An Oligoribonuclease Gene in Streptomyces griseus

Abstract

In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-␥-butyrolactone) serves as a microbial hormone that switches on many genes required for streptomycin production and morphological development. An open reading frame (Orf1) showing high sequence similarity to oligoribonucleases of various origins is present just downstream of adpA, one of the A-factor-dependent genes. Orf1 was named OrnA (oligoribonuclease A) because it showed 3-to-5 exo-oligoribonuclease activity, releasing [ 32 P]CMP from ApCpC[ 32 P]pC used as a substrate. Reverse transcription-PCR and S1 nuclease mapping analyses revealed that ornA was transcribed from two promoters; one was a developmentally regulated, A-factor-dependent promoter in front of adpA, and the other was a constitutive promoter in front of the ornA coding sequence. Transcription of ornA was thus additively enhanced at the initiation stage for secondary metabolism and aerial mycelium formation. ornA-disrupted strains grew slowly and scarcely formed aerial mycelium. ornA homologues were distributed in a wide variety of Streptomyces species, including S. coelicolor A3(2), as determined by Southern hybridization analysis. Disruption of the ornA homologue in S. coelicolor A3(2) also caused phenotypes similar to those of the S. griseus ⌬ornA strains. The OrnA oligoribonucleases in Streptomyces species are therefore not essential but play an important role in vegetative growth and in the initiation of differentiation.The filamentous, soil-inhabiting, gram-positive bacterial genus Streptomyces is characterized by the ability to produce a wide variety of secondary metabolites and by complex morphological differentiation culminating in sporulation (5). In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-␥-butyrolactone) is required for streptomycin (Sm) production and cell differentiation (13-15). A-factor at an extremely low concentration triggers Sm production and aerial mycelium formation by binding a repressor-type receptor protein (ArpA) and dissociating it from the DNA (23, 24). Recently we identified adpA, which encodes a transcriptional activator for strR, a pathway-specific regulatory gene responsible for transcription of other Sm biosynthetic genes, as one of the target genes of ArpA (22). ArpA binds the adpA promoter and represses its transcription in the absence of A-factor during early growth phase. adpA is thus developmentally regulated by A-factor. During these studies, we found an open reading frame (Orf1) showing end-to-end similarity to the oligoribonuclease of Escherichia coli (31) only 10 bp downstream from the termination codon of adpA. Because orf1 is located just downstream of adpA and was expected to be developmentally regulated by A-factor and because little is known about RNA degradation in members of Streptomyces with a complex life cycle, we analyzed the enzyme activity of Orf1 and disrupted orf1 to examine the function of the gene product.Exo-oligoribonuclease activity of Orf1. Orf1 shows high sequence similarity (44% identity) to ...

Functional and phylogenetic analysis of a plant-inducible oligoribonuclease (orn) gene from an indigenous Pseudomonas plasmid

“…Nevertheless, studies to date show that Orn is a 39 to 59 exoribonuclease and an important component of the mRNA degradation pathway in bacteria (Ghosh & Deutscher, 1999;Ohnishi et al, 2000); a similar role has been suggested for the conserved human homologue (Nguyen et al, 2000). In E. coli, Orn is one of eight known exoribonucleases and the only one known to be essential for cell viability (Zuo & Deutscher, 2001).…”

Overexpression of the polynucleotide phosphorylase gene (pnp) of Streptomyces antibioticus affects mRNA stability and poly(A) tail length but not ppGpp levels

“…The oligoribonuclease is essential in E. coli (Ghosh & Deutscher, 1999). Disruption of the gene in S. griseus or S. coelicolor produced a slow-growth phenotype but the enzyme was not essential in either species (Ohnishi et al, 2000). A gene encoding a putative RNase III homologue was identified as the absB locus in S. coelicolor (Adamis & Champness, 1992;Price et al, 1999).…”

“…An ORF with significant sequence homology to the oligoribonuclease of E. coli was cloned from Streptomyces griseus (Ohnishi et al, 2000). Overexpression of the cloned gene in E. coli verified that the product did possess oligoribonuclease activity (Ohnishi et al, 2000). The oligoribonuclease is essential in E. coli (Ghosh & Deutscher, 1999).…”

“…RNase E is a single-strand-specific endoribonuclease which in E. coli is thought to be the key enzyme involved in the initiation of mRNA decay (Coburn & Mackie, 1999;Rauhut & Klug, 1999;Regnier & Arraiano, 2000). An ORF with significant sequence homology to the oligoribonuclease of E. coli was cloned from Streptomyces griseus (Ohnishi et al, 2000). Overexpression of the cloned gene in E. coli verified that the product did possess oligoribonuclease activity (Ohnishi et al, 2000).…”

AdpA, a Central Transcriptional Regulator in the A-Factor Regulatory Cascade That Leads to Morphological Development and Secondary Metabolism inStreptomyces griseus

“…coelicolor A3(2) is the most extensively studied strain in the genus Streptomyces. As described above, this strain has one adpA ortholog, adpA-c. 35) Recently, involvement of adpA-c in morphological development has been reported. 36,37) adpA-c (bldH) is required for the onset of aerial mycelium formation.…”