Role of two novel two-component regulatory systems in development and phosphatase expression in Myxococcus xanthus

Abstract

We have cloned a two-component regulatory system (phoR2-phoP2) of Myxococcus xanthus while searching for genes that encode proteins with phosphatase activity, where phoR2 encodes the histidine kinase and phoP2 encodes the response regulator. A second system, phoR3-phoP3, was identified and isolated by using phoP2 as a probe. These two systems are quite similar, sharing identities along the full-length proteins of 52% on the histidine kinases and 64% on the response regulators. The predicted structures of both kinases suggest that they are anchored to the membrane, with the sensor domains being located in the periplasmic space and the kinase domains in the cytoplasm. The response regulators (PhoP2 and PhoP3) exhibit a helix-loop-helix motif typical of DNA-binding proteins in the effector domains located in the C-terminal region. Studies on two single-deletion mutants and one double-deletion mutant have revealed that these systems are involved in development. Mutant fruiting bodies are not well packed, originating loose and flat aggregates where some myxospores do not reshape properly, and they remain as elongated cells. These systems are also involved in the expression of Mg-independent acid and neutral phosphatases, which are expressed during development. The neutral phosphatase gene is especially dependent on PhoP3. Neither PhoP2 nor PhoP3 regulates the expression of alkaline phosphatases and the pph1 gene.

FIG. 1.

Restriction map of DNA fragments containing phoR2-phoP2 (A) and phoR3-phoP3 (B).

FIG. 2.

Alignment of PhoR2 with PhoR3 (A), and PhoP2 with PhoP3 (B). Identical amino acids are boxed in black, and functionally similar amino acids are boxed in gray. ▾, putative cleavage site of the signal peptide in both kinases; ♦, amino acids of the transmembrane domain; *, amino acids that are conserved in response regulators; , amino acids that constitute the two helixes of the helix-loop-helix motif.

FIG. 3.

Southern blot hybridization of M. xanthus chromosomal DNA digested with different restriction enzymes. The fragment NcoI-ApaI(b) encompassing phoP2 (Fig. 1A) was used as a probe. The arrows within the lanes point to the second band that hybridizes with this probe in each digestion. The arrows to the left of the gel indicate molecular sizes in kilobases.

FIG. 4.

Morphologies of fruiting bodies and myxospores of M. xanthus DZF1 and deletion mutants. (A) The shapes and sizes of fruiting bodies on different starvation media are shown; the pictures were taken on a light microscope. Bar, 1 mm. (B) Fruiting bodies of M. xanthus DZF1, JM2, and JM3 on MCM medium are shown; the pictures were taken in a scanning electron microscope. Bar, 50 μm. (C) Wild-type and JM322 myxospores are shown; the pictures were taken on a transmission electron microscope. Bar, 1 μm.

FIG. 5.

Determination of phosphatase activities in M. xanthus DZF1 (♦), JM2 (▪), JM3 (▴), and JM322 (•). Results for Mg-independent acid phosphatase (A) and Mg-independent neutral phosphatase (B) are shown. Specific activity is expressed as nmol of p-nitrophenol per mg of protein per min. The results shown are the averages of results of three experiments.