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. 2003 Sep;185(18):5452-64.
doi: 10.1128/JB.185.18.5452-5464.2003.

TodK, a putative histidine protein kinase, regulates timing of fruiting body morphogenesis in Myxococcus xanthus

Anders A Rasmussen  1 , Lotte Søgaard-Andersen
Affiliations

TodK, a putative histidine protein kinase, regulates timing of fruiting body morphogenesis in Myxococcus xanthus

Anders A Rasmussen et al. J Bacteriol. 2003 Sep.

Abstract

In response to starvation, Myxococcus xanthus initiates a developmental program that results in the formation of spore-filled multicellular fruiting bodies. Fruiting body formation depends on the temporal and spatial coordination of aggregation and sporulation. These two processes are induced by the cell surface-associated C signal, with aggregation being induced after 6 h and sporulation being induced once cells have completed the aggregation process. We report the identification of TodK, a putative histidine protein kinase of two-component regulatory systems that is important for the correct timing of aggregation and sporulation. Loss of TodK function results in early aggregation and early, as well as increased levels of, sporulation. Transcription of todK decreases 10-fold in response to starvation independently of the stringent response. Loss of TodK function specifically results in increased expression of a subset of C-signal-dependent genes. Accelerated development in a todK mutant depends on the known components in the C-signal transduction pathway. TodK is not important for synthesis of the C signal. From these results we suggest that TodK is part of a signal transduction system which converges on the C-signal transduction pathway to negatively regulate aggregation, sporulation, and the expression of a subset of C-signal-dependent genes. TodK and the SdeK histidine protein kinase, which is part of a signal transduction system that converges on the C-signal transduction pathway to stimulate aggregation, sporulation, and C-signal-dependent gene expression, act in independent genetic pathways. We suggest that the signal transduction pathways defined by TodK and SdeK act in concert with the C-signal transduction pathway to control the timing of aggregation and sporulation.

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Figures

FIG. 1.
FIG. 1.
Developmental phenotypes of todK and dotR mutants. The indicated strains were exposed to starvation on CF agar for the indicated periods of time and viewed in a Leica MZ8 stereomicroscope. The genotypes of the strains are indicated below their names. Bar, 0.2 mm.
FIG. 2.
FIG. 2.
Physical map of the todK-dotR region. ORFs are indicated by open arrows. Arrows indicate the directions of transcription. Coordinates are relative to +1, the transcriptional start site of todK. Below the physical map, the overlap between the todK and dotR stop codons is shown. The hatched arrowhead indicates the site of insertion of mini-Tn5(tet) Ω8846 at position 839. Plasmids containing the indicated DNA fragments of the todK-dotR locus are shown below the map.
FIG. 3.
FIG. 3.
Sequence analyses of TodK and DotR. (A) Domain organization of TodK. In the sensor domain the two PAS domains are indicated; in the kinase domain, the conserved H, N, D/F, and G boxes are indicated. The asterisk over the H box indicates the conserved histidine residue, the site of autophosphorylation in other sensor histidine protein kinases. (B) Alignment of transmitter domain of TodK with those in AsgD from M. xanthus (4), SdeK from M. xanthus (12), and ResE from B. subtilis (59). The conserved H, N, D/F, and G boxes are indicated. The asterisk over the H box indicates the conserved histidine residue, the site of autophosphorylation in other sensor histidine protein kinases. Residues on black, dark gray, and light gray backgrounds are conserved 100, 75, and 50%, respectively. The alignment was made using CLUSTALX (65); the presentation was made using GENEDOC (K. B. Nicholas and H. B. Nicholas, Jr., 1997). (C) Alignment of DotR with receiver domains in PhoP from B. subtilis (38), PhoB from C. crescentus (13), and PilG from P. aeruginosa (9). The asterisks indicate the highly conserved signature residues of receiver domains (67). Conserved residues are indicated as in panel B. The alignment and presentation were prepared as for panel B.
FIG. 4.
FIG. 4.
Regulation of transcription of todK. (A) Mapping of the 5′ end of the todK transcript by primer extension analysis. C, T, A, and G show the sequence ladders. Total RNA was isolated from DK1622 and SA1634 that had been starved for the indicated periods of time on TPM starvation agar. The 5′ end of the todK transcript is indicated by the arrow. The sequence around the transcriptional start site is indicated to the left. The broken arrow indicates the transcriptional start site (+1). (B) DNA sequence of the todK promoter. The broken arrowindicates +1. The putative −10 and −35 sequences are indicated in boldface. The putative start codon of todK is indicated in boldface together with the putative ribosomal binding site (RBS). The underlining indicates the location of the primer used in the primer extension analyses. (C) Quantitative RT-PCR analysis of the todK transcript. Total RNA was isolated from DK1622 and SA1634 that had been starved for the indicated periods of time on TPM starvation agar. The level of todK transcript detected is expressed as relative units per nanogram of total RNA.
FIG. 5.
FIG. 5.
Effect of todK mutation on developmental gene expression. (A) Expression of the indicated lacZ fusions in wild-type DK1622 cells and in todK SA1634 cells. Cells were starved on TPM agar, samples were withdrawn at the indicated time points, and specific activities of β-galactosidase were determined. The experiments were done in triplicate. Error bars indicate standard deviations. Specific activities of β-galactosidase are given as nanomoles of orthonitrophenyl minute−1 milligram of protein−1. (B) Immunoblot analysis of the csgA proteins in wild-type DK1622 and in todK SA1634 cells. Cells were starved on TPM agar, and total cell lysates were prepared from samples withdrawn at the indicated time points (in hours), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and reacted with anti-CsgA antibodies (35). The closed arrow indicates the 25-kDa full-length CsgA protein, and the open arrow indicates the 17-kDa CsgA protein.
FIG. 6.
FIG. 6.
Epistasis analysis of the relationship between todK and sdeK. The indicated strains were exposed to starvation on CF agar for the indicated time points and viewed in a Leica MZ8 stereomicroscope. The genotypes of the strains are indicated below their names. Bar, 0.2 mm.
FIG. 7.
FIG. 7.
Model of the C-signal transduction pathway. The signaling event in the pathway is the interaction between C signal exposed on the surface of one cell and the hypothetical C-signal sensor on a second cell. Starvation results in a RelA- and (p)ppGpp-dependent transcription of the csgA gene and in the accumulation of the intercellular A signal as well as in the induction of sdeK transcription. Moreover, starvation results in a RelA-independent inhibition of todK transcription. The A and E signals induce transcription of fruA (11, 43). C-signal transmission activates csgA transcription, possibly by activating the proteins in the act operon (14), and FruA, conceivably by stimulating the activity of the cognate FruA histidine protein kinase (labeled HPK); subsequently the phosphoryl group is transferred from the kinase to FruA. Phosphorylated FruA—directly or indirectly—alters the activity of the Frz signal transduction system in such a way that aggregation is induced. In addition phosphorylated FruA activates transcription—directly or indirectly—of C-signal-dependent genes, here exemplified by the dev operon. The possible sites of action of the SdeK kinase and the TodK kinase are indicated by the shaded box (see text for explanation). All the components in the cell to the right are also present in the cell to the left; for simplicity, only csgA is shown in the cell to the left.
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