We have shown that B. subtilis
is capable of forming robust biofilms on the roots of tomato plants and that it does so in a manner that depends on production of extracellular matrix. We have also identified a particular sensor kinase, KinD, that is required for efficient biofilm formation. We hypothesize that KinD responds to one or more chemical signals released from plant roots that stimulates the activity of the kinase. KinD feeds phosphoryl groups into the phosphorelay that phosphorylates Spo0A, which in turn activates the regulatory circuitry leading to matrix production (Jiang et al., 2000
). KinD differs from the other kinases contributing to biofilm formation in that it exhibits an extracellular domain that resembles CACHE domains of histidine kinases in other bacteria that are known to respond to small molecules such as succinic acid, alanine, and citric acid (Wu et al., 2008
, Anantharaman & Aravind, 2000
). Reinforcing the hypothesis that KinD senses one or more plant-released, chemical signals through its CACHE domain, we created amino acid substitutions in residues believed to be involved in ligand recognition and found that cells producing the mutant kinase were impaired in biofilm formation.
We do not know the identity of the signaling molecule(s) but an exudate from tomato plant roots was capable of strongly stimulating pellicle formation in a medium that otherwise does not support efficient biofilm formation. We surmise that the root exudates contain one or more signaling molecules that are recognized by KinD. We identified a variety of small molecules in the exudate, one of which, l-malic acid, was able to stimulate biofilm formation in a KinD-dependent manner but only at high concentrations. The stereo isoform of malic acid was important in that d-malic acid exhibited no activity in stimulating biofilm formation under similar conditions.
-malic acid was active only at millimolar concentrations, we suspect that it principally functions as a carbon source and that growth on l
-malic acid alters the metabolism of B. subtilis
in a manner that favors biofilm formation. Indeed, in keeping with this idea, l
-malic acid is one of the most abundant small molecules released from plant roots (Kamilova et al., 2006a
, Kamilova et al., 2006b
, Rudrappa et al., 2008
). As a precedent, Photorhabdus
species enable their nematode hosts to parasitize insect larvae in response to high concentrations of l
-proline in the insect hemolymph (Crawford et al., 2010
). Growth on l
-proline shifts the metabolism of the entomopathogenic bacteria in a manner that upregulates the production of various virulence factors important in antibiosis.
-malic acid serves both as a carbon source and as a ligand that is recognized by the CACHE domain of KinD. Alternatively, l
-malic acid and KinD might function independently with the sensor kinase recognizing and responding to an unrelated and yet-to-be-identified small molecule released from plant roots. It is also conceivable that the CACHE domain of KinD senses a small molecule produced by B. subtilis
itself in response to a shift in metabolism induced by growth on l
-malic acid and other nutrients in plant exudates. In an effort to investigate the possibility that l
-malate serves as a signaling molecule independently of its role as a carbon source, we built single and double mutants of the putative malate transport genes maeN
(Doan et al., 2003
, Tanaka et al., 2003
, Wei et al., 2000
). In our hands neither the single nor the double mutants were substantially impaired in growth with l
-malate as the sole carbon source (unpublished results). Evidently, an additional unknown transporter contributes to uptake of l
-malic acid. Nonetheless, the double mutant responded to l
-malate at 10-fold lower concentration (500 µM) than the wild type (5 mM).
Interestingly, in other work we have found that in addition to its inferred capacity to sense a small molecule, KinD functions as an osmosensor (S., Rubenstein, I. Kolodkin-Gal, L. Chai, A. McLoon, R.K., R.L. and D. Weitz, unpublished results). We have found that KinD activity is stimulated by increasing osmolarity from the production of exopolysaccharide during biofilm formation. Osmosensing occurs in a manner that is independent of the CACHE domain and instead requires on a transmembrane segment.
Finally, we note that KinD functions in conjunction with the membrane-anchored lipoprotein Med, and evidence suggests that KinD functions in a complex with Med (although this has not been shown directly) (Banse et al., 2011
). Interestingly, Med is predicted to contain a ligand-binding domain that strongly resembles known sugar- or nucleoside-binding domains of other proteins (unpublished observation, Chen et al.), including LuxP from V. harveyi
(a 99.9% probability based on HHpred search). In V. harveyi
, LuxP binds directly to the quorum-sensing molecule autoinducer-2 (AI-2, a borate-containing analog of ribose) and interacts with the membrane histidine kinase LuxQ in quorum-sensing signal transduction (Ng & Bassler, 2009
). LuxQ is a structural homolog of KinD as discussed above. Thus, Med and KinD may function in a somewhat analogous manner to that of LuxP and LuxQ, which act together in sensing the bacterial interspecies quorum-sensing signal AI-2. (A key difference is that LuxQ does not itself recognize a signal (Neiditch et al., 2006
).) Although we do not know whether Med directly senses a plant-released signal, it is appealing to imagine that Med and the CACHE domain of KinD sense distinct signals, and together they activate KinD kinase activity in inducing biofilm formation. A key challenge for the future will be to identify signaling molecules released from tomato plant roots that activate KinD and to determine whether both Med and KinD are involved in signal recognition.