Reciprocal Control of Motility and Biofilm Formation by the PdhS2 Two-Component Sensor Kinase of Agrobacterium tumefaciens

A core phosphorelay pathway that directs developmental transitions and cellular asymmetries in Agrobacterium tumefaciens putatively includes two overlapping, integrated phosphorelays. One of these phosphorelays putatively includes at least four histidine sensor kinase homologues, DivJ, PleC, PdhS1, and PdhS2, and at least two response regulators, DivK and PleD. Previously we demonstrated that PdhS2 reciprocally regulates biofilm formation and swimming motility. In the current study we further dissect the role and regulatory impact of PdhS2 in A. tumefaciens revealing that PdhS2-dependent effects on attachment and motility require the response regulator, DivK, but do not require PdhS2 autokinase or phosphotransfer activities. We also demonstrate that PdhS2 regulation of biofilm formation is dependent upon multiple diguanylate cyclases, including PleD, DgcA, and DgcB, implying that PdhS2 regulation of this process intersects with pathways regulating levels of the second messenger cyclic diguanylate monophosphate (cdGMP). Finally, we show that upon cell division a GFP fusion to PdhS2 dynamically localizes to the new pole of the bacterium suggesting that PdhS2 controls processes in the daughter cell compartment of predivisional cells. These observations suggest that PdhS2 negatively regulates DivK, and possibly PleD, activity to control developmental processes in the daughter cell compartment of predivisional cells, as well as in newly released motile daughter cells. IMPORTANCE Bacterial developmental processes, including morphological transformations as well as behavioral transitions, are tightly regulated. In many Alphaproteobacteria cell division and development are coordinated by a suite of conserved histidine kinases and their partnered regulatory proteins. Here we describe how the histidine kinase PdhS2 genetically interacts with a single-domain response regulator, DivK, and the intracellular signal cyclic diguanylate monophosphate. PdhS2 dynamically localizes to the new pole of recently divided cells and negatively regulates processes that ultimately lead to attachment and subsequent biofilm formation in Agrobacterium tumefaciens. These findings expand our understanding of the links between cell division and developmental control in A. tumefaciens and related Alphaproteobacteria.

GFP fusion to PdhS2 dynamically localizes to the new pole of the bacterium suggesting 48 that PdhS2 controls processes in the daughter cell compartment of predivisional cells. 49 These observations suggest that PdhS2 negatively regulates DivK, and possibly PleD,   . PleC and PdhS1, as well as the A. 136 tumefaciens DivK homologue, all manifested marked effects on both cell division and   Swim ring diameters of the ΔdivK and ΔdivKΔpdhS2 mutants were decreased by 175 roughly 20% compared to wildtype while the decrease in ΔpdhS2 swim ring diameters 176 was roughly 40% compared to wildtype, suggesting that the nature of the defect in 177 swimming motility differs between these two classes of mutants and that loss of divK 178 partially restores motility in the absence of pdhS2. Indeed, it was earlier noted that while 179 both the ΔdivK and the ΔpdhS2 single deletion mutants produce polar flagella few 180 ΔpdhS2 mutant bacteria were observed to be motile under wet-mount microscopy 181 implying that the swimming defect is due to diminished flagellar activity rather than  for phosphatase activity (T275 of PdhS2) (Fig. 1B).

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To test the requirement of the conserved phospho-accepting histidine for PdhS2 209 activity we mutated this residue to alanine (H271A) and evaluated the ability of this 210 mutant pdhS2 allele to complement ΔpdhS2 phenotypes (Fig. 2B). Ectopic expression 211 of PdhS2 H271A (P lac -pdhS2 on a low copy plasmid) efficiently complemented the 212 attachment and motility phenotypes of the ΔpdhS2 mutant. These data indicate that this 213 histidine residue is not required for PdhS2 regulation of swimming motility and biofilm 214 formation, and imply that PdhS2 autokinase and phosphotransfer are not required to 215 regulate these phenotypes. Instead, we hypothesize that PdhS2 acts primarily as a 216 phosphatase towards its cognate response regulators, including DivK.

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The conserved HisKA dimerization/phosphoacceptor domain is also primarily 218 responsible for the phosphatase activity of these two-component system kinases (41).

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Phosphatase activity requires a conserved threonine residue roughly one α-helical turn 220 (4 residues) downstream of the phospho-accepting histidine residue. To test the 221 requirement for PdhS2 phosphatase activity in regulating developmental phenotypes we 222 mutated this conserved threonine residue to alanine (Thr275A) and evaluated the ability 223 of this mutant pdhS2 allele to complement ΔpdhS2 phenotypes. In contrast to the 224 PdhS2 H271A mutant protein, equivalent ectopic expression of the PdhS2 T275A allele failed 225 to complement the ΔpdhS2 motility and attachment phenotypes, and in fact exacerbated 226 them (Fig. 2B). Expression of either the kinase-null or the phosphatase-null allele of 227 PdhS2 in the ΔdivK background had no effect on biofilm formation or swimming motility 228 (Fig. 2C). A PdhS2 double mutant with both the histidine and threonine residues 229 mutated had no effect on biofilm formation or swimming motility (Fig. S1). Together    CtrA is 84% identical to C. crescentus CtrA at the amino acid level and purified C. 292 crescentus CtrA binds to a site upstream of the A. tumefaciens ccrM gene (46).

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Furthermore, computational analysis of multiple Alphaproteobacterial genomes 294 uncovered numerous cell cycle regulated genes preceded by a consensus CtrA binding 295 site (47). We therefore evaluated CtrA activity by determining the transcriptional activity 296 of several known and hypothesized CtrA-dependent promoters from both C. crescentus 297 and A. tumefaciens. The ccrM, ctrA, and pilA promoters from C. crescentus were 298 chosen to represent CtrA-activated promoters that we predicted would be similarly 299 regulated in A. tumefaciens (13,14,48,49). In the ΔpdhS2 background, expression 300 levels from both the ctrA and pilA promoters from C. crescentus were significantly 301 reduced while transcription from the C. crescentus ccrM promoter was unchanged 302 (Table 1). In the A. tumefaciens ΔdivK background the C. crescentus ccrM and ctrA 303 promoters exhibited increased activity while the pilA promoter was unchanged (Table   304 1). These data are largely consistent with A. tumefaciens CtrA regulating transcription of 305 known CtrA-dependent promoters, and with PdhS2 and DivK inversely regulating CtrA 306 activity in A. tumefaciens. 307 From A. tumefaciens the ccrM promoter is the only promoter for which 308 experimental data suggest CtrA-dependent regulation, thus this promoter was selected 309 for analysis (46). Curiously, the same computational analysis that identified possible   (Table 1). These data are congruent with the above data for C. crescentus  PleD is a GGDEF motif-containing diguanylate cyclase, and thus it is likely that 336 the attachment phenotype of the ΔpdhS2 mutant requires increased levels of cdGMP.

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Earlier work from our lab identified three additional diguanylate cyclases that are 338 relevant to attachment and biofilm formation: DgcA, DgcB, and DgcC (50) (Wang 339 unpublished). As seen in wild-type C58, deletion of dgcA or dgcB in the ΔpdhS2 340 background significantly decreased attachment and biofilm formation, whereas loss of 341 dgcC did not (Fig. 5A). These data suggest that increased biofilm formation by the 342 ΔpdhS2 mutant is dependent on cdGMP pools, generated through PleD, DgcA, and 343 DgcB. Swimming motility was equivalent in either wild-type C58 or ΔpdhS2 344 backgrounds in combination with mutations in pleD, dgcA, dgcB, or dgcC (Fig. S3A).   Table 2). Of these, 24 genes were significantly upregulated,  To determine whether any of these 39 genes were putatively regulated by CtrA 408 we scanned a sequence window from 500 bp upstream of the start codon to 100 bp into  (Table 2).

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To verify our microarray results we measured transcription of translational 417 fusions to β-galactosidase from two selected genes, dgcB and Atu3318, in wild-type and 418 ΔpdhS2 backgrounds (Table 1). In both cases beta-galactosidase activity increased in  Our data support altered CtrA activity, and not abundance, as primarily responsible for  were similar to those in our own study (39, 68). All three datasets uncovered a restricted 576 set of differentially regulated genes, several of which were common to one or more 577 experiment ( Table 2). Transcriptional assays of the dgcB and Atu3318 promoters are 578 consistent with the microarray data in both directionality and magnitude. It is telling that 579 reporter activity of these potential CtrA-regulated promoters in the ΔpdhS2 mutant is two 580 to three times higher than the remaining regulated promoters (Table 1). This lower 581 overall activity may explain why more putative CtrA-dependent promoters were not    (Table S2)    Each upsream region was amplified by PCR using the primers listed in Table S3 using      Band intensities were quantified using the Odyssey Classic software.

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Proteolytic turnover of CtrA was evaluated using translational shut-off assays.              expression of a kinase-and phosphatase-null allele of pdhS2 (p-pdhS2 (K -P -)) to complement the pdhS2 phenotypes was compared against the wild-type pdhS2 allele (p-pdhS2). Biofilm formation (black bars) and swimming motility (white bars) were evaluated as in Figure 2.

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This study