Burkholderia cenocepacia strain K56-2, a representative of the Burkholderia cepacia complex, is part of the epidemic and clinically problematic ET12 lineage . The strain produced plant tissue watersoaking [ptw] on onion tissue, which is a plant disease-associated trait. Using plasposon mutagenesis, mutants in the ptw phenotype were generated . The translated sequence of a disrupted gene [ptwD4] from a ptw-negative mutant showed homology to VirD4-like proteins. Analysis of the region proximal to the transfer gene homolog identified a gene cluster located on the 92-kb resident plasmidthat showed homology to type IV secretion systems . The roleof ptwD4, ptwC, ptwB4, and ptwB10 in the expression of ptw activity was determined by conducting site-directed mutagenesis . Theptw phenotype was not expressed by K56-2 derivatives with adisruption in ptwD4, ptwB4, or ptwB10 but was observed in a derivative with a disruption in ptwC . Complementation of ptw-negative K56-2 derivatives in trans resulted in complete restoration of the ptw phenotype . In addition, analysis of culture supernatants revealed that the putative ptw effector[s] was a secreted, heat-stable protein[s] that caused plasmolysis of plant protoplasts . A second chromosomally encoded type IV secretion system with complete homology to the VirB-VirD system was identified in K56-2 . Site-directed mutagenesis of key secretory genes in the VirB-VirD system did not affect expression of the ptw phenotype . Our findings indicate that in strain K56-2, the plasmid-encoded Ptw type IV secretion system is responsible for the secretion of a plant cytotoxic protein[s].

 
The Burkholderia cepacia complex [Bcc] consists of nine genomovars recently elevated to species status [11, 65, 66, 68] . Membersof the Bcc include plant and animal pathogens as well as catabolically active soil saprophytes [64] . Some Bcc members can cause life-threateningrespiratory infections, particularly in persons with cysticfibrosis [CF] [43] . Studies indicate that 85 to 90% of strainsisolated from infected CF patients are B . cenocepacia or B.multivorans, with other Bcc species being infrequently isolated[41] . Several epidemic clonal lineages of B . cenocepacia havebeen identified [12], including the ET12 lineage, which is responsiblefor infecting many CF patients in Canada and the United Kingdom[32, 39]; the PHDC lineage, responsible for nearly all Bcc infectionsin the mid-Atlantic region of the United States [8]; and theMidwest lineage, which is responsible for infecting numerous patients in CF centers in the midwestern region of the United States [42] . Factors that account for the apparent enhanced capacity of epidemic clones for human infection are unknown.

Postulated clinically associated virulence determinants forB . cenocepacia include hemolysins [30, 67], siderophores [62],and cable pili [56] . The hemolysin induces apoptosis and degranulationof phagocytes [30], and siderophore production, regulated byquorum sensing [40], plays a role in the primary colonizationprocess of animal lung and spleen tissues [62] . Cable pili areimportant in adherence to the respiratory mucosal blanket andepithelial cells [57].

In B . cepacia type strain ATCC 25416, a plant pathogenic representativeof the Bcc, a plasmid-encoded pectate hydrolase [Peh] is a virulencefactor necessary for maceration of onion tissue [24] . Derivativesof ATCC 25416 that have been cured of the Peh-encoding plasmiddo not macerate onion tissue . However, the Peh-negative derivativesremain capable of causing a plant tissue watersoaking [ptw]phenotype that results from loss of cell membrane integrity and the accumulation of fluids in the intracellular spaces of plant tissue [22] . In a survey of isolates from the Bcc experimentalstrain panel [47], we found that all of the B . cepacia and B.cenocepacia strains tested and one B . vietnamiensis strain producedthe ptw phenotype, suggesting that members of these genomicspecies may produce a cytotoxic factor[s] that affects plantcell integrity [50].

In recent years, a body of research has substantiated the concept that bacterial pathogens share common secretion mechanisms forthe delivery of virulence determinants [9, 54] . There are numerousexamples of the importance of type III [54] and type IV secretionsystems [10] in the infection process for both plant and animalpathogens, with evidence that elements of the secretion systemsshow an ancestral relationship to bacterial transport machinery.It has been hypothesized that type III secretion systems arederived from flagellar assembly constituents modified to functionas a transport mechanism for virulence factors [46] . The typeIV secretion system from Agrobacterium tumefaciens was the firstto be identified and is used to deliver oncogenic transfer DNAand effector proteins to plant cells during infection [10, 37].Presently, type IV secretion systems can be categorized as [i]conjugation systems that mediate DNA transfer to recipient cells, [ii] effector translocator systems that transfer molecules termed effectors to eukaryotic cells during infection, or [iii] DNA uptake or release systems mediating exchange of DNA with themilieu [18] . The Legionella pneumophila Dot/Icm transporteris an example of a type IV secretion system that is capableof both conjugation and effector translocation [e.g., RalF][52] . Helicobacter pylori possesses two type IV secretion systemswith differing roles . The Cag secretory apparatus functionsin the translocation of CagA into host cells, and the Com systemis used to uptake DNA to facilitate genetic variation and enhancecell survival [16, 18].

In this study we report on the presence of both a plasmid anda chromosomally encoded type IV secretion system in B . cenocepacia strain K56-2 and describe the involvement of the plasmid-encoded system in the export of a putative protein[s] responsible for the ptw phenotype.

 
Media and growth conditions. Descriptions of plasmids and bacterial strains used in thisstudy are listed in Tables 1 and 2, respectively . Luria Bertani [LB] medium was used for routine maintenance of cultures . Minimal medium Vogel Bonner [69] amended with 1.0% glucose [VBG] wasused in mating experiments . Minimal medium M9 [59] containing1.0% glucose was used for culturing bacteria for supernatantanalysis . B . cenocepacia and Escherichia coli strains were grownat 37°C . Media were supplemented with appropriate concentrationsof antibiotics as needed for selection . Antibiotics were addedto media at the following concentrations: 50 µg of tetracycline[Tc]/ml and 100 µg of trimethoprim [Tp]/ml for B . cenocepaciaand 10 µg of Tc/ml, 10 µg of gentamicin [Gm]/ml, 40 µg of ampicillin/ml, and 100 µg of Tp/ml forE . coli.

 
Plant tissue assay and growth study. Plant tissue watersoaking activity was determined as previouslydescribed [24] . Onion cultivar 1015Y was used for plant tissueassays . Onion bulb scales were inoculated, in triplicate, with10 µl of an aqueous suspension of the isolate being testedas previously described [24] . Aqueous bacterial suspensionswere adjusted spectrophotometrically [A₄₂₅ = 0.5] to yield afinal concentration of 10⁶ CFU/scale . Sterile double-distilleddeionized water was used as a negative control and strain K56-2served as the positive control in all experiments . Scales wereplaced on a sheet of aluminum foil that had been surface sterilizedwith 70% ethanol in containers layered with paper towels thatwere moistened with double-distilled deionized water, sealed,and incubated at 37°C . Plant tissue watersoaking activitywas assessed at 24, 48, and 72 h postinoculation by measuringthe vertical and horizontal diameters of the zones for triplicatesamples in three independent experiments . Data were reportedas the average area of tissue watersoaking ± standard deviations [SD] for the three independent experiments in triplicate.

To obtain a quantitative measure of growth in plant tissue for strains K56-2, AE307, and AE310, bulb scales were inoculatedwith an aqueous suspension of the strains as described above.At 0, 24, 48, and 72 h postinoculation watersoaked zones weremeasured and tissue was processed to determine the bacterialpopulation . Scales were commuted and crushed in 0.0125 M phosphatebuffer [pH 7.1] containing Triton X-100 [0.01%] by using a sterilemortar and pestle . A dilution series of each tissue slurry wasplated to LB agar containing Gm [10 µg/ml] and Tc [10µg/ml] and was incubated at 37°C for 48 h . Populationdata for each time interval was expressed as the average CFU/gramof tissue ± SD for three independent experiments in triplicate.Tissue watersoaking data were reported as the average area ofwatersoaking ± SD for three independent experiments in triplicate.

Activity testing of culture supernatants. Cultures of strains K56-2, AE307, and AE310 were grown at 37°Cand harvested at late exponential phase [A₄₂₅ = 0.9] by centrifugation [16,000 x g, 15 min, at 5°C] . Supernatants [1 liter] werefilter sterilized using 0.22 µM filters [Pall] and wereconcentrated to 3 ml by using an Amicon ultrafiltration stirredcell [molecular weight cutoff, 10,000] . Protein concentrationof supernatant concentrates was determined by the method ofKoch and Putnam [36] . Concentrated uninoculated medium [1 liter]served as the negative control.

Plant protoplasts were used as the plant tissue system to obtaina quantitative measurement of activities of concentrated culture supernatants . Initial testing demonstrated that onion and carrot protoplasts were similar in sensitivity, and carrots were chosen because the presence of chromoplasts facilitated a more accurate evaluation of plasmolytic activity . Protoplasts were obtainedusing the following procedure [33] . Young carrots were placed in a 10% sodium hypochlorite solution for 5 min and then were rinsed four times with sterile double-distilled deionized water.The epidermis was resected with a sterile scalpel and discarded.Cortical and phloem tissues were obtained, diced into fragments 2 mm in width, and placed in a sterile petri dish containing20 ml of a filter-sterilized enzyme solution . The enzyme solutioncontained 10% mannitol [Fisher], 1.5% cellulase [CalBiochem],0.25% macerase [CalBiochem], and 0.75% bovine serum albuminfraction V [Sigma] [33] . Tissue was digested in the dark for5 h at 28°C with gentle shaking [50 rpm] . The carrot tissuewas strained through nylon mesh and centrifuged [825 x g for5 min at 25°C] . The supernatant was removed using a pipette,avoiding disturbance of the protoplasts . The protoplasts werewashed with 20 ml of 10% mannitol by resuspending with gentleswirling and centrifuged . The supernatant was removed and thepellet was gently resuspended in 10 ml of 10% mannitol, anda 5-ml 20% sucrose cushion was carefully layered in the bottomof the tube . The sample was centrifuged and the protoplastswere collected from the interface and counted with a hemacytometer[Hausser Scientific] . On average, 1.8 x 10⁶ protoplasts/ml were obtained.

The plasmolytic activity of supernatants was determined by using concentrates standardized for protein concentration and adjustedto 10% mannitol . The standardized concentrates, as well as dilutions [1:1, 1:5, and 1:10], were added to protoplasts . Assay mixturesand controls were incubated in 96-well microtiter plates [Corning]with an average concentration of 50 protoplasts per well . Assays,done in triplicate, were observed for changes in protoplastintegrity at 2-h intervals for 8 h using a Zeiss inverted microscope.In addition, the effect of temperature on plasmolytic activitywas tested by heating concentrates to 100°C for 10 min orat 80, 65, or 37°C for 1 h . Concentrates were also individuallytreated with 1-mg/ml final concentrations of proteinase K [BoehringerMannheim] dissolved in 0.05 M Tris-HCl, pH 8; DNase I [3,200U/mg; Sigma] in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, and 50µg of bovine serum albumin/ml; or RNase A [100 U/mg; Sigma]in 0.25 M Tris-HCl, pH 6.8 . All reaction mixtures were incubatedat temperatures optimal for each enzyme's activity and werethen adjusted to 10% mannitol before being mixed with protoplasts.Each incubation duration was performed in duplicate in threeindependent experiments with an average of 50 protoplasts examinedat 0, 2, 4, 6, and 8 h . Data were expressed as percent plasmolysis± SD for three independent experiments . Plasmolysis was analyzed as a function of time, dilution, treatment, and their interactions . Level of statistical significance of the different variables was determined with an analysis of variance . Statistical analysis was performed using SAS software and the general linear model procedure [SAS Institute Inc.].

Mating experiments. For triparental matings, donor, mobilizer, and recipient suspensionswere made in LB broth from cultures grown on solid media underselective conditions, as appropriate, for 18 h . Bacterial suspensionswere adjusted spectrophotometrically [A₆₀₀ = 0.5], mixed atan equal ratio [1:1:1], and transferred to a positively chargedsterile membrane layered on a 100- by 15-mm LB agar petri dish.Following a 6-h incubation period, the cells from the matingand respective controls were washed twice in phosphate buffer[0.125 M, pH 7.1] by centrifugation [12,096 x g for 10 min at5°C] . The bacterial pellets were suspended in phosphate buffer and dilution plated to media with appropriate antibiotic selection . Single-colony isolates of individual transconjugantswere obtained by streaking on selective media . Cultures weresubjected to survey lysis followed by agarose gel electrophoresis[23] to confirm plasmid transfer.

Plasposon mutagenesis. The plasposon pTnMod-RTp' was employed to obtain initial ptw-negativemutants of strain K56-2 [15] . Transconjugants were selectedon VBG supplemented with Tp . After 48 h of incubation at 37°C,isolated colonies were transferred to homologous media to confirmselection . Trimethoprim-resistant transconjugants were evaluatedfor ptw activity by using the onion tissue assay . Transconjugantsthat no longer demonstrated the ptw phenotype were single-colonypurified, retested, and retained for further analysis.

DNA isolation and sequence analysis. Genomic DNA was extracted from plasposon-generated ptw-negativeK56-2 mutants using a DNeasy kit [QIAGEN] . Three microgramsof DNA were digested with PstI and self ligated by using T4DNA ligase [New England Biolabs] . The resulting plasmids weretransformed into E . coli CC118pir by CaCl₂ transformation [59]and were selected on LB agar amended with Tp . Survey lysis andagarose gel electrophoresis were conducted to confirm the presenceof plasmid DNA in the transformants . Plasmid DNA for sequencingwas extracted using a HiSpeed Plasmid Midi kit [QIAGEN] . Primersdesigned from the 5' and 3' termini of the plasposon insertwere designated RTp1 and RTp2 [Table 3], respectively, and wereused to sequence the cloned genomic DNA from B . cenocepaciastrain K56-2 . Sequencing was conducted by the Institute of Developmentaland Molecular Biology, Gene Technologies Laboratory, Texas A&M University . MacVector 7.0 [Oxford Molecular, Ltd.] was usedfor DNA sequence analysis . DNA sequence for B . cenocepacia strain J2315 was produced by the Pathogen Sequencing Group at the Sanger Institute, Hinxton, Cambridge, and can be obtained from http://www.sanger.ac.uk/Projects/B_cenocepacia/.

 
Site-directed mutagenesis. Using PCR analysis, ptwD4, ptwC, ptwB4, and ptwB10 were foundto be located on two cosmids from a previously constructed K56-2library [62] . Primer sets PD4 [PD4-1 and PD4-2], PC [PC-1 andPC-2], PB4 [PB4-1 and PB4-2], and PB10 [PB10-1 and PB10-2] [Table3] were designed based on sequence data from the B . cenocepaciaGenome Project . Each PCR [50 µl] was performed using aTaq PCR Core kit [QIAGEN] and a GeneAmp 2700 [Applied Biosystems].Reactions were run for 25 cycles, and parameters were as follows:denaturation at 95°C for 30 s; annealing at 55, 52, 56.5,and 54°C for 1 min for primer sets PD4, PC, PB4, and PB10,respectively; and extension at 72°C for 75 s . Resultingproducts were analyzed by agarose gel electrophoresis . It wasdetermined that cosmid pSBC-9F5 contained ptwD4 and ptwC andthat pSBC-3H3 harbored ptwB4 and ptwB10, respectively . GenesptwD4, ptwC, ptwB4, and ptwB10, were disrupted using site-directed mutagenesis . A 10-kb BamHI fragment from pSBC-9F5 and an 11-kbBamHI fragment from pSBC-3H3 were individually inserted intothe multiple cloning site [MCS] of pJQ200SK [55] to construct pASE102 and pASE103, respectively . Plasmids pASE102 and pASE103were transformed into E . coli DH10B by electroporation [25 µF,2.5 kV, 200 ] followed by selection on LB agar amended withGm . Orientation of inserted DNA was determined by restrictionenzyme digest analysis using SmaI . Only plasmids containinginsertions that were in frame were used in the subsequent experiments.Using pASE102, plasmids pASE106 and pASE107 were constructedby inserting a Tc-resistance cassette, obtained as a 1.9-kbfragment from pBR325 [4], into the ScaI and EcoRV sites of ptwD4and ptwC, respectively . Plasmids pASE108 and pASE109 were constructedby inserting the Tc cassette into the ApaI and SacI sites ofptwB4 and ptwB10 of pASE103, respectively . Constructs pASE106, pASE107, pASE108, and pASE109 were electroporated into E . coli DH10B, and transformants were selected on LB agar amended withTc and Gm . Insertion of the Tc resistance cassette was confirmedby restriction enzyme digest analysis using BamHI and PCR usingprimer sets PD4, PC, PB4, and PB10 to confirm a 1.9-kb increasein plasmid size and PCR product, respectively . Plasmids pASE106,pASE107, pASE108, and pASE109 were individually mobilized intoK56-2 by utilizing pRK2013 in triparental matings to yield geneticexchange mutants . Tetracycline-resistant transconjugants weresingle-colony purified and retested for Tc^r . PCR using primersets PD4, PC, PB4, and PB10 and plasmid survey lysis were usedto confirm allelic exchange, reflected by the increase in PCRproduct and loss of the suicide vector . Single-colony isolateswere tested for the ptw phenotype.

Genes virB6 and virB11, from the chromosomally located type IV secretion system, were disrupted by site-directed mutagenesis. Primer sets VB6 [VB6-1 and VB6-2] and VB11 [VB11-1 and VB11-2] [Table 3] were designed based on sequence data from the Sanger Centre . Each PCR was performed as described above, except that the cycle parameters were as follows: denaturation at 95°Cfor 30 s; annealing at 57.5 and 56°C for 1 min for primersets VB6 and VB11, respectively; and extension at 72°C for75 s . PCR products were individually ligated into vector pDrive[QIAGEN] and were transformed into E . coli DH10B by electroporationfollowed by selection on LB agar amended with Ap to yield pASE110and pASE111, respectively . Genes virB6 and virB11 were excisedfrom pASE110 and pASE111, respectively, by double digestionusing restriction enzymes PstI and XhoI and were individuallyinserted into the MCS of pJQ200SK to construct pASE112 and pASE113,respectively . Plasmids pASE112 and pASE113 were transformedinto E . coli DH10B by electroporation followed by selectionon LB agar amended with Gm . Using pASE112 and pASE113, plasmidspASE114 and pASE115 were constructed by inserting a Tc resistancecassette, from pBR325, into the ScaI site of virB6 or virB11,respectively . Constructs pASE114 and pASE115 were individuallyelectroporated into E . coli DH10B and transformants were selectedon LB agar amended with Tc and Gm . Insertion of the Tc resistancecassette was confirmed by restriction enzyme digest analysisusing BamHI and PCR primer sets VB6 and VB11 to confirm a 1.9-kbincrease in plasmid size and PCR product, respectively . PlasmidspASE114 and pASE115 were individually mobilized into K56-2 bytriparental matings to yield genetic exchange mutants . Tetracycline-resistanttransconjugants were single-colony purified and retested forTc^r . PCR using primer sets VB6 and VB11 and plasmid survey lysiswere used to confirm allelic exchange, reflected by the increasein PCR product and loss of the suicide vector . Single-colonyisolates were tested for the ptw phenotype.

Complementation of disrupted genes. Complementation of the disrupted ptwD4, ptwB4, or ptwB10 was accomplished in trans . Plasmid pURF047 [63] was amended by insertinga Tp resistance cassette, obtained from R388 as a BamHI fragment,into the ScaI site in the bla gene to obtain pASE101 . A 10-kbBamHI fragment from pSBC-9F5 that contained ptwD4 and an 11-kbBamHI fragment from pSBC-3H3 that contained ptwB4 and ptwB11were individually inserted into the MCS of pASE101, resultingin pASE104 and pASE105, respectively . Plasmids pASE104 and pASE105were electrotransformed into E . coli DH10B, followed by selectionon LB agar amended with Tp and Gm . Orientation of inserted DNAwas determined by restriction enzyme digest analysis using ScaI.Only plasmids containing in-frame insertions were used in thesubsequent experiments . Plasmid pASE104 was mobilized into AE307,and pASE105 was mobilized into AE321 and AE322 to complementinsertionally disrupted ptw genes . Tetracycline-resistant andTp^r transconjugants were single-colony purified and retestedfor antibiotic resistance . Plasmid survey lysis and PCR wereused to confirm plasmid transfer, which was reflected by thepresence of both the resident and complement plasmid as wellas inserted and uninserted PCR products.

 
Isolation and characterization of ptw-negative mutants. A total of 5,000 transconjugants of strain K56-2 were obtainedby using the plasposon mutagenesis system . Genomic DNA from20 randomly selected transconjugants were digested with PstI,which does not cleave the TnMod-RTp' insert . The DNA fragmentswere separated by agarose gel electrophoresis, and Southernblots were probed with the Tp cassette to determine the randomnessof insertion . It was determined that the plasposon system wassuitable for the generation of random mutations in the genomeof B . cenocepacia strain K56-2 [data not shown].

From the pool of 5,000 transconjugants, 56 mutants were verified as ptw-negative using the plant tissue assay [Fig . 1] and demonstratedno other apparent phenotypic differences compared to phenotypeof the parental strain . The DNA flanking the plasposon insertionsite of 10 ptw-negative mutants was cloned and sequenced, anda BLAST search was performed . The translated gene products disruptedin clones GM237 and GM242 [Table 2] showed homology to VirD4-likeproteins, whereas GM241 and GM243 [Table 2] showed homologyto a putative regulator and promoter, respectively . Thus, ourinitial inquiry was focused on determining if strain K56-2 possessedand/or utilized a similar transfer system . Characterizationof the remaining ptw-negative mutants will be addressed in aseparate report.

 
Using sequence data from the B . cenocepacia Genome Project,a 60-kb region proximal to the identified virD4-like gene was analyzed . Strain J2315, the subject of the genome sequence project, is a member of the ET12 clonal lineage, as is strain K56-2 [47], and hence the arrangement of genes and their sequence was expected to be similar . A cluster of 11 genes potentially involved in the delivery of the ptw effector[s] was identified and designatedthe ptw cluster [Fig . 2] . Southern analysis of K56-2 lysates[data not shown] determined that the ptw cluster was locatedon a 92-kb resident plasmid . Other isolates of the ET12 lineage[J2315, BC7, and C5424] were also shown to harbor the ptw clusteron their resident plasmids [data not shown] . The resident plasmid,designated pK56-2, has a G-C ratio of 62.7%, and the ptw clusterhas a G-C ratio of 61%, whereas the value for the whole genomeis 66.9%.

 
DNA sequence and protein similarities of the ptw cluster. The encoded products of the ptw cluster showed homology to various translocation and/or conjugation related proteins from other bacteria, including Agrobacterium tumefaciens, Salmonella enterica serovar Typhi, Vibrio cholerae, Novosphingobium aromaticivorans, and E . coli . Based on amino acid sequence similarity to TraD and other VirD4-like proteins, the predicted function of ptwD4 could involve nucleoside triphosphatase activity [NTPase] and may therefore serve as an active motor for secretion [60, 61].PtwB4 and its homolog VirB4 of the VirB-VirD system may alsoexhibit NTPase activity [2] . Based on homology to R388 TrwC,PtwC may function as a component of a relaxosome [44] . PtwB7,PtwB8, PtwB9, and PtwB10 were predicted to form complexes inthe periplasm and/or membrane to create the pore [74] . PtwB11was also predicted to have NTPase activity and is part of theTraG subfamily, which belongs to the large superfamily of typeII/IV secretion NTPases [53] . PtwN, as well as PtwU, had noVirB-VirD homologs; however, they possessed homology to TraNand TraU of the F plasmid, respectively . In the F plasmid systemTraN is an adhesin and TraU is involved in mating pair stabilization[38].

Based on analysis of gene products with sequence similarityand the probable involvement in the expression of the ptw phenotype, ptwD4, ptwC, ptwB4, and ptwB10 were chosen for further analysis.Translation of the ptwD4 sequence resulted in a protein thatwas 640 amino acids in length, with a predicted molecular sizeof 72 kDa and a pI of 6.6 . Further analysis of the PtwD4 aminoacid sequence, using reverse position specific BLAST [rpsBLAST]and the conserved domain database, revealed a predicted WalkerA [P-loop] and B site for nucleotide binding [70] that is characteristicof type IV secretion ATP-binding proteins [74] . The predictedP-loop nucleotide binding motif extends from 117 to 124 in theamino acid sequence, and the Walker B motif extends from 338 to 344 . The translated sequence of ptwB4 was 1,013 amino acids in length, with a predicted molecular size of 108 kDa and apI of 6.0 . Further analysis of the PtwB4 amino acid sequencefor hydrophobicity revealed four putative transmembrane domains,which is characteristic of type IV secretion NTPases [74] . Translation of the ptwB10 sequence resulted in a protein that was 278 amino acids in length, with a predicted molecular size of 30 kDa and a pI of 5.2 . Using rpsBLAST and the conserved domain database,PtwB10 was shown to contain a well-conserved C-terminal hydrophobicregion [74] . Translation of the ptwC sequence resulted in aprotein that was 940 amino acids in length, with a predictedmolecular size of 104 kDa and a pI of 6.0 . A Pustell proteinmatrix analysis comparing PtwC and TrwC from R388 illustratedregions of similarity between the sequences . At the N terminus[amino acids 1 to 200], PtwC and TrwC showed 42% identity andsignature motifs [2 and 3] that are conserved in proteins withDNA-nicking activities [44] . An analysis of the PtwC C-terminalsequence indicated that PtwC contained 7 motifs [I, Ib, II,III, IV, V, and IV] characteristic of helicases [28].

Site-directed disruption of the ptw or virB-virD4 clusters and its effect on the ptw phenotype. Translated products of the ptw cluster showed homology to proteinsfrom known type IV secretion systems . Based on their predictedfunction, the role of ptwD4, ptwC, ptwB4, and ptwB10 in the expression of the ptw phenotype was determined . Watersoakingmutants of K56-2 with a disrupted ptwD4 were obtained in theinitial screening of the plasposon generated mutants and bysite-directed mutagenesis [Fig . 1 and Table 2] . Site-directedmutagenesis of ptwD4, ptwC, ptwB4, and ptwB10 was accomplishedby individual mobilization of pASE106, pASE107, pASE108, andpASE109, respectively, into K56-2 . Tetracycline-resistant transconjugantsshowed a 1.9-kb increase in PCR product, which reflected theinsertion of the Tc cassette [data not shown] . Insertions inptwD4, ptwB4, and ptwB10 resulted in loss of the ptw phenotype,whereas disruption of ptwC did not . Representative insertionallyinactivated ptw-negative mutants of ptwD4, ptwB4, and ptwB10were designated AE307, AE321, and AE322, respectively, whereasthe inserted ptwC derivative was designated AE320.

Complementation of ptw-negative mutants was accomplished by mobilization of pASE104 into AE307 and pASE105 into AE321 orAE322 and selecting for Tc^r and Tp^r transconjugants . All transconjugantstested were ptw positive [Fig . 3] . Plasmid pURF047 derivativeswere used in complementation studies, because pURF047 is a derivativeof pURF034 that contains the par locus and is maintained stablywithout selective pressure [>95% retention over 36 generations],which was a desirable parameter for plant tissue studies [14].A survey of transconjugant lysates confirmed plasmid transfer[data not shown] . Representatives of ptw-positive complementedptwD4, ptwB4, and ptwB10 mutants were designated AE310, AE323,and AE324, respectively [Fig . 3].

 
Genomic analysis of the J2315 sequence from the Sanger Centre identified a second cluster on chromosome II, which showed homology to the VirB-VirD type IV secretion system [Fig . 4] . Using PCR analysis, the presence of virB1, virB4, virB6, virB11, and virD4[Table 3], which span the entire virB-virD cluster, were confirmedin K56-2 [data not shown] . In A . tumefaciens, virB6 is thoughtto mediate assembly of the pilus and a functional secretionmachine through its effects on virB7 and virB9 multimerization [31], and virB11 is predicted to function as an NTPase, whichprovides the necessary energy for secretion [74] . Based on theirpredicted function, the role of virB6 and virB11 in the expressionof the ptw phenotype was assessed . Site-directed mutagenesisof virB6 and virB11 was accomplished by individual mobilizationof pASE114 and pASE115 into K56-2, yielding AE360 and AE361,respectively . Tetracycline-resistant transconjugants showeda 1.9-kb increase in PCR product, which reflected the insertionof the Tc cassette [data not shown] . Disruption of virB6 orvirB11 did not result in loss of the ptw phenotype.

 
Partial characterization of watersoaking effector[s] and population dynamics. Plant tissue assays indicated that the parental strain K56-2 and the complemented mutant [AE310] caused watersoaking, whereas the ptwD4 mutant [AE307] did not [Fig . 3] . Growth and watersoakingwere monitored over a 72-h period using the plant tissue assayto determine the contribution of ptw activity in survival andgrowth . Over the 72-h period both K56-2 and AE310 showed anapproximate 100-fold increase in CFU/gram of tissue and an increasein the watersoaking area, whereas AE307 showed no watersoakingactivity and an approximate 1,000-fold decrease in CFU/gramof tissue over the same period [Table 4].

 
Based on analysis of the ptw cluster and the putative role of a type IV secretion system in the export of an effector molecule[s], it was of interest to determine if culture supernatant concentrates showed effector activity . Over an 8-h duration, carrot protoplasts exposed to M9 medium concentrate did not experience significant plasmolysis compared to that of the protoplast controls [P > 0.05] [Table 5] . However, significant [P 0.05] plasmolysiswas observed for protoplasts exposed to the K56-2 or complementedmutant [AE310] concentrates as early as 2 h, and plasmolysisincreased at a significant rate for dilutions tested [up to1:5] over the 8-h time period compared to that of medium controls. Concentrated supernatant from the ptwD4 mutant [AE307] caused 6.7% plasmolysis after 8 h, which was not statistically different [P > 0.05] from plasmolysis resulting from treatment with medium controls . Dilution of the K56-2 and AE310 concentrates to a protein concentration of 0.02 µg/ml reduced activityto levels which approximated those of the control and AE307.Temperatures of 37, 65, and 80°C for 1 h or 100°C for10 min did not affect the plasmolysis activity of the K56-2concentrate . Complete inactivation of plasmolysis activity wasobserved when the K56-2 concentrate was treated with proteinaseK; however, incubation with DNase I or RNase A showed no effecton activity.

 
Previously, the role of a polygalacturonase [PehA] as a virulence factor in B . cepacia strain ATCC 25416 was investigated [24]. Derivatives cured of a resident plasmid that codes for the PehA no longer macerated onion tissue; however, the derivatives still produced a phenotype described as ptw . Of interest in the present study was the observation that all B . cepacia and B . cenocepacia isolates and a B . vietnamiensis strain from the Bcc experimental strain panel [47] produced the ptw phenotype in onion tissue[50] . In this study K56-2, a B . cenocepacia strain that producedthe plant disease-associated phenotype, was genetically analyzedand found to contain a plasmid-borne gene cluster, designatedptw, that encodes a type IV secretion system responsible forthe translocation of the ptw effector protein[s].

The identified gene products in the ptw cluster had homology to proteins from known type IV secretion systems . Bacteria have evolved type IV secretion systems to transfer DNA or protein macromolecules to a wide array of target cell types [2, 10,52] . Originally, the nomenclature referred to the VirB-VirD-encodedtranslocation system of A . tumefaciens and two closely relatedsystems encoded by the transfer region of the IncN plasmid pKM101and the ptl operon of Bordetella pertussis [10] . In the past decade, the type IV family has expanded considerably in number with the relaxation of defining criteria . Currently, type IV secretion systems are defined as translocation systems ancestrally related to any conjugation system of gram-negative or -positive bacteria [10, 25] . However, it is important to distinguish functionaltranslocation machines from mobile elements characteristicallyfound in bacterial genomes . Ding et al . [18] have suggestedthat mutagenesis of a putative type IV system should yield aphenotype at least consistent with a translocation defect.

Christie [10] has suggested subclassification of type IV secretionsystems based on ancestral lineage . Thus, the VirB-VirD typehas been designated IVA, and the ColIb-P9 Tra and L . pneumophilaDot-Icm types have been designated IVB . This subclassificationleft open the possibility of further division of gram-negativeand -positive secretion systems that differ from IVA and IVB.Therefore, an alternative grouping scheme has been suggested by Ding et al . [18] that separates systems based on functionand does not replace the one previously described but rather expands its usefulness . This classification method groups type IV secretion systems as [i] conjugation systems, [ii] effector translocator systems, or [iii] DNA uptake or release systems[18] . By definition, the conjugation systems deliver DNA substratesby establishing direct physical contact with target cells . Examples include the well-studied A . tumefaciens T-DNA transfer system and the F, RP4, and R388 plasmid transfer systems . Althoughthe conjugation systems are known mainly for their role in distributing DNA among bacterial populations, they can also translocate protein substrates independently of DNA [73] . There is also a subset of these systems that can transfer DNA and protein substrates to a range of eukaryotic cell types, including plant, fungal,and human [3, 71, 72] . Most of the members of the type IV effectortranslocator group inject their substrates directly into theeukaryotic cytosol . This type of translocation is now recognizedas the dominant virulence mechanism of the phytopathogen A.tumefaciens and of several medically important pathogens, includingH . pylori, L . pneumophila, Brucella spp., and Bartonella spp.[7] . Also included in this subfamily is the Ptl system of B.pertussis, even though this system exports its protein substrateindependently of host-cell contact . Presently, at least 10 typeIVA and several type IVB systems can be grouped as effectortranslocators and are thought to be essential for infection[18] . The DNA uptake and release group, which presently containsthree members, translocates DNA substrates across the cell envelopeto or from the extracellular milieu [18] . Neisseria gonorrhoeae uses a system encoded by the gonococcal genetic island to export DNA [17, 26] . Recent studies have established that this systemis very closely related to the F plasmid conjugation systemof E . coli, even though it is not performing the same function[17] . The two other members of this subfamily, Campylobacterjejuni and H . pylori, translocate DNA in the opposite direction,promoting genetic variation and cell survival [1, 29].

A notable feature of the type IV secretion systems is their extreme versatility . These systems can recognize a wide arrayof DNA and protein substrates, translocate substrates by both cell-contact-dependent and -independent mechanisms, and deliver substrates to an exceptionally wide range of prokaryotic and eukaryotic taxa [18] . The ptw cluster contains homologs to componentsof multiple type IV secretion systems [Fig. 2] . With respectto function, the plasmid-encoded type IV secretion system wehave identified in B . cenocepacia strain K56-2 was involvedin export of an effector[s] that was responsible for the ptwphenotype . The facts that the Ptw system was involved in thetranslocation of a protein[s], that it did not contain all ofthe necessary genetic components to support conjugation, andthat no oriT homolog was identified indicate that functionallyit is a member of the effector translocator subfamily . The differencein the G-C ratio between pK56-2 harboring the ptw cluster [62.7%]and the G-C ratio for the entire B . cenocepacia genome [66.9%]suggests acquisition by horizontal transfer [34].

The presence of a second type IV secretion system in B . cenocepacia is not unprecedented, because many bacteria, such as H . pylori and A . tumefaciens, have been found to harbor multiple secretion systems [18, 35] . The type IV secretion system located on chromosomeII showed most similarity to that of the A . tumefaciens VirB-VirDtranslocation system with respect to arrangement and gene productsimilarity [Fig. 4] . The difference in the G-C ratio of the chromosomally located type IV secretion system [63%] and theG-C ratio of the entire genome [66.9%] suggests horizontal acquisition[34], as with the Ptw translocation system . Evidence suggeststhat the Ptw system is responsible for export of the protein[s]involved in expression of the ptw phenotype; however, the substrate[s] translocated by the VirB-D4 system is unknown.

Identification of a cluster of genes that encoded proteins with similarities to components of a type IV secretion system allowedfor strategic generation of K56-2 mutants to support the hypothesisthat such a system was involved in expression of the ptw phenotype.The genes selected for functional characterization were ptwD4, ptwC, ptwB4, and ptwB10 . VirD4-like proteins [PtwD4 homologs] are cytoplasmic membrane NTP-binding proteins that are essential for coupling the relaxosome to the macromolecular transport system [51, 61] . It is thought to interact with the oriT-boundrelaxosome, which is made up of TrwC and TrwA, to facilitateDNA transfer [19, 45] . In the Ptw system, however, there isno TrwA homolog . The ptwD4 product appears to have functionssimilar to those of Hp0524 of H . pylori . The hp0524 productis critical for the transfer of CagA from the bacterium to epithelial cells [21] . Further analysis revealed that the PtwD4 amino acidsequence contained P-loop and Walker B sites, which are characteristicof type IV secretion ATP-binding proteins . The ptwC producthad homology to TrwC, which is both a relaxase that cleaves at the nic site within the oriT in a strand- and sequence-specificmanner, and a helicase, which is essential for transfer [48,49] . Analysis of PtwC revealed that the N terminus possessedsignature motifs [2 and 3] conserved in proteins with DNA-nickingactivities [44], and the C terminus possessed signature motifs[I, Ib, II, III, IV, V, and VI] conserved in DNA helicases [28];however, there is no identified oriT to facilitate the cleavageand thus linearization required for DNA transfer . VirB10, thePtwB10 homolog, is a protein located in the periplasmic spacethat is believed to form the core of the transfer machineryand is possibly part of the pore that spans the inner and outermembranes [74] . PtwB10 was shown to contain the C-terminal hydrophobicregion that is characteristic of VirB10-like proteins . PtwB4showed similarity to VirB4 of A . tumefaciens, which is a putativenucleoside triphosphatase that may also serve as an active motorfor secretion [74] . The PtwB4 amino acid sequence was shownto possess four transmembrane regions that are characteristicof VirB4-like NTPases [74] . The ptw phenotype was not expressedby K56-2 derivatives with a disruption in ptwD4, ptwB4, or ptwB10but was expressed by a derivative with a disruption in ptwC.These results correlated with the predicted function of thegene products, because successful secretion of the ptw effectorprotein[s] would likely involve the following: a protein thatpotentially couples the moiety to the secretion system and suppliesthe necessary energy for export [PtwD4]; a protein that actsin the assembly of the pore necessary for the passage of thesubstrate and provides energy necessary for the system to function[PtwB4]; and a protein involved in structural formation of apore [PtwB10], but not one involved in the generation of a relaxosome[PtwC].

By combining mutational analysis with cytotoxic assays employing both plant tissue and protoplasts, we have identified a typeIV secretion system that appears to export an effector molecule[s]that is proteinaceous in nature and is responsible for the activity.This activity plays an important role in the ability of B . cenocepacia strain K56-2 to cause watersoaking and to grow in plant tissue [Fig . 2, Table 4] . It appears that in the plant tissue assaythe role of the effector[s] is to provide bacterial cells withneeded nutrients by causing leakage of the plant cell cytosol.Presently, the role of the putative virulence effector protein[s]in contributing to growth and infection in a pulmonary environmentis unknown; however, the ptw phenotype appears to be commonamong isolates belonging to B . cenocepacia that infect CF patients.

Protein DspE of Erwinia amylovora and WtsE of Pantoea stewartii subsp . stewartii are involved in elicitation of the watersoaking phenotype in their respective hosts . The dspE product is a 198-kDa protein that is required for the production of fireblight disease symptoms that include watersoaking of apple and pear tissue[5] . A disruption in wtsE renders P . stewartii subsp . stewartii incapable of eliciting an observable watersoaking phenotypeand causing a systemic corn leaf infection [22] . In contrast to the ptw effector molecule[s], a type III secretion systemis presumed to export DspE and WtsE from the phytopathogen tothe plant hosts.

There are two types of substrates typically transported by both type III and type IV secretion systems [52, 58] . The first groupalters host processes by mimicking the function of a eukaryoticprotein . Substrate proteins in this category, however, do nothave significant sequence similarities with the eukaryotic factor they mimic, which makes identification difficult . The second class are genes that were "stolen" from eukaryotic host cellsand then reshaped . The RalF protein of the L . pneumophila Dot/Icm system falls into the latter group [52] . Substrates in this category are relatively easy to identify with genomic analysis. Secreted substrates from either group can vary in size from 22 kDa for A . tumefaciens VirF to 145 kDa for H . pylori CagA.They also vary in composition, from monomers to multisubunitstructures that may include protein and/or DNA [18] . Thus, thereare no universally conserved primary sequence motifs or physicalcharacteristics that are readily discernible for secreted effectors.

In conclusion, our study has identified a plasmid-encoded typeIV secretion system in B . cenocepacia strain K56-2 that was functionally an effector translocator . The chromosomally locatedtype IV secretion system showed modular similarity to the A. tumefaciens VirB-VirD translocation system; however, it wasnot responsible for the ptw phenotype in B . cenocepacia strain K56-2.

There are multiple examples for the role of type IV secretion systems in translocation of both plasmid DNA and proteins associated with plant and human pathogenesis [9, 10] . The role of thisputative virulence effector[s] in contributing to disease isbeing determined in studies using a murine model of pulmonaryinfection . Studies to identify the structural gene[s] for theeffector protein[s] and regulatory subunits are in progress.

 
We are grateful to Pamela Sokol for providing K56-2 genomiclibrary blots, to James Samuel for his critical reading of themanuscript, and James Starr for his statistical help . We alsothank Michael B . Willett, Bianca S . Batista, Francisco Vielma,and Arwyn Hood for their technical assistance.

This work was supported by grant GONZAL03G0 from the Cystic Fibrosis Foundation.

 
* Corresponding author . Mailing address: Department of Plant Pathology & Microbiology, Texas A&M University, 2132 TAMU, College Station, TX 77843 . Phone: [979] 845-7610 . Fax: [979] 845-6483 . E-mail: cf-gonzalez@tamu.edu .

Present address: Cotton Pathology Research Laboratory, USDA,ARS, SPARC, College Station, TX 77845.

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