Transcriptional Control by A-Factor of strR, the Pathway-Specific Transcriptional Activator for Streptomycin Biosynthesis in Streptomyces griseus

ABSTRACT A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) triggers streptomycin production by inducing the transcription of strR, encoding the pathway-specific transcriptional activator, through signal transduction in the A-factor regulatory cascade in Streptomyces griseus. AdpA, one of the key transcriptional activators in the cascade, bound two upstream activation sites, approximately at nucleotide positions −270 and −50 with respect to the transcriptional start point of strR, as determined by gel mobility shift assays and DNase I footprinting. Transcriptional analysis of the strR promoter with mutated AdpA-binding sites showed that both sites were required for full transcriptional activation of strR by AdpA. Potassium permanganate footprinting showed that AdpA assisted RNA polymerase in forming an open complex at an appropriate position for transcriptional initiation of strR. Nine transcriptional units within the streptomycin biosynthesis gene cluster, including the strR-aphD operon, depended on StrR, indicating that StrR is the pathway-specific transcriptional activator for the whole gene cluster. Consistent with this, expression of strR under the control of a constitutively expressed promoter in an adpA null mutant caused the host to produce streptomycin.

Transfer of Streptomycin Biosynthesis Gene Clusters within Streptomycetes Isolated from Soil

ABSTRACT Streptomyces strains isolated from soil were found to possess various numbers of genes from the streptomycin biosynthesis cluster. The strains missing genes from the cluster also lacked the ability to produce streptomycin. Two of the isolates which contain only part of the cluster are apparently recipients of a gene transfer event. The implications for the role of gene transfer in antibiotic evolution are discussed.

Dependence on reporter gene of apparent activity in gene fusions of a Streptomyces griseus streptomycin biosynthesis promoter

The adjacent genes strR–strA–strB1 lie within the large cluster of genes of streptomycin biosynthesis and resistance in Streptomyces griseus. sirR encodes a pathway-specific activator StrR, suggested by previous work to be either an antiterminator or a conventional activator, binding to its DNA target via a helix-turn-helix motif. strB1 is transcribed in an StrR-dependent fashion from a promoter (PstrB1) that lies downstream from strA; between PstrB1 and sirB1 there is a 300-bp leader region containing numerous inverted repeats that could represent modulatable transcription termination sites. Hybrid plasmids were constructed in vitro with transcriptional fusions in which fragments containing PstrB1 and either the entire leader region ("long" fragments) or a small part of it (the "short" fragment) were cloned upstream of (i) aph as reporter gene, in a high copy number plasmid background, or (ii) xylE as reporter gene, in a low copy number plasmid background. The short fragment directed high levels of APH (aminoglycoside 3′-phosphotransferase) whether StrR was present or not, while the long fragments did not do so in the absence of StrR; one long fragment directed high levels in wild-type S. griseus, in which StrR would be present. Insertion of an extraneous fragment into PstrB1 in the short fragment construct led to loss of APH activity, demonstrating that no adventitious promoter had been formed in the short construct. In vitro deletion of part of the leader region in a long fragment construct led to high APH expression with or without StrR present. Although these results are consistent with the target of StrR being within the leader region, and thus with an antiterminator role, it was found that both long and short fragments in the low copy number background failed to direct high expression of catechol oxygenase (the product of xylE) unless strR was also present on a compatible plasmid. Transfer of PstrB1-xylE fragments to the high copy number vector did not increase catechol oxygenase expression. We interpret these results in terms of an effect, in the hybrid constructs, of one of the reporter genes on promoter function, possibly by affecting local DNA topology.Key words: gene fusions, reporter genes, Streptomyces, streptomycin, regulation of secondary metabolism.