Characterization of ssfR and ssgA, two genes involved in sporulation of Streptomyces griseus

Abstract

In the presence of cefoxitin, which inhibits septum formation during sporulation, Streptomyces griseus is unable to sporulate, retaining the sonication sensitivity of nonsporulating hyphae. Cefoxitin- and sonication-resistant mutant SKK2600 was isolated and showed many morphological differences from its parental strain. A 3.6-kb DNA fragment that complemented the mutations of SKK2600 contained two open reading frames (ORFs), either of which could complement SKK2600. One ORF, designated ssfR, encoded a protein containing a potential DNA-binding helix-turn-helix motif close to its N terminus. SsfR is similar to members of a large family of transcriptional regulators, particularly IclR of Escherichia coli. The second ORF was identified as ssgA, a previously described sporulation gene from S. griseus (S. Kawamoto and J. C. Ensign, Actinomycetology 9:136-151, 1995). A point mutation of C to T seven nucleotides upstream of the UGA stop codon of ssfR was responsible for the phenotype of isolated mutant strain SKK2600. Surprisingly, this mutation should not change the primary structure of SsfR. The ssfR and ssgA disruption mutants were constructed and showed the "white" mutant phenotype, with some growth medium dependence. In addition, the ssfR null mutant sporulated ectopically in phosphate starvation medium.

FIG. 1

Restriction map of a 10-kb BamHI DNA segment of S. griseus containing ssfR and ssgA and nucleotide sequences of part of this region. (A) Segments of DNA subcloned into various plasmids are indicated by shaded bars. DIG, digoxigenin. (B) The nucleotide sequences of the part containing the intergenic region between ssfR and ssgA and deduced amino acid sequences. Arrow, point mutation of C to T; asterisk, stop codon of ssfR.

FIG. 2

Putative HTH motif and comparison with other DNA-binding proteins. For each protein, the 22-amino-acid alignment that constitutes the HTH motif is presented. The DNA-binding proteins which are selected for this alignment are PobR, the transcriptional activator of the Acinetobacter calcoaceticus pobA gene (AcPobR), IclR, the repressor of the E. coli aceBAK operon (EcIclR), PcaU, a regulatory protein of Acinetobacter sp. strain ADP1 (AcPcaU), and KsdR, a transcriptional regulator of Arthrobacter simplex (AsKsdR). Amino acids conserved with SsfR (SgSsfR) are shaded grey.

FIG. 3

Comparison of colony morphology between the wild-type strain and mutants grown on SpM. Wild-type strain NRRL B-2682 shows grey. Mutant SKK2600 shows light yellow. ssfR and ssgA null mutants SKK2663-3 and SKK2664-1 show white.

FIG. 4

Phase-contrast micrographs of impression preparations of the wild-type strain and S. griseus mutants growing on different media. NRRL B-2682 (A to C), wild-type strain; SKK2663 (D to F), ssfR null mutant; SKK2664 (G to I), ssgA null mutant. Arrows in panels A to C, F, and I, spores; arrows in panels D, E, G, and H, aerial hyphae; arrowhead (I), empty mycelium shell. Bar, 4 μm.

FIG. 5

Phase-contrast micrographs of the wild-type strain and S. griseus mutants during sporulation induced by phosphate starvation in submerged culture. NRRL B-2682 (A to C), wild-type strain; SKK2663 (D to F), ssfR null mutant; SKK2664 (G to I), ssgA null mutant. Cells were starved for 0 (A, D, and G); 6 (B, E, and H), and 24 h (C, F, and I). Arrows in panels A and G, vegetative hyphae; arrows in panels B, D, E, and H, sporogenic hyphae; arrows in panels C and F, spores; arrow in panel I, deformed sporogenic hypha. Bar, 5 μm.

FIG. 6

Fluorescence micrographs of the wild-type strain and ssfR null mutant SKK2663 after phosphate starvation in submerged culture. Cells of S. griseus that had been induced to sporulate for 4 h were exposed to only PI or both Flu-APA and PI. (A) Wild-type strain treated with PI; (B) ssfR null mutant SKK2663 treated with both Flu-APA and PI; (C) ssfR null mutant SKK2663 treated with only PI. Yellow arrow (A), sporogenic hypha; blue arrows (B), sporulation septum labeled with Flu-APA; green arrows (C), sporulation compartments. Bar, 5 μm.