Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of extracellular proteins that were obtained by TCA precipitation of the culture broth of S. griseus, followed by Resource-S cation exchange column chromatography. Lane M, molecular size standards; lane 1, TCA precipitate of the wild-type strain S. griseus IFO13350; lane 2, TCA precipitate of the A-factor-deficient S. griseus mutant HH1; lane 3, flowthrough fraction of the TCA precipitate of strain IFO13350 from the Resource-S cation exchange column; and lane 4, flowthrough fraction of the TCA precipitate of mutant HH1 from the Resource-S cation exchange column. Each lane contained appropriate amounts of proteins. Four proteins, A, B, D, and E, are observed only in the wild-type strain, and one protein, C, is observed only in mutant HH1.

Transcriptional analysis of sprT and sprU. (A) The time courses of sprT and sprU transcription were followed by low-resolution S1 mapping with RNA prepared from cells grown at 28°C for the indicated hours on cellophane on the surface of YMPD medium. As controls, transcription of adpA and hrdB was also determined with the same RNA samples. hrdB, which is transcribed throughout growth, was used to check the purity and amount of mRNA used. The wild-type (wt) strain grew as substrate mycelium (SM) at 24 h, as a mixture of substrate and aerial mycelium (AM) at 48 h, and as a mixture of aerial hyphae and spores (SP) at 72 h. (B) The transcriptional start points of sprT and sprU were determined by high-resolution S1 nuclease mapping. RNA (40 μg each) prepared from wild-type cells grown at 28°C for 72 h on YMPD solid medium was used. The arrows indicate the positions of S1 nuclease-protected fragments.

DNase I footprinting for determining the AdpA-binding sites in front of sprT (A) and sprU (B). DNase I footprinting assays were performed on the sense and antisense strands. The amounts of AdpA-H used in lanes 1, 2, 3, 4, and 5 were 0, 0.2, 0.4, 0.8, and 0 μg, respectively. The DNase I digests were run with the same probes that were chemically cleaved (G+A lanes).

Disruption of the chromosomal sprT and sprU genes and trypsin activity in the disruptants. (A and B) Schematic representationof disruption of sprT and sprU on the chromosome of S. griseus IFO13350. Restriction enzymes: N, NcoI; B, BamHI; K, KpnI; P, PvuII; Sm, SmaI; H, HindIII; and E, EcoRI. (C) Southern hybridization to check the correct disruption of sprT and sprU with the indicated sprT probe against the BamHI-digested chromosome. The sprT probe also hybridized to the sprU sequence. ΔsprTU, doubly disrupted sprT and sprU mutant. (D) Trypsin activities of the ΔsprT, ΔsprU, and ΔsprTU mutants as a function of cultivation time. Strains were grown at 28°C for 72 h on YMPD agar medium.

Gene organization in the cloned fragments containing sprT and sprU (A), amino acid sequences of SprT and SprU (B), and nucleotide sequences of the promoter and operator regions of sprT and sprU (C). (A) The positions and directions of open reading frames predicted by FramePlot analysis (10) of the nucleotide sequence are indicated by arrows. Restriction sites used in the construction of plasmids are also shown. Abbreviations for restriction enzymes: A, Aor51HI; B, BamHI; H, HindIII; N, NcoI; S, SalI; and Sc, SacI. The numbers on the restriction sites are the nucleotide positions, taking the transcriptional start point as +1. (B) The amino acid sequences of SprT and SprU were deduced from the nucleotide sequences and aligned. The amino acid sequence of SprT, starting at Val-37, determined with the purified protein is shown with a dotted line. The amino acids that form a His-Asp-Ser catalytic triad are indicated by stars. The N terminus of SprT is indicated by an open triangle, and sites in SprT and SprU that are probably cleaved by a signal peptidase are also shown by solid triangles. (C) The promoter and operator sequences of sprT and sprU are shown. The transcriptional start points determined by high-resolution S1 mapping (see Fig. 3B) are taken as +1. The AdpA binding sites determined by DNase I footprinting (see Fig. 5) are indicated. Probable −35 and −10 sequences for the sprT and sprU promoters are indicated with dotted lines (31). Purine-rich sequences, which may serve as ribosome-binding (Shine-Dalgarno) sites, are also shown.

Binding of AdpA-H to the operator regions of sprT (A) and sprU (B), as determined by gel mobility shift assays. (A) The amounts of AdpA-H used were 0.04 μg (lane 2), 0.2 μg (lane 3), and 0.4 μg (lane 4). Lane 1 is a control to which no AdpA-H was added. The probes used were T1 (positions −448 to +61), T2 (positions −112 to +7), T3 (positions −448 to −61), T4 (positions −34 to +61), T5 (positions −238 to −61), and T6 (positions −448 to −219). AdpA-H was found to bind to a single site, as shown. (B) The amounts of AdpA-H used were the same as in A. The probes used were U1 (positions −444 to +96), U2 (positions −200 to +277), U3 (positions +77 to +566), U4 (positions −444 to −181), U5 (positions −193 to −67), U6 (positions −24 to +277), and U7 (positions −24 to +96). AdpA-H was also found to bind to a single site, as shown.

Effects of a mutation in the AdpA-binding sites on the promoter activities of sprT and sprU. (A) Mutations were introduced in the AdpA-binding sites for sprT and sprU. The consensus AdpA-binding sequences are underlined. The sequences indicated by bold letters in the consensus sequence were changed to generate a KpnI cleavage sequence. (B) Gel mobility shift assays for determination of AdpA binding to the mutated sequences. Probe TW (positions −162 to +77) contained the intact operator for sprT, and probe TM contained the mutated operator. Probe UW (positions −164 to +76) contained the intact operator for sprU, and probe UM contained the mutated operator. The amounts of AdpA-H used in lanes 1, 2, 3, 4, and 5 were 0, 15, 45, 150, and 300 ng, respectively. (C) Transcriptional analysis of sprT and sprU with mutated AdpA-binding sites by reverse transcription-PCR. RNA was prepared from cells grown at 28°C for 72 h on YMPD agar medium. The primers for reverse transcription were designed within the sequences that were deleted from the chromosome in the ΔsprT and ΔsprU hosts so that only mRNA derived from the plasmids was reverse transcribed into cDNA. After PCR of the cDNA in a total of 25 cycles, the amplified fragments were separated by 2% agarose gel electrophoresis. The lanes for sprT contained 10 μl each of the amplified solution in a total of 100 μl, and the lanes for sprU contained 20 μl each of the same volume of the amplified solution.