A Bacillus subtilis sensor kinase involved in triggering biofilm formation on the roots of tomato plants

Abstract

The soil bacterium Bacillus subtilis is widely used in agriculture as a biocontrol agent able to protect plants from a variety of pathogens. Protection is thought to involve the formation of bacterial communities - biofilms - on the roots of the plants. Here we used confocal microscopy to visualize biofilms on the surface of the roots of tomato seedlings and demonstrated that biofilm formation requires genes governing the production of the extracellular matrix that holds cells together. We further show that biofilm formation was dependent on the sensor histidine kinase KinD and in particular on an extracellular CACHE domain implicated in small molecule sensing. Finally, we report that exudates of tomato roots strongly stimulated biofilm formation ex planta and that an abundant small molecule in the exudates, (L) -malic acid, was able to stimulate biofilm formation at high concentrations in a manner that depended on the KinD CACHE domain. We propose that small signalling molecules released by the roots of tomato plants are directly or indirectly recognized by KinD, triggering biofilm formation.

Figure 1. B. subtilis forms biofilms on the tomato root surfaces

Panels A–D. B. subtilis cells that harbor a consistently expressed mkate2 reporter gene at amyE were co-cultured with tomato plantlets in a defined medium. After two days of incubation, root-associated cells were visualized by CLSM. Wild type cells (CY49) formed robust biofilms on the root surfaces (Panel A). A 50 × 50 µm framed area in Panel A was enlarged in Panel B to show details of the chained cells in the root-associated biofilms. A mutant of the epsA-O operon (CY209) exhibited severely decreased attachment of the cells to the root surfaces and virtually no biofilm-like structures (Panel C). Panel D shows a plant root grown in the absence of B. subtilis. Panels E-H show results of biofilm assays of the B. subtilis cells on root surfaces using either heat-killed (80°C for 10 min; Panels E and F) or cold-killed (−80°C overnight; Panels G and H) tomato plantlets. Wild type cells did not form biofilms on the surfaces of either heat-killed or cold-killed tomato plants. Images in panels F and H are enlarged ones from the panels E and G, respectively. Scale bars in Panels A, C, D, E, and G are 20 µm, and the scale bars in Panels F and H are 50 µm.

Figure 2. Tomato root exudates induce pellicle formation by B. subtilis in LB broth in a KinD-dependent manner

In Panel A, wild type cells and various single or double kinase mutants [WT (3610); ∆kinA (RL4562); ∆kinB (RL4563); ∆kinA ∆kinB (RL4573); ∆kinC (RL4262); ∆kinD (RL4552); ∆kinC ∆kinD (RL5273) were inoculated into LB standing culture in 12-well plates with (top panels) or without (bottom panels) addition of 1% (v/v) tomato root exudates. Samples were incubated at room temperature for 2 days. Panel B compares the ability of wild type (3610), the kinD null mutant (RL1927), and two kinD complementation strains, ∆kinD amyE::kinD^wt (CY78) and ∆kinD amyE::kinD^mut (CY79) to form pellicles in LB medium with the addition of 1% root exudate.

Figure 3. Tomato root exudates induce expression of the matrix genes and the sdpABC operon

Panels A and B show luciferase activities for the two wild type reporter strains harboring either P_epsA-lux (strain ALM89 in A) or P_tapA-lux (strain ALM90 in B) that were grown in LB shaking culture with (squares) or without (diamonds) addition of 1% root exudate. Panel C shows luciferase activities of the wild type (CY136) and the ∆kinD mutant (CY137) that harbored the P_sdpA-lux reporter fusion in the presence (squares for WT and triangles for ∆kinD) or absence (diamonds for WT and circles for ∆kinD) of 1% root exudate. The arrows indicate the start of stationary phase. The luciferase activities were presented in arbitrary units (AU).

Figure 4. Biofilm formation by various kinase mutants on tomato roots

Cells of the indicated mutants [∆kinA (CY204); ∆kinB (CY205); ∆kinA ∆kinB (CY206); ∆kinC (CY207); ∆kinD (CY127); ∆kinC ∆kinD (CY126); ∆kinD amyE::kinD^mut (CY190); ∆kinD amyE::kinD^wt (CY189)] that harbored a constitutively expressed mKate2 reporter gene were incubated with tomato plantlets for two days at room temperature and were visualized by CLSM. Scale bar, 50 µm.

Figure 5. The CACHE domain of KinD

Panel A is a cartoon representation of KinD. Panel B shows similarity between the three-dimensional structures of the CACHE domains of McpX (3C8C) from V. cholerae and of KinD (3FOS) from B. subtilis. In particular, the predicted small molecule-binding pocket in the CACHE domain in KinD is strikingly similar to that in the CACHE domain of McpX. Panel C shows the amino acid sequence alignment of the CACHE domains from KinD and nine related proteins. All residues in direct contact with a ligand are highlighted in red in the proteins that were known to bind ligands based on published protein structures. The red box indicates a four-amino-acid-residue sequence that is predicted as most critical residue in ligand binding. Amino acid substitutions of these residues were created in KinD. The protein IDs in the Protein Data Bank (PDB) for each of the sequences in Panel C are as follows: Bs_KinD (3FOS), Mm_HK1s-Z2 (3LI9), Mm_HK1s-Z3 (3LIB), Vc_McpX (3C8C), So_HK1s-Z6 (3LIC), Kp_CitA (1P0Z), Vc_DctB (3BY9), Sm_DctB (3E40), Rp_HK1s-Z16 (3LIF), and Hp_HK1s-Z8 (3LID).

Figure 6. l-Malic acid stimulates biofilm formation

Panels (A–C). Identification of l-malic acid in tomato root exudates by HPLC/MS. (A) Separation by HPLC of an active subfraction from tomato root exduate. mAU in Y-axis represents "milli-absorbance units".X-axis is the retention time. (B) An abundant molecule whose retention time is at 17.2 min (indicated by arrow in panel A) was identified as l-malic acid based on MS analysis (M-H Ion Extraction at 132). Values in Y-axis represent relative TIC (Total Ionization Current). (C) Commercially obtained l-malic acid (Sigma) was used as a standard in HPLC (not shown) and MS analysis (M-H Ion Extraction at 132). (D) Luciferase activities of the wild type strain (CY136) that harbored a P_sdpA-lux reporter and was grown in LB shaking culture in the absence (diamonds), or presence of 5 mM l-malic acid (squares) or 5 mM d-malic acid (triangles). (E) Luciferase activities of the ∆kinD mutant (CY137) and the two kinD complementation strains, ∆kinD amyE::kinD^wt (CY185) and ∆kinD amyE::kinD^mut (CY186) that harbored the P_sdpA-lux reporter and were grown in LB shaking culture in the presence or absence of 5 mM l-malic acid. Symbols are designated as follows: ∆kinD amyE::kinD^wt, +malic acid (squares); ∆kinD amyE::kinD^wt, −malic acid (diamonds); ∆kinD amyE::kinD^mut, +malic acid (stars); ∆kinD amyE::kinD^mut, −malic acid (crosses); ∆kinD, +malic acid (triangles); ∆kinD, −malic acid (circles). (F) 3610 cells were inoculated into a minimal medium (see Materials and Methods) that does not support pellicle formation. l-malic acid when added in a gradient from 250 µM to 5 mM, induced pellicle formation after 48 hours of incubation at the room temperature.