Novel developmental genes, fruCD, of Myxococcus xanthus: involvement of a cell division protein in multicellular development

Abstract

Myxococcus xanthus is a gram-negative soil bacterium that undergoes multicellular development upon nutrient starvation. In the present study, two novel developmental genes, fruC and fruD, of M. xanthus were identified and characterized. The FruD protein has significant amino acid sequence similarity to the DivIVA proteins of many bacteria including Bacillus subtilis. Vegetative cells of the fruD mutant exhibited a filamentous phenotype. The fruC and fruD mutants displayed similar delayed-development phenotypes. The formation of tightly aggregated mounds by fruC and fruD mutants was slower than that by the wild-type strain. Spore formation by the fruC and fruD mutants initiated after 30 h poststarvation, whereas wild-type M. xanthus initiated spore formation after 18 h. The fruCD genes were constitutively expressed as an operon during vegetative growth and development. S1 mapping revealed that transcription initiation sites of the fruCD operon were located 114 (P1) and 55 bp (P2) upstream of the fruC initiation codon. Only the P1 promoter was active during vegetative growth, while both the P1 and P2 promoters were active during development. The FruD protein was produced as a cytoplasmic protein and formed an oligomer during vegetative growth and development.

FIG. 1.

(A) Gene organization of the fruACD region of M. xanthus. Top horizontal line, restriction map. Circles and triangles, locations of Km^r and Tc^r insertion mutations, respectively; open and filled symbols, insertional mutations promoting proficient development and those producing deficient development, respectively; stippled bar, location of ΔfruCD1 mutation; arrows below the lines, fruACD genes and open reading frames deduced from the nucleotide sequence. The lower part of diagram shows an expanded view of the fruC region and the inserts of plasmids used for complementation experiment and promoter activity analysis. Bent arrows, P1 and P2 transcription initiation sites. The indicated DNA segments were synthesized by PCR and inserted into pTC1 or pSI1403attP. Nucleotide A of the fruC ATG initiation codon is indicated as +1. (B) FruC amino acid sequence deduced from the nucleotide sequence. Numbers on the right indicate residues from the N-terminal end. (C) Alignment of amino acid sequences of M. xanthus FruD and various bacterial DivIVA proteins. White lettering on a black background, identical amino acid residues in three or more sequences. Mxa, M. xanthus; Cpe, C. perfringens; Bha, B. halodurans; Lin, L. innocua; Bsu, B. subtilis.

FIG. 2.

(A) Morphogenesis during the development of M. xanthus DZF1 (wild type) and fruC::Km Ω10, fruD::Km Ω12, and ΔfruCD1 mutants. Vegetative cells of each strain were spotted on CF agar plates. The spots were photographed through a dissecting microscope at the indicated times. (B) Sporulation of M. xanthus DZF1 (wild type) and fruC::Km Ω10, fruD::Km Ω12, and ΔfruCD1 mutants. Vegetative cells (2 × 10⁸) of each strain were spotted on CF agar. At the indicated times, the spots were scraped off the agar surface and sonicated. The refractile spores were counted in a counting chamber in triplicate. Open circles, DZF1 (wild type); solid squares, fruC::Km Ω10 mutant; solid triangles, fruD::Km Ω12 mutant; solid diamonds, ΔfruCD1 mutant.

FIG. 3.

Detection of the fruD transcript during vegetative growth and development of M. xanthus. Shown is RT-PCR analysis of fruCD expression in M. xanthus. Total RNAs prepared from vegetative cells (V) and developing cells at 6 (D6), 12 (D12), and 24 h (D24) poststarvation were treated with (+RT) or without (−RT) RT and subjected to PCR with appropriate primers. C, PCR product amplified from the M. xanthus DZF1 chromosome DNA; M, molecular weight standards, sizes of which are given on the left. Arrow, position of the 133-bp PCR product.

FIG. 4.

(A and B) Transcription initiation sites of the fruCD mRNA. Total RNAs were prepared from M. xanthus DZF1 vegetative cells (V) and developing cells at 6, 12, and 24 h poststarvation and subjected to S1 mapping analysis. Arrows, positions of the S1-protected fragments; G, A, T, and C, sequence ladder generated by the dideoxy chain termination method with appropriate primers and with pFC1 DNA as the template. Panels A and B represent different locations of the gel for S1 mapping analysis. (C) Nucleotide sequence of the fruCD promoter region. Bent arrows, transcription initiation sites. The −35 and −10 regions and the putative Shine-Dalgarno sequence (SD) are underlined. The start codon of fruC is boxed. The N-terminal amino acid sequence of FruC is also indicated.

FIG. 5.

(A) Western blot analysis of the fruD product. Total cell proteins from vegetative cells (V) and developing cells at 24 (D24) and 48 h (D48) after starvation of M. xanthus DZF1 (wild type) and fruC::Km Ω10 and fruD::Km Ω12 mutants were separated by SDS-PAGE. The FruD protein was detected by Western blot analysis using anti-GST-FruD antiserum. Molecular mass standards are given on the left. Arrow, position of the FruD protein. (B) Subcellular localization of the FruD protein. To determine the localization of the FruD protein, M. xanthus DZF1 cells from a vegetative culture were disrupted and separated to periplasmic, cytoplasmic, and total-membrane fractions. After SDS-PAGE, the FruD protein was detected by Western blot analysis using anti-GST-FruD antiserum. Molecular mass standards are given on the left. Arrow, position of the FruD protein. Lanes: W, whole cell; P, periplasm; C, cytoplasm; M, total membrane.

FIG. 6.

Oligomer formation of the FruD protein. (A) Gel filtration chromatography of the FruD protein. Soluble proteins were prepared from M. xanthus vegetative cells (Veg) and developing cells (Dev) at 6 h poststarvation and subjected to TSK-Gel G2000 gel filtration chromatography. After SDS-PAGE of eluate fractions, the FruD protein was detected by Western blot analysis using anti-GST-FruD antiserum. The elution positions of molecular mass standards are indicated at the top (290 kDa, glutamate dehydrogenase; 142 kDa, lactose dehydrogenase; 67 kDa, enolase; 32 kDa, adenylate kinase; 12.4 kDa, cytochrome c). (B) FruD-FruD interaction in the yeast two-hybrid system. S. cerevisiae PJ69-4A cells containing pGADT7 and pGBDT7 with or without the fruD gene were grown at 30°C on SD medium lacking tryptophan, leucine, histidine, and adenine. The β-galactosidase activity (units) of each transformant is shown in parentheses.