The core dimerization domains of histidine kinases contain recognition specificity for the cognate response regulator

Abstract

Histidine kinases DivJ and PleC initiate signal transduction pathways that regulate an early cell division cycle step and the gain of motility later in the Caulobacter crescentus cell cycle, respectively. The essential single-domain response regulator DivK functions downstream of these kinases to catalyze phosphotransfer from DivJ and PleC. We have used a yeast two-hybrid screen to investigate the molecular basis of DivJ and PleC interaction with DivK and to identify other His-Asp signal transduction proteins that interact with DivK. The only His-Asp proteins identified in the two-hybrid screen were five members of the histidine kinase superfamily. The finding that most of the kinase clones isolated correspond to either DivJ or PleC supports the previous conclusion that DivJ and PleC are cognate DivK kinases. A 66-amino-acid sequence common to all cloned DivJ and PleC fragments contains the conserved helix 1, helix 2 sequence that forms a four-helix bundle in histidine kinases required for dimerization, autophosphorylation and phosphotransfer. We present results that indicate that the four-helix bundle subdomain is not only necessary for binding of the response regulator but also sufficient for in vivo recognition specificity between DivK and its cognate histidine kinases. The other three kinases identified in this study correspond to DivL, an essential tyrosine kinase belonging to the same kinase subfamily as DivJ and PleC, and the two previously uncharacterized, soluble histidine kinases CckN and CckO. We discuss the significance of these results as they relate to kinase response regulator recognition specificity and the fidelity of phosphotransfer in signal transduction pathways.

FIG. 1.

Polypeptide fragments of kinases DivJ, PleC, and DivL that interact with DivK in the yeast two-hybrid screen. An alignment of translated inserts in representative DivJ, PleC, and DivL clones is shown. The positions of the N- and C-terminal residues are indicated for each fragment, except those that extended to the C-terminal end of the proteins. Locations of the conserved sites of phosphorylation, histidine (H) in DivJ and PleC and tyrosine (Y) in DivL, are shown. Shaded areas of each kinase represent overlapping sequences common to the respective DivJ, PleC, and DivL clones. The numbers in parentheses following plasmid names identify the clones tested in the spot test shown in Fig. 3. β-Galactosidase (B-gal) assays (17) were performed on the cultures of the same clones that were grown in SC medium lacking leucine and uracil to mid-exponential phase. At least three independent cultures of each strain were assayed. Including the clones shown here, the fusion joints of 76 clones were sequenced. Among 34 pleC clones sequenced, the most frequent fusion points were either at amino acid 456 (7 clones) or between amino acids 492 and 495 (21 clones). Of the 42 DivJ clones sequenced, a total of 21 DivJ fragments started between residues 190 and 196 and 6 started between residues 328 and 331. The C-terminal ends of most clones were not sequenced.

FIG. 2.

Amino acid sequence alignment of the helix 1, helix 2 sequences in histidine kinases. The amino acid sequences of conserved DivJ, PleC, CckN, and DivL fragments identified in the yeast two-hybrid screen with DivK as the bait protein (shaded) are aligned with the core helix 1, helix 2 domain of the E. coli EnvZ protein. CckA and CckO are included for comparison, although cckA clones were not isolated in the screen and cckO clones did not encode the helix 1, helix 2 sequence (see text). Locations of helixes 1 and 2 in EnvZ were determined as described previously (33). Residues conserved in both PleC and DivJ are in bold.

FIG. 3.

Interactions of DivK with DivJ, PleC, and DivL in the yeast two-hybrid assay. Yeast strains containing both bait and prey plasmids were grown overnight at 30°C in SC medium lacking uracil and leucine. Cultures were serially diluted 10-fold and spotted onto SC plates lacking leucine and uracil (−leu −ura), histidine (−his), or adenine (−ade). All of the strains tested contained the divK bait plasmid and interacting clones, except a, b, and c. a, divK bait plus empty prey vector; b, pGDB-KAR9 plus pGAD-BIM1 (18); c, pNOR1196 plus empty bait vector. The rationale behind the method is that stronger interactions between bait and prey polypeptides result in higher enzymatic activities for the synthesis of histidine and adenine and thus permit more rapid growth on the selective His⁻ and Ade⁻ plates. The numbered clones correspond to those indicated in parentheses in Fig. 1. Before being photographed, the Leu⁻ Ura⁻, His⁻, and Ade⁻ plates were incubated for 2 days (2d), and the Ade⁻ plate was incubated for 4 days (4d), at 30°C.

FIG. 4.

Alignment of CckO clones. Alignment of translated inserts in cckO clones with the translated CckO sequence (CC0026). The unshaded area represents the kinase domain, and the shaded area represents the receiver domain, of the hybrid kinase. The predicted sites of phosphorylation at H537, D821, other conserved motifs in the kinase domain (N, D, and G boxes [reviewed in reference 5]), and conserved residues in the receiver domain are shown. β-Galactosidase (B-gal) assays on each of the CckO clones underlined were performed as described in the legend to Fig. 1.

FIG. 5.

FACS analysis of divJ, cckN, and cckO null mutants. Cultures were grown in PYE medium supplemented with appropriate antibiotics to optical densities at 650 nm of ca. 0.1 to 0.3, and a portion of each culture was further incubated with rifampin (rif) for 3 h at 30°C before harvesting. Samples were prepared for flow cytometry as described in Materials and Methods, and DNA content (chromosome number) was measured. Normally, swarmer cells (G₁ phase) contain one chromosome per cell and predivisional cells (G₂ phase) contain two chromosomes per cell (3). wt, wild type.