The&IT Agrobacterium tumefaciens&IT CheY-like protein ClaR regulates biofilm formation

The switch from a motile, planktonic existence to an attached biofilm is a major bacterial lifestyle transition that is often mediated by complex regulatory pathways. In this report, we describe a CheY-like protein required for control of the motile-to-sessile switch in the plant pathogen Agrobacterium tumefaciens. This regulator, which we have designated ClaR, possesses two distinct CheY-like receiver (REC) domains and is involved in the negative regulation of biofilm formation, through production of the unipolar polysaccharide (UPP) adhesin and cellulose. The ClaR REC domains share predicted structural homology with characterized REC domains and contain the majority of active site residues known to be essential for protein phosphorylation. REC1 is missing the conserved aspartate (N72) residue and although present in REC 2 (D193), it is not required for ClaR-dependent regulation suggesting that phosphorylation, which modulates the activity of many CheY-like proteins, appears not to be essential for ClaR activity. We also show that ClaR-dependent negative regulation of attachment is diminished significantly in mutants for PruA and PruR, proteins known to be involved in a pterin-mediated attachment regulation pathway. In A. tumefaciens, pterins are required for control of the intracellular signal cyclic diguanylate monophosphate through the DcpA regulator, but our findings suggest that pterin-dependent ClaR control of attachment can function independently from DcpA, including dampening of c-di-GMP levels. This report of a novel CheY-type biofilm regulator in A. tumefaciens thus also adds significant details to the role of pterin-mediated signalling.