Identification of an activator protein required for the induction of fruA, a gene essential for fruiting body development in Myxococcus xanthus

Abstract

Myxococcus xanthus exhibits social behavior and multicellular development. FruA is an essential transcription factor for fruiting body development in M. xanthus. In the present study, the upstream promoter region was found to be necessary for the induction of fruA expression during development. A cis-acting element required for the induction was identified and was located between nucleotides -154 and -107 with respect to the transcription initiation site. In addition, it was found that two binding sites exist within this element of the fruA promoter. By using DNA affinity column chromatography containing the cis-acting element, a fruA promoter-binding protein was purified. The purified protein was shown by N-terminal sequence analysis to be identical to MrpC, a protein identified previously by transposon insertion mutagenesis as an essential locus for fruiting body development [Sun, H. & Shi, W. (2001) J. Bacteriol. 183, 4786-4795]. Furthermore, fruA mRNA was not detectable in the mrpC::km strain, demonstrating that MrpC is essential for fruA expression. Moreover, mutational analysis of the binding sites for MrpC in the fruA promoter indicates that binding of MrpC activates transcription of fruA in vivo. This report provides evidence for a direct molecular interaction involved in temporally regulated gene expression in M. xanthus.

Fig. 1.

fruA expression during development. (A) The map of the 9.5-kbp fragment containing fruA (S, StuI; H, HincII; C, ClaI). (B) The promoter region of fruA. The transcription initiation site is indicated by an arrow. The DNA-binding site identified by footprint analysis shown in Fig. 2B is double-underlined. The sequences corresponding to oligonucleotide primers a–d are underlined. (C) lacZ fusion analysis. The promoter regions from nucleotides –185 to +270 (squares) and from nucleotides –40 to +270 (circles) were fused to lacZ, and β-galactosidase activity was measured during development. Triangles represent pZKAT without the promoter.

Fig. 2.

Identification of the DNA-binding site. (A) Shown is a DNA-binding assay. The probe contains the promoter region from nucleotides –185 to –41. AS fractions prepared from vegetative (lane 2) and 4-, 8-, and 12-h developmental cells (lanes 3, 4, and 5, respectively) were used for the protein source. Note that the same number of initial cells is harvested at each time point. Lane 1 contains no AS fraction. (B) Shown is footprint analysis. DNA-binding reactions were performed under the same conditions as described for A. After the binding reaction and gel electrophoresis, complex I, complex II, and free probe were excised from the gel and subjected to the treatment with 1,10-phenanthroline-copper. Lane 1, free probe; lane 2, complex I; lane 3, complex II. Lanes G, A, T, and C represent sequence ladders generated by a primer 5′-GATCCCCAGCCCCAATGGGAGTG-3′, which was labeled at the 5′ end with [γ-³²P]ATP by T4 polynucleotide kinase and can hybridize just upstream of the –35 region boxed in Fig. 1B. (C) Shown are the sequences of the DNA-binding site. (D) Sequence comparison between regions a and b.

Fig. 3.

Identification of the fruA promoter-binding protein. (A) Shown is purification of FBP from M. xanthus. After the second round of DNA affinity column chromatography, the sample was applied to SDS/15% PAGE and visualized by silver staining. (B) Purification of MrpC2 from E. coli. After the second round of DNA affinity column chromatography, the sample was applied to SDS/15% PAGE and visualized by silver staining. (C) Shown are the sequences of the upstream region of mrpC and N-terminal end of MrpC. The previously assigned ribosome-binding site (nucleotides 1–5) and the initiation codon (nucleotides 13–15) are underlined (10). The N-terminal sequence determined for FBP is double-underlined. The newly assigned initiation codon and the ribosome-binding site are indicated by bold letters and underlined.

Fig. 4.

Footprint analysis. MrpC2 (20 ng) purified from E. coli, FBP (20 ng) from M. xanthus, and the AS fraction (200 μg) were used. The DNA-binding reactions were carried out in a 100-μl volume, and, after gel electrophoresis, complex I, complex II, and free probe were excised from the gel and subjected to the treatment of 1,10-phenanthroline-copper. Lane 1, free probe; lane 2, complex I with FBP; lane 3, complex II with FBP; lane 4, complex I with MrpC2; lane 5, complex II with MrpC2; lane 6, complex II with the AS fraction.

Fig. 5.

Expression of fruA in M. xanthus DZF1 and mrpC::km. fruA expression was examined by primer extension analysis as described (18). Total RNA was prepared from DZF1 and mrpC::km during vegetative growth (0 h) and fruiting body development (6 and 12 h). As a control, vegA expression was examined. Oligonucleotide primers 5′-TTGACTTTCAGCTACTCCTGACG-3′ and 5′-GCTTTATCCACGGACATT-3′ were used for fruA and vegA, respectively.

Fig. 6.

DNA-binding analysis. (Upper) Three kinds of probes (10 fmol), an HindIII (H)–BamHI (B) fragment containing both regions a and b (lanes 1–3), an SmaI (S)–BamHI fragment containing region a (lanes 4–6), or an HindIII–SmaI fragment containing region b (lanes 7–9) were used for DNA-binding reactions in a 10-μl volume. Note that the HindIII and BamHI sites are from the cloning vector. No protein was added for lanes 1, 4, and 7. Purified MrpC2 was added for lanes 2, 5, and 8 (1 ng) and lanes 3, 6, and 9 (2 ng). MrpC–DNA complexes are indicated by arrowheads. (Lower) The fruA promoter region from nucleotides –185 to –41 is shown.

Fig. 7.

Mutational analysis. (A) DNA-binding assay. Probes (10 fmol) contain the promoter region from nucleotides –185 to –41 without mutations (lanes 1 and 2) and with mutations TC-150GA (lanes 3 and 4), TT-145AC (lanes 5 and 6), GA-140TC (lanes 7 and 8), TC-126GA (lanes 9 and 10), CT-121AC (lanes 11 and 12), GA-116TC (lanes 13 and 14), and GA-140TC/TC-126GA (lanes 15 and 16). Purified MrpC2 (1 ng) was added to DNA-binding reaction mixtures in a 10-μl volume for lanes 2, 4, 6, 8, 10, 12, 14, and 16; no protein was added for lanes 1, 3, 5, 7, 9, 11, 13, and 15. MrpC-DNA complexes are indicated by arrows. Changes of sequences are shown below DNA-binding patterns. (B) lacZ fusion analysis. The promoter region from nucleotides –185 to +270 containing the WT or mutant sequences was fused to lacZ, and β-galactosidase activity was measured 12 h after the initiation of development. The activity is shown as a percentage of the activity of the WT promoter.

Fig. 8.

A model for the signal transduction pathway including FruA during fruiting body development of M. xanthus. See Discussion for details.