Wendy Champness

Regulation of the Streptomyces coelicolor Calcium-Dependent Antibiotic by absA, Encoding a Cluster-Linked Two-Component System

The Streptomyces coelicolor absA two-component system was initially identified through analysis of mutations in the sensor kinase absA1 that caused inhibition of all four antibiotics synthesized by this strain. Previous genetic analysis had suggested that the phosphorylated form of AbsA2 acted as a negative regulator of antibiotic biosynthesis in S. coelicolor (T. B. Anderson, P. Brian, and W. C. Champness, Mol. Microbiol. 39:553-566, 2001). Genomic sequence data subsequently provided by the Sanger Centre (Cambridge, United Kingdom) revealed that absA was located within the gene cluster for production of one of the four antibiotics, calcium-dependent antibiotic (CDA). In this paper we have identified numerous transcriptional start sites within the CDA cluster and have shown that the original antibiotic-negative mutants used to identify absA exhibit a stronger negative regulation of promoters upstream of the proposed CDA biosynthetic genes than of promoters in the clusters responsible for production of actinorhodin and undecylprodigiosin. The same antibiotic-negative mutants also showed an increase in transcription from a promoter divergent to that of absA, upstream of a putative ABC transporter, in addition to an increase in transcription of absA itself. Interestingly, the negative regulation of the biosynthetic transcripts did not appear to be mediated by transcriptional regulation of cdaR (a gene encoding a homolog of the pathway-specific regulators of the act and red clusters) or by any other recognizable transcriptional regulator associated with the cluster. The role of absA in regulating the expression of the diverse antibiotic biosynthesis clusters in the genome is discussed in light of its location in the cda cluster.

Genetic and transcriptional analysis of absA, an antibiotic gene cluster-linked two-component system that regulates multiple antibiotics in Streptomyces coelicolor.

In Streptomyces coelicolor, the AbsA1–AbsA2 two‐component system regulates the expression of multiple antibiotic gene clusters. Here, we show that the response regulator encoded by the absA2 gene is a negative regulator of these antibiotic gene clusters. A genetic analysis shows that the phosphorylated form of the AbsA2 response regulator (phospho‐AbsA2), generated by the cognate AbsA1 sensor histidine kinase, is required for normal growth phase regulation of antibiotic synthesis. In the absence of phospho‐AbsA2, antibiotics are produced earlier and more abundantly. Overexpression of AbsA1 also deregulates antibiotic synthesis, apparently shifting the AbsA1 protein from a kinase‐active to a phospho‐AbsA2 phosphatase‐active form. The absA1 and absA2 genes, which are adjacent, are located in one of the antibiotic gene clusters that they regulate, the cluster for the calcium‐dependent antibiotic (CDA). The absA genes themselves are growth phase regulated, with phospho‐AbsA2 responsible for growth phase‐related positive autoregulation. We discuss the possible role and mechanism of AbsA‐mediated regulation of antibiotic synthesis in the S. coelicolor life cycle.

Identification of Streptomyces coelicolor genes involved in regulation of antibiotic synthesis

To define genetic elements that regulate antibiotic synthesis, we screened for mutations that visibly blocked synthesis of Streptomyces coelicolors two pigmented antibiotics and found mutant strains in which all four antibiotics were blocked. The responsible mutations defined two loci, absA and absB. Two additional approaches to defining genes have been taken: isolation of cloned genes with a dominant negative effect on antibiotic synthesis and isolation of genes which, in multicopy, can compensate for specific mutational blocks. These genes apparently function in a global regulatory pathway (or network) for control of antibiotic synthesis.

The gol site: A Cis-acting bacteriophage T4 regulatory region that can affect expression of all the T4 late genes☆

Abstract We have shown that mutations in the Escherichia coli lit gene can prevent the expression of the late genes of bacteriophage T4 at temperatures below 34 °C. The defect in late gene expression occurs, at least partially, at the level of transcription, and neither DNA replication nor DNA encapsidation into phage heads is significantly affected. Rare T4 “ gol ” mutations overcome the defect in late transcription. Refined mapping experiments place gol mutations within gene 23, but an altered gene 23 protein is not responsible for the phenotype. Rather, gol mutations seem to alter a cis -acting site in T4 DNA, the wild-type form of which interferes with late transcription in lit − hosts.

Genetic suppression analysis of non-antibiotic-producing mutants of the Streptomyces coelicolor absA locus.

The absA locus in Streptomyces coelicolor A3(2) was identified because mutations in it uncoupled sporulation from antibiotic synthesis: absA mutants failed to produce any of the four antibiotics characteristic of S. coelicolor. These mutants are now shown to contain point mutations in the absA1 gene which encodes the histidine kinase sensor-transmitter protein of a two-component signalling system. The absA1 non-antibiotic-producing mutants, which are unpigmented, spontaneously acquire pigmented colony sectors. Genetic analysis established that the pigmented sectors contain second-site suppressive mutations, sab (for suppressor of abs). Phenotypic characterization showed that sab strains produce all four antibiotics; some overproduce antibiotics and are designated Pha, for precocious hyperproduction of antibiotics. A set of sab mutations responsible for suppression was localized by plasmid-mediated and protoplast fusion mapping techniques to the vicinity of the absA locus. DNA cloned from this region was used to construct phage that could transduce sab mutations. Sequence analysis of sab strains defined sab mutations in both the absA1 gene and the absA2 gene; the latter encodes the two-component systems response regulator.