Agrobacterium tumefaciens twin-arginine-dependent translocation is important for virulence, flagellation, and chemotaxis but not type IV secretion

Abstract

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 (approximately 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.

FIG. 1.

Functionality of the A. tumefaciens TAT pathway. (A) Percent identities between the A. tumefaciens Tat proteins and those of related α-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 P_tac-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 P_lac-tatC during anaerobic growth with glycerol as a carbon source and nitrate as an electron acceptor; replicates of these experiments yielded similar results.

FIG. 2.

Effect of the tatC mutation on aerobic growth and cell division. (A) Representative growth curves of A348, PC2000, and PC2000(pZDB36) expressing P_lac-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).

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 (P_lac-tatC) or pZDB44 (P_virB-tatC) with A348 (wild type) and A136 (Ti plasmidless) as positive and negative controls; middle leaf, PC2000 mixed with LBA4404 (ΔT-DNA) or At12516 (ΔvirE2) to monitor the capacity of the tatC mutant to secrete T-DNA or VirE2, respectively; bottom leaf, PC2000 mixed with PC1000 (ΔvirB) 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 P_lac-tatC.

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 (P_tac-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).

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.

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).