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. 2005 Feb 22;102(8):3010-5.
doi: 10.1073/pnas.0409371102. Epub 2005 Jan 24.

Enhancer-binding proteins with a forkhead-associated domain and the sigma54 regulon in Myxococcus xanthus fruiting body development

Lars Jelsbak  1 , Michael Givskov, Dale Kaiser
Affiliations

Enhancer-binding proteins with a forkhead-associated domain and the sigma54 regulon in Myxococcus xanthus fruiting body development

Lars Jelsbak et al. Proc Natl Acad Sci U S A. .

Abstract

In response to starvation, Myxococcus xanthus initiates a developmental program that results in the formation of spore-filled, multicellular fruiting bodies. Many developmentally regulated genes in M. xanthus are transcribed from sigma(54) promoters, and these genes require enhancer-binding proteins. Here we report the finding of an unusual group of 12 genes encoding sigma(54)-dependent enhancer-binding proteins containing a forkhead-associated (FHA) domain as their N-terminal sensory domain. FHA domains in other proteins recognize phosphothreonine residues. An insertion mutation in one of these genes, Mx4885, caused a cell autonomous aggregation and sporulation defect. In-frame deletion mutants showed that the FHA domain is necessary for proper Mx4885 function. The altered pattern of developmental gene expression in the mutant implied that Mx4885 is on the pathway of response to the morphogenetic C-signal. Immunoblots specific for C-signal and FruA imply that the site of Mx4885 action is downstream of FruA synthesis on the C-signal transduction pathway. Mx4885 may help to coordinate the level of intracellular phosphorylated FruA (FruA-P) with the level of C-signal displayed on the signal donor cell. Because FHA domains respond to phosphothreonine-containing proteins, these results suggest a regulatory link to the abundant Ser/Thr protein kinases in M. xanthus.

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Figures

Fig. 1.
Fig. 1.
Domain organization of a generic EBP and of putative FHA-EBPs in M. xanthus. Domains are color-coded as follows: green, FHA domains (Pfam accession no. PF00498); yellow, GAF domains (Pfam accession no. PF00072); blue, σ54-dependent EBP domains (Pfam accession no. PF00158); red, helixturn-helix (HTH) DNA-binding domains (Pfam accession no. PF02954). (Scale bar, 100 aa.)
Fig. 2.
Fig. 2.
Amino acid sequence alignment of the FHA domains of the putative M. xanthus EBPs with those of RAD53FHA1 from Saccharomyces cerevisiae (GenBank accession no. A39616) and EspA from M. xanthus (GenBank accession no. AAD47812). The conserved GR, SXXH, and NG motifs are indicated above the alignment. Residues on black, dark gray, and light gray backgrounds indicate 100%, 75%, and 50% amino acid similarity, respectively.
Fig. 3.
Fig. 3.
Mx4885. (A) Shown is a physical map of the Mx4885 neighborhood. ORFs are indicated by large arrows. The +1 map coordinate is the first nucleotide in the translation start codon of Mx4885, and 1380 is the last nucleotide of the translation stop. Plasmids containing the indicated DNA fragments of the Mx4885 region are shown on the lines below the physical map. (B) Shown is the aggregate morphology of wild type (DK1622) and Mx4885::Km mutant (DK12702) starved on CF agar for the indicated periods of time. (Scale bar, 0.5 mm.)
Fig. 4.
Fig. 4.
Aggregation phenotypes of Mx4885 mutants. The strains indicated above the images were starved on CF agar plates for 24 h before photography. The strains used were DK1622 (wild type), DK12702 (Mx4885::Km), DK12703 (ΔMx488548–426), DK12704 (ΔMx488548–123), DK12705 (Mx4885::Km, attB::pJEL4885.11), and DK12706 (Mx4885::Km, attB::pSWU30). (Scale bar, 0.5 mm.)
Fig. 5.
Fig. 5.
Effect of the Mx4885 mutation on developmental gene expression. Expression of the indicated Tn5lac reporter fusions in wild-type DK1622 cells (•) and in Mx4885::Km DK12702 cells (○) on CF agar. Culture samples were collected at the indicated time points and assayed for specific activity β-galactosidase, given as nanomoles of o-nitrophenol produced per minute per milligram of total protein.
Fig. 6.
Fig. 6.
Assays to find the site of action of Mx4885 in the C-signal transduction pathway. (A) C-signal transmission measured by extracellular complementation for sporulation of the csgA mutant DK5208 by a Mx4885::Km mutant (DK12702; white bar) or wild type (DK1622; black bar). The sporulation efficiency of the csgA test strain is given as the percentage of complementation by wild type. (B) Accumulation of CsgA and FruA proteins measured on immunoblots of Mx4885::Km and wild type. Total cell lysates were prepared from cells at the indicated time of development on CF agar and reacted with polyclonal anti-CsgA or anti-FruA antibodies. In each lane, 10 and 4 μg of total protein was loaded for CsgA and FruA immunoblots, respectively.
Fig. 7.
Fig. 7.
A model of the C-signal transduction pathway. C-signaling increases csgA transcription directly or indirectly by means of the proteins of the act operon. A-signal is required for developmental accumulation of the FruA response regulator. Downstream of the act pathway, C-signaling induces phosphorylation of FruA (red lines). Rippling, aggregation, sporulation, and C-signal-dependent gene expression are induced by increasing levels of FruA-P. Different levels are represented by the degree of shading of the open vertical bar. Expression of reporter genes is induced by the corresponding levels of FruA-P. The proposed site of action of Mx4885 is indicated by red lines, which are downstream of the act feedback loop and downstream of FruA accumulation but upstream of FruA-P.
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