then, one set of seedling roots was excised and observed under a Zeiss LSM510 Meta laser scanning confocal microscope (Carl Zeiss MicroImaging, Thornwood, NY); the tips of all seedling roots were scanned and the images were captured by a charge-coupled device camera and analyzed by using a Zeiss LSM imager browser. A matched set of seedling roots were excised, weighed, ground in sterile water, and dilution plated on TZC to quantify the total bacteria adhering to roots. The cell numbers were normalized to seedling root fresh weight. Biolm assay. The polyvinylchloride (PVC) microtiter plate assay was used to quantify biolm formation in R. solanacearum with a minor modication (46). Briey, overnight cultures of R. solanacearum were collected by centrifugation and resuspended in fresh CPG broth; the OD600 of the suspensions was adjusted to 0.1. A total of 5 l of this bacterial suspension was used to inoculate 95 l of CPG in the well of a PVC microtiter plate, and the plates were sealed with plastic wrap and incubated without shaking for 24 h at 28C. Crystal violet staining and biolm quantication were performed as previously described (46) except that the absorbance was determined at 530 nm using a Wallac Victor2 microplate reader (Perkin-Elmer, Waltham, MA). Data analysis. The aerotaxis index, each days disease index in disease assays, the number of cells attached to the seedlings, and biolm formation data were all analyzed by using analysis of variance (ANOVA) at the 95% level. In some cases, the Fisher least signicant differences were calculated and applied to compare the differences among the treatments at the 95% condence level. All statistical analyses were performed by using MINITAB 14 (Minitab, State College, PA).

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FIG. 1. Two probable aerotaxis transducers in the R. solanacearum strain K60 genome. (A) The genomic regions of aerotaxis transducer genes aer1 and aer2 in R. solanacearum strain K60. The arrows indicate the direction of transcription, the solid arrows represent the intact predicted gene, and the dotted arrows represent partial predicted genes. Two aer mutants were created by inserting a gentamicin resistance cassette (accC1, solid triangle) into aer1 (to make strain K770) or a kanamycin resistance cassette (nptII, open triangle) into aer2 (to make strain K773). B, BamHI; Bs, BstEII; Bg, BglII; EI, EcoRI; EV, EcoRV; N, NdeI; No, NotI; S, SacI; Sa, SacII; Sm, SmaI; Sp, SphI. (B) Predicted protein domain architectures of Aer1 and Aer2 as dened by the SMART database (37, 52): PAS and PAC domains are for FAD binding and signal transduction (10, 49, 57), and the HAMP domain is for transmitting signals (5). T, transmembrane domain, which was further analyzed by the DAS program (16); MA, methyl-accepting chemotaxis-like domains, which are required for response to stimuli during bacterial taxis (24).

Nucleotide sequence accession number. The nucleotide sequences determined in the present study have been deposited in the GenBank database under the accession numbers EF450772 and EF450771.

RESULTS R. solanacearum strain K60 possesses two aerotaxis transducers. The genomes of R. solanacearum strains GMI1000 and UW551 have been sequenced, and more than 20 putative MCPs were identied (25, 50). Further analysis of R. solanacearum genomes using the E. coli aerotaxis transducer Aer (EcAer) sequence (11, 48) and the BLASTP program (4) revealed that two of these MCPs (RS03168 and RS03711 in GMI1000 and RRSL_01121 and RRSL_01710 in UW551) have signicant similarity to EcAer with expect values less than e 89. The genes encoding RS03168 in GMI1000 and its homolog RRSL_01121 in UW551 and RS03711 in GMI1000 and its homolog RRSL_01710 in UW551 were named aer1 and aer2, respectively. R. solanacearum Aer1 and Aer2 are similar to each other with an expect value less than e 80. The genome of R. solanacearum strain K60 has not been sequenced, but K60 is much more closely related to UW551 than to GMI1000 (data not shown). Using sequences from GMI1000 and UW551, we designed primers that amplied aer1 and aer2 from strain K60 DNA. The resulting PCR products were cloned as a 1,465-bp fragment in pSJYaer1 and a 2,309-bp fragment in pSJYaer2. These served as probes to screen a cosmid library of R. solanacearum strain K60 by colony hybridization. Cosmids p13-6 and p18-9 contained full-length copies of K60 aer1 and aer2, respectively. The aer loci were further subcloned and sequenced. Sequencing and analysis of these inserts disclosed microsynteny of

the aer1 and aer2 genomic regions in strains K60, UW551, and GMI1000 (Fig. 1A) (25, 50). In K60 a putative ipt gene encoding for isopentenyl transferase is located 5 of aer1 with a 361-bp gap. A putative narK gene encoding a nitrate transporter is located 3 of aer1 with a 379-bp gap. An open reading frame (ORF) encoding a hypothetical protein transcribed in the opposite orientation and a putative viuB gene encoding a siderophore-interacting protein in the same orientation were located 5 of aer2. Another ORF encoding a hypothetical protein was located 3 of aer2 and transcribed in the opposite orientation (Fig. 1A). The deduced K60 Aer1 and Aer2 proteins have predicted molecular masses of 55.1 and 56.1 kDa, respectively. Analysis with the SMART and DAS algorithms (16, 52) revealed that Aer1 and Aer2 have protein domain architectures similar to the E. coli Aer protein (Fig. 1B) (11, 48). The signature N-terminal PAS (for period circadian protein, Ah receptor nuclear translocator protein, single-minded protein) and PAC (for PAS-associated Cterminal) domains are required for FAD binding and redox status sensing in Aer from E. coli (49, 64). Aer1 and Aer2 have membrane topologies with two transmembrane domains, similar to E. coli Aer (11, 48). The C-terminal HAMP (for histidine kinases, adenylyl cyclases, methyl binding proteins and phosphatases) and MA (for methyl-accepting chemotaxis-like) domains (Fig. 1B) also resemble those of E. coli Aer and form a module typical of bacterial taxis sensory transducers for signal transmission in response to stimuli (5, 24). R. solanacearum Aer1 and Aer2 both function as aerotaxis transducers in E. coli. To assay for heterologous function, the strain K60 aer1 and aer2 genes were cloned in frame into pTrc99A under the control of the Ptac promoter. The resulting

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FIG. 2. Cloned R. solanacearum Aer proteins can function as taxis transducers in E. coli. (A) Colony morphology of E. coli aer mutant UU1117 on soft-agar plate containing 40 mM succinate; (B) colony morphology of E. coli multiple MCP knockout BT3388 on soft-agar plate containing 10 mM glycerol. The strains harbor the following plasmid constructs: pTrc99A, empty vector as a negative control; pGH1, vector expressing E. coli Aer as a positive control; pTJYaer1, vector expressing R. solanacearum Aer1; and pTJYaer2, vector expressing R. solanacearum Aer2. The plates were photographed after incubation at 32C for 24 h. The assays were replicated at least four times, and the images shown are typical.

plasmids, pTJYaer1 and pTJYaer2, expressed Aer1 and Aer2 proteins in E. coli strains after induction by IPTG (isopropyl-D-thiogalactopyranoside) (data not shown). Both constructs were transformed into E. coli aer strain UU1117 (11) and into E. coli BT3388, a mutant lacking all ve taxis transducers (63). We included BT3388 because it lacks both Aer and the Tsr taxis transducer, which also has an energy taxis function. The behavior of these transformed E. coli strains was examined by using soft-agar swarm plate assays. Both pTJYaer1 and pTJYaer2, as well as pGH1 containing the E. coli aer gene, restored the ability of aer mutant strain UU1117 to produce aerotactic swarms on succinate media (Fig. 2A). This indicates that R. solanacearum strain K60 Aer1 and Aer2 proteins can function as aerotaxis transducers in E. coli. Interestingly, the complete transducer knockout strain BT3388 displayed a much stronger tactic response on glycerol media when it carried pTJYaer1 than when it carried either pTJYaer2 or pGH1 (Fig. 2B). This stronger tactic response may reect a higher sensitivity of Aer1 to changes in redox levels in E. coli in response to glycerol concentrations (energy taxis).

R. solanacearum mutants lacking aer1, aer2, or both genes are no longer aerotactic. To further investigate the function of Aer1 and Aer2 in R. solanacearum, we constructed three sitedirected aer mutants of strain K60. R. solanacearum aer1 mutant strain K770 and aer2 mutant strain K773 were generated by inserting gentamicin and kanamycin resistance gene cassettes into the aer1 and aer2 ORFs, respectively (Fig. 1A). These two mutations were combined to create an aer1 and aer2 double-mutant strain, K774. All three mutants grew as well as parent strain K60 in rich medium (CPG), buffered minimal medium (BMM plus 10 mM glucose or malate), and in planta (in tobacco leaves) (data not shown). All three aer mutant strains displayed normal swimming motility under the microscope and were chemotactic toward malate on plates, indicating that their core motility and general chemotaxis functions were not affected (data not shown). Aerotaxis along an oxygen gradient was measured by a nested chemotaxis chamber method. In this 30-min assay, 8.1% of wild-type strain K60 cells migrated up the oxygen gradient into the upper well, but the upper wells contained signicantly fewer cells of aer1 mutant K770 (3.4%), aer2 mutant K773

FIG. 3. Both aer1 and aer2 genes are required for aerotaxis in R. solanacearum. A modied nested chemotaxis well method was used to quantify R. solanacearum aerotaxis. Aerotactic ability is represented as the proportion of bacterial cells in the lower well that migrated through an 8- m-pore-size lter into the upper well in response to an oxygen gradient. (A) R. solanacearum aer mutants had signicantly reduced aerotactic ability; (B) The aer mutations were complemented with a functional copy of the relevant gene in trans. Each column represents the mean of three individual experiments with two replicates per treatment. The error bars represent the standard error of the mean. Columns with different letters above are signicantly different according to the Fisher least signicant difference test (P 0.05).

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FIG. 4. Disease progress of R. solanacearum aer mutants on tomato. Unwounded 16-day-old tomato plants (cv. Bonny Best) were inoculated by pouring bacterial suspensions near the crown to a nal concentration of about 3 107 CFU/g of potting mix. Plants were rated daily on a disease index scale from 0 to 4 (see Materials and Methods for details). Each point represents the mean disease index of four individual experiments each containing 16 plants per treatment. For the time points marked with asterisks, wild-type strain K60 and aer1/aer2 mutant K774 are signicantly different according to ANOVA (P 0.05).

(1.2%), and aer1/aer2 double mutant K774 (0.7%) (Fig. 3B). This result demonstrates that Aer1 and Aer2 function as aerotaxis transducers in R. solanacearum. Moreover, the signicant difference in the ability of aerotaxis between K770 and K774, but not between K773 and K774 (Fig. 3A) suggests that Aer2 plays a more important role than Aer1 in guiding R. solanacearum toward favorable redox status. An intact copy of aer1 or aer2 inserted into the attTn7 site fully restored the aerotactic ability of K770 and K773, respectively (Fig. 3B). However, the aerotactic ability of double mutant K774 was not restored to full wild-type levels when the same complementation strategy was applied, although the aerotaxis index of the complemented strain did increase signicantly, from 0.98% to 5.1% (Fig. 3B). R. solanacearum aer2 mutants have delayed disease development in tomato. We previously found that tactic motility plays a key role in the early stage of wilt disease development (62). However, since both nontactic strains studied are impaired in the entire taxis signal transduction pathway, that study could not identify the role of specic types of taxis, such as aerotaxis. To determine the role of aerotaxis in wilt disease development, we performed two types of virulence assay on a susceptible tomato host. In the naturalistic soil soak assay, which requires bacteria to locate and invade host roots from soil, wild-type strain K60 caused a mean disease index of 3.84 by 14 days postinoculation. This was slightly greater than the nal disease indices of aer1 mutant K770 (3.68), aer2 mutant K773 (3.55), or aer1/aer2 double mutant K774 (3.68), although the differences were not signicant (Fig. 4). However, the overall shape of the disease progress curves were different and from day 5 to day 7 postinoculation, K773 and K774, which both lack functional Aer2, caused signicantly lower disease severity compared to that of the wild-type parent (P 0.05 [ANOVA]) (Fig. 4). There was no signicant difference between K60 and K770 at any time point (Fig. 4). This result suggests that Aer2 but not Aer1 makes a small contribution to rapid wilt development in

this assay, possibly because aerotaxis plays a critical role in initial location of plant roots by the pathogen (see below). In contrast, there was no signicant difference among these strains at any time in a cut-petiole assay, where bacteria are introduced directly into tomato xylem tissue (data not shown). This result is consistent with our previous nding that taxis in general is not required for virulence when R. solanacearum is directly introduced into the plant (62). The aer1/aer2 mutant cannot rapidly locate and aggregate on tomato seedling roots. A green uorescent protein (GFP)tagged R. solanacearum aer1/aer2 mutant, K774GFP, was created by integrating a well-characterized constitutively expressed Tn5::gfp construct into the strain K774 chromosome. This allowed us to observe early interactions between a dilute suspension of uorescent R. solanacearum cells and tomato seedling roots under a confocal microscope in real time. Typical results are shown in Fig. 5A to C. After incubation with tomato seedling roots for 30 min, many more wild-type R. solanacearum strain K60GFP cells were present on the surface of the roots (Fig. 5A) than cells of either nonaerotactic strain K774GFP (Fig. 5B) or fully nontactic strain K760GFP (Fig. 5C). The bacteria preferentially clustered on root tips and in the elongation zone immediately behind the root cap. Cells of nonaerotactic strain K774GFP seemed to be slightly better than nontactic strain K760GFP at localizing on tomato root surfaces, but the difference was not dramatic in this qualitative microscopic assay. We used direct plating to determine the number of bacterial cells attached to the plant roots after 30 min of incubation. In this quantitative assay, signicantly more wild-type K60GFP cells were present on rinsed roots than aer1/aer2 mutant K774GFP or nontactic mutant K760GFP (Fig. 5D). These results suggest that aerotaxis facilitates rapid localization and colonization of host roots by R. solanacearum. Moreover, the difference between nonaerotactic mutant K774GFP and wholly nontactic mutant K760GFP (Fig. 5D) indicates that other types of taxis also contribute to this behavior. Both aer1 and aer2 affect biolm formation on an abiotic surface. We observed that aerotaxis mutants K770, K773, and K774 frequently formed cell aggregates in CPG broth culture under agitation. Microscopic study revealed that these cell aggregates had structural similarity to biolms as described in other bacterial systems (17). We used a standard PVC microtiter plate assay to study the role of aerotaxis in R. solanacearum biolm formation. After R. solanacearum strains were incubated without shaking in CPG broth at 28C for 24 h, biolm bands formed at the air-liquid interface (Fig. 6A). Interestingly, R. solanacearum aerotaxis mutants K773 and K774, nontactic mutant K760, and nonmotile mutant K701 all formed signicantly more biolm than wild-type strain K60 (Fig. 6). However, although aer1 mutant K770 consistently produced more biolms than K60, the two strains were not signicantly different when evaluated by the Fisher least-signicant-difference test (Fig. 6B). A pilQ mutant (36), which lacks type IV pili and was included as a control, produced very little biolm. DISCUSSION Taxis plays an important role in the life cycle of R. solanacearum (62), but the role of specic types of taxis such as aerotaxis was unknown. We identied and characterized two Aer homologs in R. solanacearum strain K60. Both R. solanacearum

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FIG. 5. An R. solanacearum aer1/aer2 double mutant is impaired in rapid localization of tomato seedling roots. Aseptic tomato seedling roots were incubated with 5 ml of the different GFP-tagged R. solanacearum strains at a cell density of about 107 CFU/ml. After 30 min at room temperature, the seedling roots were rinsed with sterile water, blotted dry on tissues, excised, and either directly examined under a laser scanning confocal microscope or ground and dilution plated on TZC medium. (A to C) Seedling root surfaces were visualized under the microscope with the indicated strain. Each strain constitutively expressed GFP, visible as a green color on the root surface. Assays were repeated three times; the images shown are typical results. (D) Number of bacterial CFU recovered from tomato seedling roots. Each column is the mean of three individual experiments with two replicates per treatment. The error bars represent the standard error of the mean. Columns with different letters are signicantly different according to Fisher least signicant difference test (P 0.05).

FIG. 6. R. solanacearum aer mutants overproduce biolm on PVC surfaces. PVC microtiter plate biolm assays were performed as previously described (46). (A) R. solanacearum formed biolms on PVC surfaces, visible as purple crystal violet stains. The assays were repeated at least three times and photos shown are typical. (B) R. solanacearum biolm formation was quantied by measuring A530 of crystal violet-stained wells rinsed with ethanol. Each column is the mean of three individual experiments with two replicates per treatment. The error bars represent the standard error of the mean. Columns with different letters are signicantly different according to the Fisher least signicant difference test (P 0.05).

Aer proteins have protein domain architectures (Fig. 1B) resembling those of E. coli energy taxis MCPs (11, 23, 48, 60). R. solanacearum Aer1 and Aer2 both restored aerotaxis to an E. coli aer mutant (Fig. 2A). However, Aer1, which also restored taxis to an E. coli strain lacking all MCPs on a gradient of the oxidizable substrate glycerol, appeared to be a better transducer in this condition than either Aer2 or E. coli Aer (Fig. 2B). This difference could be due to greater stability of Aer1 in E. coli, to a greater sensitivity of Aer1 to cellular redox changes in the presence of the energy source glycerol, or possibly to a broader substrate sensitivity of Aer1. A single taxis transducer often responds to multiple signals, and Aer1 may do this. Wild-type K60 cells migrated up an oxygen gradient at a signicantly higher frequency than the motile aer mutant cells, a nding consistent with a previous report that R. solanacearum exhibits aerotaxis (35). Since neither motile but nontactic cheW mutant K760 nor nonmotile iC mutant K701 moved up the oxygen gradient to a signicant degree, this migration was not due to either random swimming or simple diffusion (Fig. 3A). Interestingly, aer1 mutant K770 was slightly more aerotactic than aer2 mutant K773 (Fig. 3A), suggesting that Aer2 contributes differently to aerotaxis behavior. Adding back a copy of the wild-type gene restored aerotactic ability to aer1, aer2, and aer1/aer2 mutant strains. However, we could not fully complement the aer2 mutant phenotype (Fig. 3B). It is possible that because aer2 was inserted into the att site using a Tn7 complementation construct rather than into its original chro-

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mosomal location, the expression level was different from the wild-type; another study found that in E. coli accurate expression of Aer is important for normal function (27). We adapted a nested-well method (54) to quantify aerotaxis in R. solanacearum since the traditional capillary tube aerotaxis assay (48), an air bubble assay, and a soft-agar swarm plate assay (11) all failed to reproducibly quantify R. solanacearum aerotaxis (data not shown). R. solanacearum did not form a distinct aerotaxis band in the capillary tube assay nor aggregate near air bubbles; in fact, R. solanacearum apparently responds to oxygen gradients by changing behaviors such as increasing cell swimming speed or increasing the proportion of swimming cells in the population (data not shown). Further, we noticed that the bacterium aggregates in a diffuse cloud a few millimeters away from the opening of capillary tubes and just below the surface of a soft agar stab tube, suggesting a preference for moderate rather than maximal oxygen levels. This may correlate with its adaptation to the microaerophilic environments in plant rhizospheres and xylem tissue. Our evidence demonstrates that Aer1 and Aer2 function as aerotaxis transducers in R. solanacearum, and the absence of detectable aerotactic ability in the aer1/aer2 mutant in the well assay suggests that at least under these conditions aer1 and aer2 are the only genes contributing to the behavior. However, since more than 20 genes encode putative chemotaxis-like proteins in the R. solanacearum strain GMI1000 and UW551 genomes (25, 50), others could also serve as aerotaxis transducers under different conditions. Each genome contains a homolog of Tlp1 (RS00467 in GMI1000 and RRSL_03970 in UW551), a novel aerotaxis transducer recently identied and characterized in A. brasilense (28). Neither R. solanacearum genome contains an obvious homolog of E. coli Tsr, which also functions as aerotaxis transducer (48). R. solanacearum aer2 and aer1/aer2 mutant strains had slightly delayed disease development on susceptible tomato plants in a biologically representative soil soak inoculation assay (Fig. 4) but not in a cut-petiole inoculation assay (data not shown). This is consistent with our previous nding that once bacteria enter the plant vascular system, tactic motility is no longer needed for disease development. The altered virulence is not due to poor growth, since strains K773 and K774 grew as well as the wild-type strain in synthetic media and in planta (data not shown). We speculate that sensing optimal energy status may lead R. solanacearum to parts of the root where nutrients are leaking out, such as the elongation zone, where we observed rapid intense aggregation in our microscopic assay, or to sites of secondary root emergence, which are known to be a primary infection court for this pathogen (19). However, in a similar assay the completely nontactic cheA or cheW mutants were much more dramatically reduced in virulence than the aer2 mutants (62). This indicates that at least one other type of taxis besides aerotaxis attracts R. solanacearum to plant roots and possibly to optimal locations inside the host. Given the relatively large number of putative MCPs encoded by the genome, it seems likely that the pathogen has adapted to recognize, integrate, and respond to a complex set of environmental signals. The possible ecological role of aerotaxis during microbial interactions with hosts and nonliving environmental habitats has been intensively discussed (2, 58), and energy taxis has

been shown to be required for efcient attachment and colonization of wheat roots by the biocontrol bacterium A. brasilense (28). To better understand the potential role of bacterial aerotaxis in the early stage of host root localization and colonization by R. solanacearum, we used GFP-expressing R. solanacearum strains to visualize the process in vivo. When tomato seedling roots were incubated with bacterial cells, within 30 min wild-type cells clustered thickly on root surfaces. The aer1/aer2 double mutant K774 did not exhibit this behavior, although K774 was slightly better able to nd the roots than the completely nontactic mutant K760 (Fig. 5A). It seems likely that the reduction in virulence that we observed in aer2 mutants results from their poor ability to locate and colonize host roots in the earliest stages of pathogenesis. Biolms are assemblages of microorganisms embedded in a matrix of extracellular polymers that adhere to each other and to a surface, thereby adapting to uctuating environmental conditions in a social manner (17, 41). Many plant-associated bacteria form tissue-like biolms in contact with biotic or abiotic environments (17, 41). Although biolms are suspected to play a role in R. solanacearum-host interaction (41), few studies have been done (33). We have observed that R. solanacearum forms biolm-like aggregations on the surface of tomato seedling roots (62), and we speculate that, inside the plant, biolms could help the pathogen remain anchored to xylem vessel walls and effectively lter nutrients from the dilute ow of xylem uid. However, the factors that affect biolm formation in this organism are still unknown. We found that R. solanacearum wild-type strain K60 forms biolms on PVC wells at the liquid-air interface (Fig. 6A) that are similar to those formed by R. solanacearum AW1 (33) and another plantassociated bacterium, P. uorescens (46). Interestingly, R. solanacearum aerotaxis mutants overproduced biolms compared to wild-type strain K60 (Fig. 6A and 6B). This result was unexpected, since nonmotile and nontactic mutants of the wellcharacterized biolm-forming aerobic bacterium P. aeruginosa are impaired in biolm formation (36, 45). The nonaerotactic strains (which are motile and retain general taxis) had biolm phenotypes indistinguishable from those of entirely nonmotile or generally nontactic strains, suggesting that the lack of aerotaxis is specically responsible for the thicker biolms produced by all of these mutants and that wild-type strains use aerotaxis to regulate biolm formation. Since R. solanacearum is a microaerophilic organism, we speculate that the dense biolms formed at the liquid-air interface in this assay result from the toxic effect of the high oxygen concentrations there. Aerotaxis may lead the majority of wildtype bacterial cells to avoid the liquid-air interface, and any cells trapped there may form biolms for protection from the higher oxygen concentrations. Aerotaxis mutants, which cannot sense and avoid high oxygen levels, may thus be more likely to form biolms. It seems likely that in this pathogens natural habitat, aerotaxis behavior is typical of planktonic cells rather than those anchored in biolms. Additional experiments are needed to observe biolm formation in planta by wild-type and nonaerotactic strains. Many questions remain unanswered about R. solanacearum aerotaxis. Do Aer1 and Aer2 have other signal transducing functions in R. solanacearum? How does R. solanacearum regulate expression of aerotaxis-related genes? What are the spe-

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cic signals from plant roots or within host plants that trigger signaling by the aerotaxis transducers? Does aerotaxis play a role in biolm formation on biotic surfaces? Our results suggest that aerotaxis and/or energy taxis contributes to R. solanacearums successful navigation of the complex and poorly understood microenvironments on root surfaces and inside plant tissues.
ACKNOWLEDGMENTS This research was supported by the National Science Foundation (IBN-0090692), by USDA-NRI (35319-13851), by a USDA Floral and Nursery Industry Task Force Specic Cooperative Agreement (581230-3-174), and by the University of WisconsinMadison College of Agricultural and Life Sciences. We thank Timothy Denny (University of Georgia) for helpful discussions and suggestions and for R. solanacearum strain K60-Q and the original Tn5-GFP construct; Mark Johnson (Loma Linda University) for the gift of E. coli strain BT3388, plasmid pGH1, and anti-EcAer antiserum; John S. Parkinson (University of Utah) for E. coli strain UU1117; Mark Goulian (Princeton University) for plasmid pTrc99A; and Herbert Schweizer (Colorado State University) for the mini-Tn7 delivery system.
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