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Journal of Bacteriology, February 2003, p. 760-771, Vol. 185, No. 3
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.3.760-771.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Agrobacterium tumefaciens Twin-Arginine-Dependent Translocation Is Important for Virulence, Flagellation, and Chemotaxis but Not Type IV Secretion

Zhiyong Ding and Peter J. Christie*

Department of Microbiology and Molecular Genetics, The University of Texas-Houston Medical School, Houston, Texas 77030

Received 10 September 2002/ Accepted 31 October 2002


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study characterized the contribution of the twin-arginine translocation (TAT) pathway to growth, motility, and virulence of the phytopathogen Agrobacterium tumefaciens. In contrast to wild-type strain A348, a tatC null mutant failed to export the green fluorescent protein fused to the trimethylamine N-oxide reductase (TorA) signal sequence or to grow on nitrate as a sole electron acceptor during anaerobic growth. The tatC mutant displayed defects in growth rate and cell division but not in cell viability, and it also released abundant levels of several proteins into the culture supernatant when grown in rich medium or in vir induction minimal medium. Nearly all A348 cells were highly motile in both rich and minimal media. By contrast, approximately 0.1% of the tatC mutant cells were motile in rich medium, and <0.01% were motile in vir induction medium. Nonmotile tatC mutant cells lacked detectable flagella, whereas motile tatC mutant cells collected from the edge of a motility halo possessed flagella but not because of reversion to a functional TAT system. Motile tatC cells failed to exhibit chemotaxis toward sugars under aerobic conditions or towards nitrate under anaerobic conditions. The tatC mutant was highly attenuated for virulence, only occasionally (~15% of inoculations) inciting formation of small tumors on plants after a prolonged incubation period of 6 to 8 weeks. However, an enriched subpopulation of motile tatC mutants exhibited enhanced virulence compared to the nonmotile variants. Finally, the tatC mutant transferred T-DNA and protein effectors to plant cells and a mobilizable IncQ plasmid to agrobacterial recipients at wild-type levels. Together, our findings establish that, in addition to its role in secretion of folded cofactor-bound enzymes functioning in alternative respiration, the TAT system of A. tumefaciens is an important virulence determinant. Furthermore, this secretion pathway contributes to flagellar biogenesis and chemotactic responses but not to sensory perception of plant signals or the assembly of a type IV secretion system.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Gram-negative bacteria secrete proteins across the inner membrane in an unfolded conformation by the general secretory pathway (GSP) or in a folded conformation by the sec-independent twin-arginine translocation (TAT) pathway (7, 49). This latter system was only recently discovered in bacteria and is structurally and functionally related to the {Delta}pH-dependent protein import pathway of the plant chloroplast thyalkoid membrane (6). In bacteria, the TAT system is used predominantly for secretion of cofactor-bound enzymes to the periplasm for function in various respiratory and photosynthetic electron transport pathways (7). However, the TAT system also has been shown to direct the secretion of (i) multisubunit enzyme complexes involved in respiration (37, 58); (ii) monomeric, cofactorless proteins whose functions are unrelated to energy metabolism (2, 50, 55); and (iii) integral inner membrane proteins (42). Of particular interest is that the TAT system (40, 50), but not the GSP (20), can export an active form of the green fluorescent protein (GFP) fused to a characteristic TAT signal sequence. Further, the TAT system of Pseudomonas aeruginosa secretes cofactorless hemolytic (PlcH) and nonhemolytic (PlcN) phospholipases across the cytoplasmic membrane, where, in a second translocation step, these substrates are exported via a type II secretin across the outer membrane (55).

In Escherichia coli, the TAT system is encoded by the tatA, tatB, tatC, and tatE genes (7). tatA, tatB, and tatC are cotranscribed from the same promoter, and tatE is located elsewhere in the genome. tatD is cotranscribed with tatA, tatB, and tatC but encodes a DNase with no discernible role in TAT translocation (57). Mutagenesis studies have shown that TatB and TatC are essential TAT pathway components, whereas TatA and TatE, which are ~60% identical, can functionally substitute for one another. TatA, TatB, and TatC interact with each other (9, 41), and a functional TAT system has been reconstituted in vitro (59). The TAT system recognizes secretion substrates by virtue of the presence of a characteristic RR motif, e.g., S/L-R-R-X-F-L-K, near the amino terminus of an unusually long signal sequence. Additionally, TAT signal sequences generally are less hydrophobic than those conferring sec-dependent translocation, and they often possess a positively charged Sec avoidance signal near the carboxyl-terminal end (38, 48). Sequences in the mature protein are also thought to play a role in substrate selection by the TAT system (38, 42).

Our laboratory characterizes virulence mechanisms of the phytopathogen Agrobacterium tumefaciens (16). A. tumefaciens has long served as a useful model for mechanistic studies of processes of general importance for pathogenesis, including pathogen-host signaling (25, 60), colonization (32), and macromolecular trafficking (16). The hallmark of the A. tumefaciens infection process is the interkingdom transfer of a segment of the bacterial genome, the oncogenic T-DNA, as well as several effector proteins to plant cells via a type IV secretion machine (51). For translocation of these virulence determinants, however, A. tumefaciens first must establish productive contact with the plant cell target. Attachment is mediated by an array of surface factors, including cellulose fibrils (31) and exopolysaccharide (13), as well as periplasmic ß-1,2-glucan (4). Motility and chemotaxis to plant-derived signals also contribute to the infection process (15), indicating that, as for many mammalian pathogens, the flagellum also functions as a bona fide virulence determinant. Finally, recent screens for mutants defective in biofilm formation have led to the identification of a number of loci encoding surface-associated or secreted proteins (C. Fuqua, personal communication). These observations clearly establish that A. tumefaciens pathogenesis intimately depends on elaboration of several surface structures and secreted factors operating at the bacterium-host interface.

Besides secretion of phospholipases (55), the P. aeruginosa TAT system exerts effects on motility (33). These findings raise the important question of whether the TAT system participates in the biogenesis or function of surface-located supramolecular structures such as flagella or various pili. In the present study, we identified the tat genes of A. tumefaciens, constructed a tatC null mutation, and cloned the wild-type tatC gene downstream of regulatable promoters. We demonstrate that an intact TAT system is vital for A. tumefaciens infection of plant cells. We further show that the TAT system is required for wild-type cell division, chemotaxis, and flagellar biogenesis but is completely dispensable for type IV secretion to plant and bacterial target cells. We conclude that, in addition to its role in secretion of cofactor-bound proteins and virulence factors, the TAT system participates in both the assembly and function of one cell surface machine, the flagellum, but not another, the type IV translocase-conjugative pilus.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Enzymes, chemicals, and reagents. Restriction enzymes were purchased from Promega (Madison, Wis.), New England Biolabs (Beverly, Mass.), or Gibco-BRL (Grand Island, N.Y.). Klenow fragment of E. coli DNA polymerase I and T4 DNA ligase were from Promega. Basic fuchsin used for staining flagella was from Sigma Chemical Co. (St. Louis, Mo.). Isopropyl-ß-D-thiogalactopyranoside (IPTG), L-arabinose, chloramphenicol, and kanamycin were from Sigma Chemical Co., and carbenicillin was from Gemini Bio-Products Inc. (Calabasas, Calif.).

Bacterial strains, plasmids, and growth conditions. Table 1 lists the strains and plasmids used in these studies. E. coli strains were maintained on Luria-Bertani medium, and A. tumefaciens strains were maintained on MG/L (rich) medium (essentially Luria-Bertani medium with added salts [23]) or on ABIM, a minimal medium (pH 5.5) supplemented with acetosyringone for induction of the vir genes (23). Cells were grown anaerobically in screw-cap test tubes with continuous sparging with 95% N2 and 5% CO2 (34) in M9 minimal medium supplemented with glycerol (0.4% wt/vol) as a carbon source and sodium nitrate (0.4% wt/vol) as a respiratory oxidant. The medium was supplemented with antibiotics (concentrations in micrograms per milliliter) as follows: chloramphenicol (20), carbenicillin (50), kanamycin (50), tetracycline (5), spectinomycin (400), and gentamicin (100).


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  		TABLE 1. Strains and plasmids used in this study

 
Recombinant DNA techniques. DNA manipulations and DNA electrophoresis were performed as previously described (23). PCR amplification was performed with a Perkin-Elmer Cetus DNA thermocycler using Pwo DNA polymerase (Boehringer, Mannheim, Germany). DNA sequencing was carried out at the DNA Core Facility of the Department of Microbiology and Molecular Genetics with an ABI 373A DNA sequencer (Perkin-Elmer) using Taq polymerase in a thermal cycling reaction. Oligonucleotides were purchased from Sigma-Genosys (Woodlands, Tex.).

Construction of an A. tumefaciens tatC mutant. The oligonucleotide primers 5'-AGGGATCCATATGAGCGGGGATACCGAGG-3' (BamHI and NdeI sites are underlined) and 5'-TCTCTCGAGTCAGGTCTCTTCCAGCTCCG-3' (the XhoI site is underlined) were used to PCR amplify the tatC gene from the A. tumefaciens A348 genome. pZD29 carrying wild-type tatC was constructed by cloning the PCR fragment into pCR2.1-TOPO. pZD26 carrying tatC was constructed by cloning the 800-bp BamHI/XhoI fragment from pZD29 into pBSK.NdeI. pZD31 carrying a tatC gene disruption was constructed by inserting a Kanr gene cassette from pUC4K as a 1.2-kb HincII fragment into the unique NarI site at bp 315 relative to the start site of tatC. pZD41 was constructed by inserting a ~2.3-kb PstI fragment containing the sacB gene from pBB50 into pZD31. Plasmid pZD41 was electroporated into A. tumefaciens A348 with Kanr and Crbr selection for integration at the tatC locus by a single-crossover event. These recombinants were grown in MG/L with kanamycin (50 µg/ml) to allow for excision of vector sequences by a second crossover event. The overnight cultures were streaked onto MG/L plates containing 5% sucrose and kanamycin (50 µg/ml) to select for Sucr Kanr Crbs double recombinants. Three independent recombinants were analyzed by PCR amplification across the tatC gene. In each case, PCR products of an expected size of ~2 kb were shown by sequence analysis to carry the expected tatC gene disruption.

tatC and torA::GFP expression plasmids. Plasmid pZD36 expressing Plac-tatC was constructed by digesting pZD26 with NdeI and religation to delete ~100 bp of multiple cloning site sequence. pZD44 expressing PvirB-tatC was constructed by substituting a ~0.8-kb NdeI/KpnI fragment carrying tatC from pZD36 for virB1 in similarly digested pPC914KS. pWM1487 is a pBR322 derivative expressing PBAD-torA::GFP. pZD51 expressing Ptac-torA::GFP was constructed by introducing the 950-bp NheI/HindIII fragment carrying torA::GFP from pWM1487 into the unique HpaI site of pMMB22. Plasmids utilizing a ColE1 replication origin were ligated to broad-host-range plasmid pSW172 for introduction into A. tumefaciens. Such cointegrate plasmids are given the ColE1 plasmid name plus a B to indicate broad-host-range replication.

Isolation of extracellular material. Material in culture supernatants was filtered through a 0.22-µm-pore-size cellulose acetate membrane to remove any unpelleted cells, concentrated 20-fold by centrifugation through Amicon filters (Centricon-30), and analyzed directly by silver staining of sodium dodecyl sulfate (SDS)-polyacrylamide gels. To isolate flagella and other cell surface-associated proteins, A. tumefaciens was grown at 25°C in MG/L broth with shaking to an optical density at 600 nm (OD600) of 0.5 or on MG/L agar plates incubated overnight. To isolate T pili, cells were grown to an OD600 of 0.5 in MG/L medium, harvested, diluted to an OD600 of 0.2 in ABIM, and induced for vir gene expression by shaking for 6 h at 22°C. Next, 200 µl of the acetosyringone-induced culture was spread on ABIM agar plates, and the plates were incubated for 3 days at 18°C. The procedure for isolation of flagella and T pili involved resuspension of cells harvested from broth or plates in 50 mM KH2PO4 buffer, pH 5.5. This cell suspension was passed through a 25-gauge needle 10 times, and cells were removed by centrifugation at 14,000 x g for 30 min at 4°C. The supernatant was filtered to remove whole cells and then centrifuged at 55,000 x g for 60 min at 4°C and resuspended in 50 µl of distilled water for analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Protein analysis, immunoblotting, and cell fractionation. Proteins were resolved by SDS-PAGE or with a Tricine-SDS-PAGE system as previously described (39). Total cellular or extracellular proteins were visualized by silver staining of SDS-polyacrylamide gels. Vir and ChvE proteins were visualized by SDS-PAGE and immunostaining with our collection of Vir antibodies (39) and ChvE antibodies kindly supplied by Y. Machida. Molecular size markers were obtained from Gibco-BRL. Fractionation of A. tumefaciens into soluble material (cytoplasm and periplasm) and insoluble material (cytoplasmic and outer membranes) was carried out as previously described (39). Cellular and subcellular fractions were loaded on a per-cell-equivalent basis to compare protein abundances in different strains.

Motility and chemotaxis assays. Motility assays were performed in MG/L and ABIM 0.3% soft agar medium as described previously (15). Cell cultures were normalized to an OD600 of 0.5, and 2 µl of each strain (with equivalent CFU) was inoculated onto the surface of the motility plates. Motility was examined at 12, 24, and 48 h of incubation at 18, 22, and 28°C. At least five independent motility assays were carried out for each strain and condition. Chemotaxis assays were performed on 0.3% soft agar plates without carbon sources. A Whatman paper disk saturated with 15% (wt/vol) glucose, maltose, fructose, or sucrose was placed on the surface of the medium. Two microliters of each cell culture normalized to the same OD600 was inoculated onto the swarm plate 4 cm from the paper disk. The chemotaxis plate was maintained at 18 or 25°C and examined throughout a 96-h incubation period.

Flagellum staining. Flagella were stained with a Leifson protocol modified by Clark (17). Briefly, A. tumefaciens cells were grown in MG/L medium to late logarithmic phase with shaking at 28°C. Broth cultures were formalinized by adding 50 µl of 37% formaldehyde solution per ml of culture. Cells were pelleted by centrifugation, washed once with distilled water with care, and then resuspended in distilled water. Five microliters of cell suspension was dropped onto a clean slide and allowed to air dry, and then 0.5 ml of stain solution was added to stain for 5 min. The slides were washed with tap water and allowed to air dry. Flagella were detected by light microscopy.

Microscopy and image analysis. Newly transformed cells of A. tumefaciens were grown in MG/L medium at 28°C to an OD600 of 0.5. IPTG (25 µM final concentration) was added to induce gene expression from the Ptac promoter for fluorescence analyses. Images of cells were acquired with an Olympus BX60 microscope equipped with a 100x oil immersion phase-contrast objective, a standard fluorescein isothiocyanate filter set for GFP, and an Optronics Engineering DEI-750 24-bit color video camera. Images were captured and digitized with a Scion LG3 framegrabber and manipulated with Adobe Photoshop. Care was always taken to minimize exposure of the bacteria to the blue excitation light to minimize photobleaching.

Conjugation assays. The RSF1010 derivative pML122Genr was introduced into various A. tumefaciens donor strains by electroporation. A. tumefaciens strains carrying pML122Genr were mated with a Spcr derivative of A348 or strain PC2000 (tatC) as previously described (39). Briefly, mid-log-phase (OD600 = 0.5) cells were harvested and incubated in ABIM for 6 h at 22°C to induce expression of the vir genes. Five microliters of preinduced donor and recipient cells was mixed on a nitrocellulose filter on an IM agar plate, and the plate was incubated for 3 days at 18°C. Mating mixtures were recovered from filters and plated onto media selective for transconjugants or serially diluted for determination of transconjugant and donor or recipient cell numbers. Frequencies of transfer were estimated as transconjugants recovered per donor or recipient. Experiments were repeated in triplicate, and results for a representative experiment are reported.

Virulence assays. A. tumefaciens strains were tested for virulence by inoculation of ~108 CFU of mid-logarithmic-phase cell cultures on wound sites of Kalanchoe daigremontiana leaves. The controls for the tumorigenesis assays included coinoculation of the same leaf with wild-type A348 and strain A136 lacking plasmid Ti. Virulence was scored in terms of tumor size and time course of tumor appearance. Assays were repeated at least four times for each strain on separate leaves. Tumors were photographed 4 to 6 weeks after inoculation.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A. tumefaciens tat loci. A search of the completed A. tumefaciens C58 genome revealed a tat locus composed of tatA, tatB, and tatC on the circular chromosome (Fig. 1A). There is a discernible promoter upstream of tatA and close juxtaposition of the tat genes (50 bp between tatA and tatB and a 4-bp overlap between tatB and tatC), suggestive of an arrangement as a single operon. The genome carries two possible tatD genes, one (accession no. gi_17935395) located ~200 kb from the tatABC locus and a second (gi_17938608) on pATC58, and no discernible tatE gene. The deduced TatA, TatB, and TatC proteins, of 70, 247, and 267 residues, respectively, are most highly related to their homologs of the phylogenetically related {alpha}-proteobacteria, e.g., Sinorhizobium meliloti, Brucella melitensis, and Caulobacter crescentus, and less related to those of E. coli (Fig. 1A). The TMHMM (version 2.0) algorithm predicts that the A. tumefaciens TatA, TatB, and TatC proteins are configured at the cytoplasmic membrane similarly to the corresponding E. coli proteins, with TatA and TatB spanning the membrane once and TatC spanning the membrane a possible six times.



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  		FIG. 1. Functionality of the A. tumefaciens TAT pathway. (A) Percent identities between the A. tumefaciens Tat proteins and those of related {alpha}-proteobacteria and E. coli. Accession numbers: TatA, NP_532390.1; TatB, NP_532389.1; TatC, NP_532388.1. (B) Visualization of TAT-dependent TorA::GFP export to the periplasm. Wild-type strain A348 and the tatC mutant PC2000, expressing Ptac-torA::GFP from plasmid pZD51, were grown as described in the text and examined by fluorescence microscopy. (C) Representative growth curves of wild-type A348, PC2000, and PC2000(pZDB36) expressing Plac-tatC during anaerobic growth with glycerol as a carbon source and nitrate as an electron acceptor; replicates of these experiments yielded similar results.

 
The TAT system is functional in A. tumefaciens. To assess the functionality of the TAT system in A. tumefaciens, we introduced plasmid pZD51, which produces GFP fused to the signal sequence of E. coli trimethylamine N-oxide reductase (TorA), into wild-type A348 and the isogenic tatC null mutant, designated strain 2000. In E. coli, TorA::GFP is secreted to the periplasm via the TAT system, resulting in a halo pattern of fluorescence. E. coli tat mutants fail to secrete TorA::GFP, and cells fluoresce uniformly by virtue of retention of TorA::GFP in the cytoplasm (50). Similarly, in screens of several thousand cells from cultures grown in rich MG/L medium to an OD600 of 0.5, virtually all A348(pZD51) cells expressing Ptac-torA::GFP displayed a halo pattern of fluorescence (Fig. 1B). In striking contrast, similarly grown PC2000(pZD51) cells expressing the same construct were exclusively uniformly fluorescent (Fig. 1B). tatC-dependent export of TorA::GFP strongly suggests that the TAT system is functional in A. tumefaciens.

Previous studies showed that the TAT systems of E. coli and other species export cofactor-bound proteins required for anaerobic growth in the presence of a nonfermentable carbon source, e.g., glycerol, and trimethylamine N-oxide, dimethyl sulfoxide, nitrate, or fumarate as a respiratory oxidant (27, 43). We searched the A. tumefaciens genome for TAT motifs (RRxF or RRxL) at appropriate positions in signal sequences identified by the SignalP-2.0 algorithm (http://www.cbs.dtu.dk/services/Signal P-2.0/) and compared the list of possible TAT substrates with that kindly supplied to us by the laboratory of M. Pohlschröder with a newly developed TATFIND algorithm (Table 2). Notable among the possible TAT substrates are a number of respiratory subunits, e.g., periplasmic nitrate reductase, formate dehydrogenase H alpha subunit, and ubiquinol-cytochrome c reductase FeS (Rieske) subunit. To determine the importance of the TAT system for alternative respiration during anaerobic growth, A. tumefaciens strains were incubated anaerobically in glycerol- and nitrate-containing M9 minimal medium. Wild-type A348 displayed anaerobic growth under these conditions, whereas the tatC mutant PC2000 showed no visible growth throughout a 96-h incubation period. Introduction of pZDB36, a plasmid expressing Plac-tatC, completely restored growth of strain PC2000 to wild-type levels (Fig. 1C). We conclude that the TAT system of A. tumefaciens is essential for anaerobic growth with nitrate as an electron acceptor, most likely due to a requirement for secretion of cofactor-bound redox enzymes.


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  		TABLE 2. Potential TAT substrates of A. tumefaciensa

 
The tatC mutation impairs cell growth and division. Next, we characterized the effects of the tatC mutation on aerobic growth and cell morphology. In rich (MG/L) medium, the tatC mutant exhibited a pronounced lag in growth compared to A348, whereas the complemented strain, PC2000(pZDB36), grew at wild-type levels (Fig. 2A). As noted above, this effect could be due to a deficiency in export of respiratory proteins bearing TAT signal sequences, such as the Rieske subunit. However, by 48 h the tatC mutant attained an OD600 comparable to that of strains expressing the wild-type tatC gene. Furthermore, the tatC mutant exhibited no loss in cell viability compared to A348 or PC2000(pZDB36) throughout the growth curve, as shown by CFU quantitations at several different ODs (data not shown). These findings suggest that the effect of the tatC mutation on aerobic growth is fairly modest and is limited to a reduction in growth rate during the mid-logarithmic growth phase.



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  		FIG. 2. Effect of the tatC mutation on aerobic growth and cell division. (A) Representative growth curves of A348, PC2000, and PC2000(pZDB36) expressing Plac-tatC in rich (MG/L) medium. (B) Light microscope analysis of corresponding cells from cultures grown to late logarithmic phase. tatC mutant cells are appreciably larger and more spherical and possess terminal buds or branches (arrowheads).

 
A348, PC2000, and PC2000(pZDB36) cells displayed similar cell shapes and sizes within the first 12 h following inoculation into fresh medium when examined by phase-contrast microscopy. By 20 h, however, most tatC cells were visibly larger and more spherical than wild-type cells. Additionally, nearly 10% of the tatC mutants were branched, appearing as distinct X or Y shapes (Fig. 2B). Growth in MG/L and vir induction minimal (ABIM) media yielded approximately the same percentage of cells with gross morphological defects. We never detected these aberrant morphologies among A348 or PC2000(pZDB36) cells under any growth conditions. Interestingly, these are precisely the types of morphological aberrations visualized in A. tumefaciens and S. meliloti when cell septation is blocked by cephalexin treatment or FtsZ overproduction (29). The A. tumefaciens TAT system therefore appears to influence a late stage of cell division. In E. coli, tat mutants are similarly perturbed in cell division, as also shown by an increase in cell size, but instead of branching, these mutants form long chains (47).

Twin-arginine translocation is important for A. tumefaciens virulence. We next tested the effect of the tatC mutation on the capacity of A. tumefaciens to incite tumor production on susceptible plant tissue. A348 inoculated at ~106 to 108 CFU typically induces tumors on wounded K. daigremontiana leaves within 2 to 3 weeks. In striking contrast, within this time period, the tatC mutant failed to induce any tumor formation. At 6 to 8 weeks, small tumorous foci were observed, but on only ~15% of wound sites that were exposed to a very heavy inoculum size of >1010 CFU (Fig. 3A). Complementation of the tatC mutation with plasmids bearing Plac-tatC or PvirB-tatC completely rescued the virulence phenotype (Fig. 3A). These findings establish that the TAT system functions as an important virulence determinant during the A. tumefaciens infection process.



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  		FIG. 3. Effect of the tatC mutation on A. tumefaciens virulence and type IV secretion. (A) Top leaf, virulence test of PC2000 and PC2000 bearing pZDB36 (Plac-tatC) or pZDB44 (PvirB-tatC) with A348 (wild type) and A136 (Ti plasmidless) as positive and negative controls; middle leaf, PC2000 mixed with LBA4404 ({Delta}T-DNA) or At12516 ({Delta}virE2) to monitor the capacity of the tatC mutant to secrete T-DNA or VirE2, respectively; bottom leaf, PC2000 mixed with PC1000 ({Delta}virB) or Mx355 (virD4::Tn3HoHo1) to test for TAT-dependent secretion of unidentified effectors to plant cells. (B) Western blot analysis showing accumulation of the VirB proteins listed at the right and ChvE in whole-cell extracts (lanes WC) and extracellular material (lanes E) of A348, PC2000, and PC2000(pZDB36) expressing Plac-tatC.

 
Twin-arginine translocation is not required for type IV secretion of proteins and DNA. To understand the relationship between twin-arginine translocation and virulence, we first examined whether a functional TAT system is necessary for delivery of DNA and protein effectors to plant cells by a type IV secretion system assembled from the products of VirB and VirD4 proteins. It is known that this secretion system can independently deliver the T-DNA in the form of a T-DNA/VirD2 nucleoprotein complex, as well as the VirE2 secretion substrate, to plant cells (53). In the plant cell, these molecules assemble as a T-DNA/VirD2/VirE2 complex for translocation to the nucleus (51). Transfer of these substrates can be monitored by mixed-infection assays, whereby coinoculation with a virE2 mutant (competent for T-DNA/VirD2 export) and a T-DNA deletion mutant (competent for VirE2 export) on a plant wound site results in tumor formation (36). We assessed whether the tatC mutant separately exports T-DNA/VirD2 or VirE2 by mixed infection with the T-DNA deletion mutant LBA4404 or the virE2 mutant At12516, respectively. Figure 3A shows that the tatC mutant rescued the genetic deficiencies of both strains, resulting in formation of tumors resembling those incited by wild-type A348 in size and kinetics of appearance. These findings suggest that the tatC mutant efficiently exports both T-DNA/VirD2 and VirE2 secretion substrates to plant cells.

A. tumefaciens also can utilize the type IV T-DNA secretion system to mobilize IncQ plasmids to agrobacterial recipients. Consistent with the above findings, the tatC mutant exhibited wild-type donor behavior in conjugal DNA transfer experiments (Table 3). Furthermore, this mutant also efficiently acquired the IncQ plasmid when mated with a wild-type A348(pML122Genr) donor strain (Table 3). We conclude that the TAT system does not participate in type IV-dependent transfer of substrates to plant or agrobacterial target cells or in acquisition of DNA during conjugation.


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  		TABLE 3. Effects of a tatC mutation on virB-dependent conjugal transfera

 
Previous work has established that perturbations in assembly of this type IV system are highly destabilizing for several VirB proteins (5, 23). However, the tatC mutant reproducibly accumulated VirB proteins at levels comparable to those for wild-type A348 and PC2000(pZDB36) cells (Fig. 3B). The tatC mutant also accumulated high levels of VirB2 pilin and the pilus-associated proteins VirB5 and VirB7 in the extracellular fraction, providing further evidence that the TAT pathway plays no role in assembly of either the transfer channel or the T pilus. The T pili isolated from the surfaces of A348 and PC2000 and purified by sucrose density gradient centrifugation (39) also were morphologically indistinguishable by electron microscopy (data not shown).

Notably, however, the periplasmic sugar binding protein ChvE accumulated at low levels in the exocellular fraction of the tatC mutant (Fig. 3B). We also have identified an RNase activity and additional proteins in this exocellular fraction that are missing in the corresponding fractions of A348, PC2000(pZDB36), or PC2000(pZDB44) (data not shown). We presume that these are periplasmic proteins released by the tatC mutant, because neither VirB membrane proteins or the VirE2, VirE1, or VirD1 cytoplasmic proteins were detected in the extracellular fractions of wild-type or tatC mutant strains (see below). We have further observed that the tatC mutant displays enhanced lysis upon exposure to lysozyme and EDTA treatment compared to wild-type cells and also that the mutant produces large amounts of lipopolysaccharide, including succinoglycan as monitored by Calcofluor binding (13), under specific growth conditions. Collectively, these phenotypes suggest that the A. tumefaciens tatC mutant exhibits pleiotropic defects in its outer membrane, reminiscent of a recent report for E. coli (47).

Finally, in view of our finding that the tatC mutation significantly disrupts virulence independently of an effect on type IV secretion, we considered the possibility that the TAT system might deliver an unidentified effector molecule(s) to the plant cytosol. To test this possibility, we coinoculated the tatC mutant (competent for type IV secretion) with a virB or a virD4 mutant (competent for TAT translocation) on wounded plant tissue. These mixed infections did not induce tumor formation, suggesting that the TAT system is not part of any translocation pathway involved in substrate transfer across kingdom boundaries (Fig. 3A).

The A. tumefaciens TAT system is important for motility and flagellation. Nearly all A348 and PC2000(pZDB36) cells in cultures grown to an OD600 of 0.5 in MG/L or ABIM were highly motile, as monitored by light microscopy. In striking contrast, fewer than 0.1% of PC2000 cells grown in MG/L and 0.01% of cells grown in ABIM were motile when examined throughout a 36-h period following inoculation from a frozen stock. Correspondingly, A348 and the complemented strain formed large halos within 18 h following inoculation of MG/L or ABIM motility plates (Fig. 4A). PC2000(pZDB44) expressing PvirB-tatC also exhibited wild-type motility on ABIM and, interestingly, MG/L media even though this promoter is not induced under these growth conditions. In other studies, we have determined that PvirB is expressed at a basal level in MG/L medium, suggesting that even a very low level of TatC production suffices for assembly of a functional TAT system. Strikingly, PC2000 formed only a very small motility halo on an MG/L motility plate but no detectable halo on an ABIM plate after 36 h of incubation (Fig. 4A). PC2000 formed a very small motility halo on ABIM by 64 h of incubation.



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  		FIG. 4. Effect of the tatC mutation on A. tumefaciens motility. (A) Motility of the strains listed on rich (MG/L) and minimal (ABIM) media after 36 h of incubation at 24°C. (B) Motility and corresponding fluorescence pattern of strains A348 and PC2000 bearing pZD51 (Ptac-torA::GFP), in which cells were collected from the center and edges of a motility colony, retested for motility, and examined for TorA::GFP export by fluorescence microscopy. (C) Virulence assays of motile and nonmotile tatC variants collected from the edge and center of a motility colony, respectively. A348 cells collected from these sites showed no difference in virulence (data not shown).

 
We were intrigued by the observation that a low fraction of tatC mutant cells displayed motility. To determine whether this phenotype was stably inherited, we collected tatC cells as well as wild-type cells from the outer edges and centers of colonies grown for 36 h on MG/L motility plates. The cells were suspended in MG/L broth, normalized to equivalent OD600 units, and reinoculated onto swarm plates. Interestingly, tatC cells obtained from the outer edge of the halo were appreciably more motile than their sessile counterparts collected from the center of the colony, albeit still less motile overall than wild-type cells (Fig. 4B). Wild-type A348 cells collected from the center and outer edges of the colony formed motility halos of identical sizes (data not shown).

One explanation for the observed motility phenotype is that a subpopulation of tatC mutants reverted to wild-type TAT function. To test this possibility, we propagated A348(pZD51) and PC2000(pZD51) cells producing TorA::GFP on motility plates. Microscopic observation showed that all A348 cells collected either from the center or edge of the motility colony exhibited halo patterns of fluorescence (Fig. 4B and data not shown). By contrast, despite differences in motility, PC2000 cells collected from the outer edge and center of a motility colony were exclusively uniformly fluorescent (Fig. 4B). These findings show that the motility phenotype is stably inherited, but not because of a mutagenic or adaptive event that restored TAT function. Finally, we compared the virulence of predominantly motile and nonmotile tatC subpopulations. Very interestingly, the motile tatC subpopulation reproducibly induced appreciably larger tumors than the nonmotile cells (Fig. 4C). The motile and nonmotile cells exhibited similar growth rates, excluding this as a possible cause for the observed differences in virulence.

Next, we examined the two subpopulations of tatC mutants for production of flagella. A. tumefaciens carries four flagellin genes in its genome, two of which produce abundant levels of flagellin proteins, migrating at ~31 and 33 kDa, that are easily visualized by silver staining of extracellular material (19). The 33-kDa FlaA protein is essential for motility and production of wild-type flagella (19). As shown in Fig. 5A, wild-type A348 and the complemented tatC strains possessed abundant levels of these flagellin proteins. By contrast, the tatC mutant grown directly from a frozen stock culture did not possess detectable levels of flagellins. Instead, we detected abundant levels of several proteins in the extracellular fraction of the tatC mutant that were missing from the corresponding fractions of A348 or the complemented mutant (Fig. 5A). Moreover, when we examined tatC cells derived from the center of a motility colony, we detected no extracellular flagellins and abundant amounts of the novel proteins. Conversely, the subpopulation of motile cells propagated from the edge of the motility halo possessed abundant levels of flagellins and significantly reduced levels of the novel proteins (Fig. 5A). Finally, periplasmic ChvE (Fig. 5B) and a presumptive periplasmic RNase (data not shown) were present at abundant levels in the culture supernatants of nonmotile tatC cells but at significantly reduced levels in supernatants of the motile mutants. Cytoplasmic VirE2, VirE1, or VirD1 and VirB membrane proteins were not detected in extracellular fractions from any strains or the subpopulations of nonmotile and motile tatC mutants (Fig. 5B and data not shown).



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  		FIG. 5. Effect of the tatC mutation on production of extracellular flagellin and flagella. (A) Extracellular flagellins (arrowhead) and other protein species detected by silver staining of SDS-polyacrylamide gels. Extracellular fractions from the strains listed at the top of the gel were analyzed; edge and center refer to extracellular fractions of cells propagated from the edge and center of a motility halo, respectively. MW, molecular weight markers, with sizes in thousands listed at the left. (B) Western blot analysis to monitor accumulation of periplasmic ChvE and cytoplasmic VirE2 in the extracellular fractions. Lanes are as in panel A, except that the left-hand lane shows the migration of ChvE and VirE2 upon SDS-PAGE of PC2000 whole-cell extracts. (C) Flagellum production by the strains listed grown from stock cultures (first three panels) or from the center and edge of a motility colony.

 
Cells were stained with basic fuchsin to detect flagella by microscopy. Flagella were detected on the surfaces of A348, PC2000(pZDB36), and PC2000(pZDB44) cells but not on any PC2000 cells grown directly from a stock culture (Fig. 5B). Cells propagated from the center of a motility colony also did not elaborate flagella, whereas approximately half of the motile tatC mutants propagated from the motility halos possessed flagella (Fig. 5B). Taken together, these findings establish the overall importance of the TAT system for flagellar biogenesis in A. tumefaciens, although a subpopulation of mutants defective in TAT function can produce flagella and also display phenotypes consistent with a restoration of outer membrane integrity.

Motile tatC variants fail to exhibit chemotaxis. A. tumefaciens has been shown to exhibit chemotaxis to various sugars (12) and to an optimal concentration of nitrate when grown anaerobically (30). Grown aerobically, wild-type A348 and the complemented strain, PC2000(pZDB36), exhibited chemotaxis toward glucose (Fig. 6A), as well as fucose, maltose, and sucrose (data not shown). Grown anaerobically, both strains also displayed chemotaxis to nitrate (Fig. 6B). By contrast, an enriched subpopulation of motile tatC cells showed absolutely no chemotactic responses to any of these sugars or to nitrate under aerobic and anaerobic conditions, respectively (Fig. 6). Therefore, even though a fraction of tatC mutants revert to a mot+ phenotype and elaborate flagella, these cells remain unable to transduce exogenous chemotactic signals to the flagellar motor.



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  		FIG. 6. Effect of the tatC mutation on chemotaxis. (A) Taxis by the strains listed toward glucose applied to a Whatman filter disk. (B) Taxis toward an optimal concentration of nitrate, visualized by a zone of growth surrounding the nitrate source (center), on a Whatman filter disk. Strains were grown microaerobically and normalized to the same OD600, and then equivalent CFU were mixed with M9 minimal medium supplemented with glycerol and 0.3% agar. PC2000 cells propagated from the edge of a motility halo were assayed for chemotaxis to nitrate. Motility plates were incubated under anaerobic conditions as described by Lee et al. (30).

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we characterized the contribution of the TAT translocation system of A. tumefaciens to growth, motility, and virulence. We showed that inactivation of this system completely abolishes two cellular functions, anaerobic respiration and chemotaxis. The requirement for anaerobic respiration was expected on the basis of previous work on the TAT pathway in other bacterial species (7) and our identification of characteristic signal sequences at the N termini of several redox enzymes of A. tumefaciens, including the large subunit of periplasmic nitrate reductase (Table 2). This enzyme is known to support anaerobic respiration in A. tumefaciens (30) and other denitrifying bacteria (8) with nitrate as a terminal oxidant. Although the periplasmic nitrate reductase of A. tumefaciens has not been characterized, studies of a homologous enzyme from another denitrifying species, Ralstonia eutropha, demonstrated that the large subunit binds an iron-sulfur cluster near its N terminus and is secreted by the TAT system (8). Therefore, the failure of the A. tumefaciens tatC mutant to grow anaerobically with nitrate is completely consistent with a prevailing model that the TAT system is the only pathway available to gram-negative bacteria for secretion of cofactor-bound redox proteins.

In striking contrast, the requirement for the TAT system for chemotaxis was completely unexpected. For E. coli, extensive studies have shown that the chemotaxis signaling system is composed of a complex hierarchy of protein interactions between periplasmic ligand binding proteins, inner membrane chemoreceptors, transducer kinases and response regulators, and components of the flagellar motor (10). This signal transduction pathway is considered to mediate the chemotaxis response in other bacterial species, including the {alpha}-proteobacteria, although recent work on S. meliloti has identified significant deviations from the enterobacterial paradigm with respect to the regulatory cascade governing flagellar gene expression and the flagellar structure and mode of rotation (45).

Our screen of the A. tumefaciens genome identified 77 possible TAT substrates on the basis of the appropriate positioning RRxF or RRxL motifs in classical signal sequences. However, very recently Pohlschröder and colleagues examined over 80 bacterial and archaeal genomes with their newly developed TATFIND algorithm, and an analysis of the A. tumefaciens genome identified 51 potential TAT substrates (M. Pohlschröder, personal communication). Twenty-five potential TAT substrates were identified with both screens, and, very interestingly, many of these were periplasmic ligand binding proteins (Table 2). Using TATFIND, many periplasmic ligand binding proteins also were identified as possible TAT substrates in Halobacterium sp. strain NRC-1 (38). These observations raise the possibility that the A. tumefaciens tatC mutant is defective for chemotaxis as a result of mislocalization of periplasmic ligand binding proteins. However, the tatC mutant utilized sugar substrates of ABC transport systems, e.g., arabinose, maltose, ribose, and xylose, without any detectable reduction in growth compared to wild-type A348. Additionally, the tatC mutant efficiently utilized other sugars as carbon sources, including cellobiose, dextrose, fucose, glucose, lactose, mannitol, maltotriose, melibiose, sorbitol, sucrose, and glycerol. These findings suggest that the TAT pathway is dispensable for elaboration of several sugar import pathways and that at least a subset of ligand binding proteins localize properly. It is possible, however, that redundant pathways are masking effects of a tatC mutation; therefore, direct tests for TAT-dependent export of periplasmic substrate binding proteins are warranted.

We showed that the tatC mutant exhibits a wild-type response to exogenous sugar and phenolic signals for activation of vir gene expression. This transduction pathway involves recognition of plant-derived sugars and phenolic compounds, e.g., acetosyringone, by ChvE, a homolog of the E. coli ribose and galactose/glucose-binding proteins, and the VirA sensor kinase, respectively. Sugar-bound ChvE as well as acetosyringone also mediate chemotaxis responses, presumably through interactions with VirA (1, 12, 44, 52). The fact that the tatC mutant expresses the vir regulon at wild-type levels establishes that sugar and phenolic signals are being appropriately perceived. However, we also have shown that the tatC mutant does not exhibit chemotaxis to glucose, galactose, or other sugars (Fig. 6), and we recently determined that this mutant also does not exhibit chemotaxis to acetosyringone (data not shown). Thus, at least for two types of exogenous signals, the block in chemotaxis clearly occurs downstream of ligand binding.

At the opposite end of this signaling pathway is the flagellar motor and the flagellum. Although it is clear that inactivation of the TAT system profoundly disrupted the capacity of cells to assemble flagella, none of the structural subunits for this organelle possess TAT-like signal sequences as judged by our screen and that of Pohlschröder (personal communication). This suggests that the TAT pathway probably does not participate directly in the biogenesis of this organelle. The tatC mutant clearly displayed pleiotropic effects on several surface processes, and, interestingly, these effects were diminished in a subpopulation of cells enriched by selection for motile variants. Notably, the motile tatC mutants also showed a reduction in the release of periplasmic ChvE as well as a presumptive periplasmic RNase. These findings are compatible with a model whereby inactivation of this pathway disrupts biogenesis of the outer membrane and this in turn imposes a block either on the regulatory cascade governing transcription of the flagellar genes or on the actual process of machine assembly. If the block is at the stage of machine assembly, further studies defining the role of the TAT pathway for flagellar biogenesis should be quite informative, especially in view of the fact that the TAT pathway is completely dispensable for assembly of another supramolecular surface organelle, the type IV secretion system, which, like the flagellar apparatus, also is composed of a secretion channel and an extracellular filament.

At this point, we envision several mechanisms that could account for the pseudoreversion phenotypes of a subpopulation of tatC mutants. First, they might arise by mutation. Yet, a simple suppressor mutation is unlikely based on the high frequency with which the motile variants were detected on outgrowth of nonmotile cells, e.g., ~101 to 102 cells in a culture of ~105 to 106 cells per ml. A second intriguing possibility is that a fraction of tatC mutants utilize a compensatory mechanism for restoration of some cellular processes in the absence of a functional TAT system. One such mechanism might involve gene amplification for modulating dosage or expression patterns of genes involved in membrane biogenesis, division, or motility. In E. coli, such a precedent exists, whereby the amplification of the cell division genes ftsZAQ and of the transcriptional activator sdiA serves to overcome drug-induced blocks in cell division and exposure to DNA-damaging agents, respectively (54, 56).

A third mechanism might involve use of an alternative secretion pathway for exporting TAT substrates. As yet, there is no evidence that a natural TAT substrate can be secreted by a different route, but both competitive (18) and cooperative (27) interactions between the TAT and sec-dependent pathways have been documented. It is intriguing to speculate that tatC mutants can utilize the GSP as a default pathway for secretion of TAT substrates whose functions are absolutely vital to cell survival. Of course, a complex interplay of environmental, regulatory, and posttranslational controls could have an impact on protein-folding kinetics and targeting to an alternative pathway, and this might account for the small fraction of pseudorevertants that we identified during outgrowth of the nonmotile cells.

The observed correlation between pseudoreversion to a mot+ phenotype and enhanced virulence might reflect a causal relationship, because for many mammalian pathogens motility plays an important role in trafficking within the eukaryotic host (21, 35). Additionally, for some pathogens, e.g., P. aeruginosa, flagella can promote adhesion to specialized cell types to facilitate colonization and biofilm formation (33). For A. tumefaciens, motility and chemotaxis have been shown to strongly expedite early stages of the infection process, e.g., trafficking within the rhizosphere to wounded plant cells (26). Interestingly, however, this trafficking requirement can be partially circumvented by inoculation of aflagellate mutants directly onto a wound site. These mutants incite tumor formation, albeit still slightly less efficiently than wild-type cells (11, 15). A recent study further reported that flagellar biosynthesis is downregulated in A. tumefaciens upon induction of the virulence regulon (28). Thus, once A. tumefaciens reaches the infection site, the activation of vir genes in response to plant signal perception favors colonization of a specialized environmental niche by shutting down motility.

The TAT system of P. aeruginosa also was reported to contribute to virulence and several types of motility (33). Interestingly, microscopic studies showed that a subpopulation of tatC mutants formed filamentous aggregates and that cells in these aggregates displayed swimming, swarming, and twitching motility. Ochsner et al. (33) postulated that the P. aeruginosa tatC mutants can elaborate flagella and pili, but these organelles might function abnormally as a result of a block in motor function, chemotaxis signaling, or both. The results of our studies suggest instead that these mutants might sort as subpopulations of fla mutant and fla+ cells, the latter arising through some type of mutagenic or other compensatory mechanism. Clearly, the results of both studies have firmly established that tatC mutation disrupts motility, and we have further shown that inactivation of the TAT pathway blocks chemotaxis in A. tumefaciens. Finally, it is noteworthy that P. aeruginosa tat mutants also were unable to form biofilms (33). In an initial screen, there was no disruption of the tatC mutation on A. tumefaciens biofilm formation (C. Fuqua, personal communication), but further studies are needed to explore possible effects under various growth conditions.

Currently, we are attempting to define the spectrum of TAT-dependent substrates and their roles in A. tumefaciens physiology and virulence. At this juncture, our findings argue against a role for this secretion pathway in the intercellular transmission of effector molecules to the plant cytosol. On the basis of ancestral relationships as well as a number of structure-function relationships, it also seems unlikely that the TAT pathway contributes to assembly or function of other bacterial conjugative transfer systems or to type IV secretion systems operating in mammalian pathogens. By contrast, given the extensive ancestral and structure-function relationships between flagella and type III secretion, it is tempting to speculate that the TAT pathway mediates assembly of these medically important translocation machines.


   ACKNOWLEDGMENTS
 
We thank C. Robinson for the gift of pJDT1 for use in construction of pWM1487 by the Margolin laboratory and W. Margolin for the gift of pWM1487. We thank Y. Machida for the gift of ChvE antibodies. We thank C. Fuqua and T. Day for initial studies on the effects of the tatC mutation on A. tumefaciens biofilm formation. We thank M. Pohlschröder for kindly providing results of the screen for A. tumefaciens TAT substrates with the TATFIND algorithm prior to publication. We also thank the members of our laboratory for helpful discussions and Bei Bei Song for excellent technical assistance.

This study was supported by NIH grant GM48746.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, The University of Texas-Houston Medical School, 6431 Fannin, Houston, TX 77030. Phone: (713) 500-5440. Fax: (713) 500-5499. E-mail: Peter.J.Christie{at}uth.tmc.edu. Back


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 Materials and Methods
 Results
 Discussion
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Journal of Bacteriology, February 2003, p. 760-771, Vol. 185, No. 3
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.3.760-771.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.