ATP-binding cassette transport system involved in regulation of morphological differentiation in response to glucose in Streptomyces griseus

Abstract

Streptomyces griseus NP4, which was derived by UV mutagenesis from strain IFO13350, showed a bald and wrinkled colony morphology in response to glucose. Mutant NP4 formed ectopic septa at intervals along substrate hyphae, and each of the compartments developed into a spore which was indistinguishable from an aerial spore in size, shape, and thickness of the spore wall and in susceptibility to lysozyme and heat. The ectopic spores of NP4 formed in liquid medium differed from "submerged spores" in lysozyme sensitivity. Shotgun cloning experiments with a library of the chromosomal DNA of the parental strain and mutant NP4 as the host gave rise to DNA fragments giving two different phenotypes; one complementing the bald phenotype of the host, and the other causing much severe wrinkled morphology in the host. Subcloning identified a gene (dasR) encoding a transcriptional repressor belonging to the GntR family that was responsible for the reversal of the bald phenotype and a gene (dasA) encoding a lipoprotein probably serving as a substrate-binding protein in an ATP-binding cassette (ABC) transport system that was responsible for the severe wrinkled morphology. These genes were adjacent but divergently encoded. Two genes, named dasB and dasC, encoding a membrane-spanning protein were present downstream of dasA, which suggested that dasRABC comprises a gene cluster for an ABC transporter, probably for sugar import. dasR was transcribed actively during vegetative growth, and dasA was transcribed just after commencement of aerial hypha formation and during sporulation, indicating that both were developmentally regulated. Transcriptional analysis and direct sequencing of dasRA in mutant NP4 suggested a defect of this mutant in the regulatory system to control the expression of these genes. Introduction of multicopies of dasA into the wild-type strain caused ectopic septation in very young substrate hyphae after only 1 day of growth and subsequent sporulation in response to glucose. The ectopic spores of the wild type had a thinner wall than those of mutant NP4, in agreement with the observation that the former was sensitive to lysozyme and heat. Disruption of the chromosomal dasA or dasR in the wild-type strain resulted in growth as substrate mycelium, suggesting an additional role of these genes in aerial mycelium formation. The ectopic septation and sporulation in mutant NP4 and the wild-type strain carrying multicopies of dasA were independent of a microbial hormone, A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone), that acts as a master switch of aerial mycelium formation and secondary metabolism.

FIG. 1.

Morphology of S. griseus NP4 on solid medium. (A) Mycelium of mutant NP4 and the wild-type (wt) strain S. griseus IFO13350 was spread on YMPD agar medium and grown at 28°C for 4 days. The wild-type strain forms white spores, but mutant NP4 shows a wrinkled and ragged morphology. (B) Scanning electron micrographs of the colony surfaces of the wild-type and NP4 strains grown at 28°C for 4 days. Bars, 1 μm. Transmission electron micrographs of a single spore of both strains are also shown.

FIG. 2.

Heat and lysozyme resistance of ectopic, substrate spores of S. griseus mutant NP4 and the wild-type strain S. griseus IFO13350 harboring pES1. The three strains were grown on YMPD agar for the indicated days and spread on the same medium after challenges by heat treatment at 55°C for 30 min and by lysozyme treatment at 20 μg/ml and 37°C for 1 h. At day 2, mutant NP4 and the wild-type strain grew as substrate mycelium, as did the wild-type strain harboring pIJ486 as a control, but the wild type carrying pES1 showed a wrinkled colony morphology, indicative of ectopic septation. At day 5, mutant NP4 formed substrate spores, whereas the wild-type strain formed arthrospores.

FIG. 3.

Morphology of S. griseus NP4 in liquid medium. (A) The wild-type strain S. griseus IFO13350 (wt), mutant NP4, and the wild-type strain carrying multicopies of dasA on pES1 were grown at 28°C in YMPD liquid medium, and their wet cell weights were monitored. (B) Phase-contrast photomicrographs of the wild-type and NP4 strains grown at 28°C for 4 days in YMPD medium.

FIG. 4.

Restriction map and subcloning of the cloned fragment. (A) The extents and directions of open reading frames on the cloned fragments are indicated by arrows. orf1* and orf8* are truncated. Plasmid pNS5 was harbored in one of the seven NP4 transformants showing normal development culminating in sporulation. Plasmid pES1 was harbored in the NP4 transformant showing a severe wrinkled morphology (esp*). pES1 caused the wild-type strain S. griseus IFO13350 to form ectopic, substrate spores (esp). An inverted repeat sequence downstream of dasR is TGA (termination codon of orf3)-(N)₁₀-GGGCGGTGGCCCGGGGA-(N:loop)₅-TCCCCGGGCCACCGGCCC-(N)₄₆-TCA (termination codon of dasR). An inverted repeat sequence downstream of dasA is TGA (termination codon of dasA)-(N)₆₇-TCCGGGGC-(N:loop)₂-GCCCCGGA-(N)₂₂-GTG (start codon of dasB). The insertion of the kanamycin resistance determinant (kan) into dasA and dasR is shown schematically. Abbreviations for restriction enzymes: B, BamHI; E, EcoRI; Hc, HincII: K, KpnI; N, NruI; S, Sau3AI; Sa, SacI; and Sp, SphI. (B) Mutant NP4 containing only dasR on either the high-copy-number or the low-copy-number plasmid developed normally and formed spores after growth at 28°C for 4 days on YMPD agar medium, whereas NP4 containing dasA on pIJ486 showed a more severe wrinkled morphology. Scanning electron micrographs reveals the formation of normal arthrospores by mutant NP4 harboring pHR3 and substrate spores by NP4 harboring pES1.

FIG. 5.

Homologies of DasRABC products with components of the ABC-type transport systems. (A) Amino acid alignment of DasR with the transcriptional repressors in the GntR family. TreR from P. fluorescens (DNA database accession number AAG31030), HutC from P. putida (P22773), and FarR from E. coli (S04645) are shown. Asterisks indicate similar amino acids that are conserved in three of the four proteins. (B) Probable cleavage site of the signal peptide (indicated by a triangle), the signature sequence in SBPs specific for maltooligosaccharides, multiple sugars, α-glycerol phosphate, and iron (60), and amino acid alignment of this region of DasA with MalE of S. coelicolor A3(2) and CebE of S. reticuli are shown. X represents any amino acid. The Lys residue in boldface is the highly conserved amino acid in this family. (C) The consensus sequence conserved in MSDs of ABC transporters (52) and amino acid alignment of this region of DasBC with MalFG and CebFG are shown. X represents any amino acid. The Gly residue in boldface is the highly conserved amino acid in this family. (D) Predicted topology of DasABC as components of an ABC transporter. DasA, a substrate-binding protein, is anchored to the outer surface of the membrane by a lipid attached to Cys-21. DasBC are integrated in the membrane, to each of which an ATP-hydrolyzing subunit (ABD) is associated.

FIG. 6.

Morphology of the wild-type S. griseus strain IFO13350 containing extra copies of dasA. (A) S. griseus IFO13350 harboring pES1 shows a wrinkled colony morphology on YMPD agar medium, as does mutant NP4, and forms substrate spores, as examined by scanning electron microscopy. Photographs were taken after growth at 28°C for 4 days. Bars, 1 μm. (B) The morphological defect of S. griseus IFO13350 harboring pES1 depends on glucose as a carbon source, because on YMP medium containing other sugars at a concentration of 1% instead of glucose, the wild-type strain harboring pES1 shows normal sporulation. Photographs were taken after growth at 28°C for 5 days. (C) Sporulation in the substrate mycelium of agar-grown S. griseus IFO13350 harboring pES1. Bars, 1 μm. An ultrathin section containing substrate mycelium penetrated into agar after growth at 28°C for 2 days was examined at different magnifications (left panel). A substrate spore chain of the wild type harboring pES1 is shown, together with the substrate (SM) and aerial (AM) mycelium of the wild-type strain as controls (right panel). (D) Ectopic, substrate spores of S. griseus IFO13350 harboring pES1, together with the aerial spore of the wild-type strain as a control, is shown. The samples were prepared after growth at 28°C for 4 days. Bar, 0.1 μm.

FIG. 7.

Bald phenotypes of the ΔdasA and ΔdasR mutants derived from S. griseus IFO13350. (A) Colonies of the ΔdasA and ΔdasR mutants were formed with a toothpick on YMPD medium. The growth of the mutants is the same as the wild-type strain, as seen by the diameters of the colonies, but the mutants show a bald phenotype. (B) Scanning electron micrographs of the ΔdasA mutant harboring the vector plasmid pKU209 or pLA1, containing the intact dasA gene. (C) Scanning electron micrographs of the ΔdasR mutant harboring the vector plasmid pKU209 or pLR1, containing the intact dasR gene.

FIG. 8.

Determination of the transcriptional start points of dasA and dasR by S1 nuclease mapping. (A) RNA was prepared from cells grown on YMPD medium at 28°C for 1 day for dasR and 3 days for dasA. The sequence ladders, derived from the same primer, are shown with the A+G and T+C reactions. The deduced transcriptional start points are shown by arrows. (B) The nucleotide sequence covering the promoter regions of dasA and dasR is shown, together with their NH₂-terminal amino acid sequences. Probable Shine-Dalgarno (SD) sequences and probable −35 and −10 sequences for dasA are indicated. (C) RT-PCR for determination of transcriptional linkages of dasABC. Pairs of primers were used to amplify the indicated four DNA fragments. No amplification occurred when reverse transcriptase was omitted from the reaction mixture, indicating the absence of DNA in the mRNA samples.

FIG. 9.

Transcription of dasRABC. (A) RNA was prepared from colonies formed on YMPD agar medium. The wild-type S. griseus IFO13350 strain grew as substrate mycelium at day 1, as a mixture of substrate and aerial hyphae at day 2, and as a mixture of aerial hyphae and spores at day 3. (B) Transcription of dasA is repressed in the wild-type strain harboring pHR3.