The rpoZ gene, encoding the RNA polymerase omega subunit, is required for antibiotic production and morphological differentiation in Streptomyces kasugaensis

Abstract

The occurrence of pleiotropic mutants that are defective in both antibiotic production and aerial mycelium formation is peculiar to streptomycetes. Pleiotropic mutant KSB was isolated from wild-type Streptomyces kasugaensis A1R6, which produces kasugamycin, an antifungal aminoglycoside antibiotic. A 9.3-kb DNA fragment was cloned from the chromosomal DNA of strain A1R6 by complementary restoration of kasugamycin production and aerial hypha formation to mutant KSB. Complementation experiments with deletion plasmids and subsequent DNA analysis indicated that orf5, encoding 90 amino acids, was responsible for the restoration. A protein homology search revealed that orf5 was a homolog of rpoZ, the gene that is known to encode RNA polymerase subunit omega (omega), thus leading to the conclusion that orf5 was rpoZ in S. kasugaensis. The pleiotropy of mutant KSB was attributed to a 2-bp frameshift deletion in the rpoZ region of mutant KSB, which probably resulted in a truncated, incomplete omega of 47 amino acids. Furthermore, rpoZ-disrupted mutant R6D4 obtained from strain A1R6 by insertion of Tn5 aphII into the middle of the rpoZ-coding region produced neither kasugamycin nor aerial mycelia, similar to mutant KSB. When rpoZ of S. kasugaensis and Streptomyces coelicolor, whose deduced products differed in the sixth amino acid residue, were introduced into mutant R6D4 via a plasmid, both transformants produced kasugamycin and aerial hyphae without significant differences. This study established that rpoZ is required for kasugamycin production and aerial mycelium formation in S. kasugaensis and responsible for pleiotropy.

FIG. 1.

Restriction map of pSK117 and pSK1171. Asterisks represent unique restriction sites in the plasmid, and ermE(p) indicates the promoter region of ermE harboring ermEp1 and ermEp2 (4). Both plasmids carry the hybrid tyrosinase genes melE and melE1 as described in Materials and Methods.

FIG. 2.

Restriction map of 9.3-kb cloned DNA fragment. Solid arrows above the map indicate open reading frames predicted by FramePlot analysis (19) on the 3.9-kb BamHI-SmaI sequenced region. Solid lines below the map denote the DNA regions subcloned into the respective deletion plasmids.

FIG. 3.

Kasugamycin production by transformants of mutant KSB (A) and disruptant mutant R6D4 (B). Bioassays using agar plugs were performed for detection of kasugamycin production by the transformants as detailed in Materials and Methods. The halos formed around the plugs indicate the accumulation of kasugamycin by the transformants. The transformants of wild-type strain A1R6 with pSK117 were used as a positive control. (A) From left to right, strain A1R6 carrying pSK117; and mutant KSBs harboring pSK117, pAK3521, pAK3541, pAK3371, and pAK522. (B) From left to right, strain A1R6 carrying pSK117 and mutant R6D4s harboring pSK117, pAK522, and pAK531.

FIG. 4.

Aerial mycelium formation by transformants of mutant KSB (A) and disruptant mutant R6D4 (B). Aerial hypha formation was examined qualitatively by inoculation of the transformants on GMY media as described in Materials and Methods. The transformants of wild-type strain A1R6 with pSK117 were used as a positive control. (A) Clockwise, from top left, mutant KSBs carrying pSK117, pAK3521, pAK3371, and pAK522 and strain A1R6 harboring pSK117. (B) Clockwise, from top left, disruptant mutant R6D4s carrying pSK117, pAK522, and pAK531 and strain A1R6 harboring pSK117.

FIG. 5.

Multiple alignment of the amino acid sequences of subunit of ω in S. kasugaensis. With the ClustalX program (available at inn-prot.weizmann.ac.il/software/ClustalX.html), sequences of ω subunits from the following genome-sequenced actinomycetes were analyzed: S. coelicolor (CAB93358), M. tuberculosis H37Rv (CAB02173), M. tuberculosis CDC1551 (AAK45700), and M. leprae (CAC30050). Asterisks indicate fully conserved amino acids, colons represent that one of the “strong” groups of amino acids (such as STA, MILV, NDEQ, HY, and NHQK) is fully conserved, and dots indicate that one of the “weak” groups of amino acids (such as STPA, ATV, and SGND) is fully conserved.

FIG. 6.

Restriction map of pAK3741 used for generation of disruptant mutant R6D4. A shuttle vector pAK3741 composed of 5.57- and 5.37-kb EcoRI digests of S. kasugaensis plasmid pSK21-K2 carrying tsr (31) and pAK374, whose construction is detailed in Materials and Methods. In the plasmid, bla indicates β-lactamase originating from pUC18. As illustrated, a homologous recombination was expected to occur between aphII-disrupted rpoZ and chromosomal rpoZ of wild-type strain A1R6.

FIG. 7.

Comparison of nucleotide sequences of rpoZ from strain A1R6 and mutant KSB. Hyphens in the rpoZ-encoding region from mutant KSB indicate gaps in the nucleotide sequence.