Abstract
A core regulatory pathway that directs developmental transitions and cellular asymmetries in Agrobacterium tumefaciens involves two overlapping, integrated phosphorelays. One of these phosphorelays putatively includes four histidine sensor kinase homologues, DivJ, PleC, PdhS1 and PdhS2, and two response regulators, DivK and PleD. In several different alphaproteobacteria, this pathway influences a conserved downstream phosphorelay that ultimately controls the phosphorylation state of the CtrA master response regulator. The PdhS2 sensor kinase reciprocally regulates biofilm formation and swimming motility. In the current study, the mechanisms by which the A. tumefaciens sensor kinase PdhS2 directs this regulation are delineated. PdhS2 lacking a key residue implicated in phosphatase activity is markedly deficient in proper control of attachment and motility phenotypes, whereas a kinase-deficient PdhS2 mutant is only modestly affected. A genetic interaction between DivK and PdhS2 is revealed, unmasking one of several connections between PdhS2-dependent phenotypes and transcriptional control by CtrA. Epistasis experiments suggest that PdhS2 may function independently of the CckA sensor kinase, the cognate sensor kinase for CtrA, which is inhibited by DivK. Global expression analysis of the pdhS2 mutant reveals a restricted regulon, most likely functioning through CtrA to separately control motility and regulate the levels of the intracellular signal cyclic diguanylate monophosphate (cdGMP), thereby affecting the production of adhesive polysaccharides and attachment. We hypothesize that in A. tumefaciens the CtrA regulatory circuit has expanded to include additional inputs through the addition of PdhS-type sensor kinases, likely fine-tuning the response of this organism to the soil microenvironment.
Highlights
Bacteria are sometimes considered to be elementary life forms, with simple body plans, streamlined reproductive cycles, and monolithic behavior when compared with higher eukaryotes
PleC/DivJ homologue sensor kinase (PdhS) family kinase homologues reveals a high level of conservation of this domain including the phospho-accepting histidine residue (H271 of PdhS2) and a threonine residue predicted to be important for phosphatase activity (T275 of PdhS2) (Fig. 1B)
(plasmid-borne Plac-pdhS2, kinase-negative, K; phosphatase-positive, P+) effectively complemented the attachment and motility phenotypes of the pdhS2 mutant similar to wild type pdhS2 (Fig. 2A). These data indicate that this histidine residue is not crucial for PdhS2 regulation of swimming motility and biofilm formation
Summary
Bacteria are sometimes considered to be elementary life forms, with simple body plans, streamlined reproductive cycles, and monolithic behavior when compared with higher eukaryotes. Underlying asymmetric cell division is subcellular differentiation that includes localization of specific regulatory proteins to programmed locations within each cell (15) Prominent among these in many alphaproteobacteria are components of two overlapping phosphorelays, the first which functions through the response regulators. DivJ-dependent phosphorylation of PleD at the stalk pole of the predivisional cell stimulates its diguanylate cyclase activity, resulting in higher levels of cdGMP at this end of the cell. Core components of the DivK-CtrA pathway are well conserved in A. tumefaciens, including three non-essential PleC/DivJ homologue sensor kinase (PdhS). Mutants in PleC and PdhS1, as well as the A. tumefaciens DivK homologue, all manifested marked effects on cell division, with branched and elongated cells, as well as deficiencies in motility and biofilm formation. Our findings suggest that PdhS2 activity is required for proper development of motile daughter cells
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