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 (), 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 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.Members of the genus Streptomyces produce an enormous variety of biologically active antibiotics and morphologically differentiate in a similar manner to eukaryotic fungi. Mature colonies of these gram-positive prokaryotic bacteria show an upper layer of aerial mycelia bearing spores that covers a lower layer of vegetative mycelia growing on the surface of agar medium. The production of antibiotics and formation of aerial hyphae, which appear to be independent events, have been considered genetically correlated based on the occurrence of pleiotropic mutants that neither produce antibiotics nor form aerial mycelia (8,17). Hence, the pleiotropic regulation has provided a subject of special interests in Streptomyces genetics.A variety of pleiotropic mutants such as bldA, bldB, and bldD mutants have been obtained from Streptomyces coelicolor together with many other types of bld mutants that fail to form aerial hyphae. Molecular cloning of the responsible genes has demonstrated transcriptional and translational regulation of pleiotropy. The bldA gene encodes leucyl tRNA for a rarely used UUA codon in streptomycetes (24, 26). Since the rare TTA codon is present in the regulatory genes for actinorhodin and undecylprodigiosin syntheses in S. coelicolor and for streptomycin and aerial mycelium productions in Streptomyces griseus, bldA mutations result in the pleiotropic phenotype in the streptomycetes (12, 23). Furthermor...

Deletion of the rpoZ gene, encoding the ω subunit of RNA polymerase, results in pleiotropic surface-related phenotypes in Mycobacterium smegmatis

“…Given the role identified for v in mediation of the effects of alarmone ppGpp on RNAP, the defective execution of stringent response signals for biofilm formation due to lack of v subunit in RNAP can be conceived as a possible explanation for the phenotypes we observed for mcdrz. This will be similar to the explanation that has been offered for another surprising connection observed for the v subunit: with antibiotic production and morphogenesis in Streptomyces kasugaensis (Kojima et al, 2002).…”

“…However, evidence regarding a functional role played by v has been rare in the literature. The finding that a mutation in the rpoZ gene encoding v caused pleiotropic phenotypes in Streptomyces kasugaensis was suggestive of functions which required to be further defined (Kojima et al, 2002). A more direct indication has been the recent confirmation of the requirement of the v subunit for the response of RNAP to ppGpp (Vrentas et al, 2005).…”

Response of RNA polymerase to ppGpp: requirement for the ω subunit and relief of this requirement by DksA

“…are positively controlled by ppGpp (Bibb 2005). Disruption of rpoZ drastically disrupts these processes in Streptomyces kasugaensis (Kojima et al 2002), suggesting that the effects of ppGpp on RNAP in this organism are dependent on in vivo, perhaps by the same mechanism described here for E. coli.…”

The Biosynthesis of Kasugamycin, an Antibiotic against Rice Blast Disease, with Particular Reference to the Involvement of rpoZ, a Gene Encoding RNA Polymerase Omega Subunit

“…We previously cloned a 22.4-kb chromosomal DNA region (kas cluster) containing the biosynthetic genes for KSM together with kac 338 the KSM acetyltransferase gene, kasKLM, ABC transporter genes, both of which are responsible for KSM self-resistance, and kasT, the KSM-biosynthesis-specific transcriptional activator gene [6][7][8][9]. Meanwhile, we found that disruption of rpoZ, the gene encoding the RNA polymerase (RNAP) subunit omega (w), turned the wild-type S. kasugaensis into pleiotropic mutants, which were phenotypically similar to the bldA mutation, and clarified that the gene is required for KSM production and aerial mycelium formation in the streptomycete [10] Antibiotic-producing streptomycetes generally harbor the selfresistance gene within the biosynthetic gene cluster, and a variety of such clusters have been cloned using the self-resistance genes as probe [4] . In a similar attempt, we initially cloned a 7.6-kb Pstl-Kpnl DNA fragment, including the KSM acetyltransferase gene (kac338) and eight genes (kasA-kasl) as illustrated in Figure. 4, from the chromosomal DNA of wild-type S. kasugaensis [7] .…”