Keith F. Chater

PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin

Streptomycetes are high G+C Gram-positive, antibiotic-producing, mycelial soil bacteria. The 8.7-Mb Streptomyces coelicolor genome was previously sequenced by using an ordered library of Supercos-1 clones. Here, we describe an efficient procedure for creating precise gene replacements in the cosmid clones by using PCR targeting and λ-Red-mediated recombination. The cloned Streptomyces genes are replaced with a cassette containing a selectable antibiotic resistance and oriTRK2 for efficient transfer to Streptomyces by RP4-mediated intergeneric conjugation. Supercos-1 does not replicate in Streptomyces, but the clones readily undergo double-crossover recombination, thus creating gene replacements. The antibiotic resistance cassettes are flanked by yeast FLP recombinase target sequences for removal of the antibiotic resistance and oriTRK2 to generate unmarked, nonpolar mutations. The technique has been used successfully by >20 researchers to mutate around 100 Streptomyces genes. As an example, we describe its application to the discovery of a gene involved in the production of geosmin, the ubiquitous odor of soil. The gene, Sco6073 (cyc2), codes for a protein with two sesquiterpene synthase domains, only one of which is required for geosmin biosynthesis, probably via a germacra-1 (10) E,5E-dien-11-ol intermediate generated by the sesquiterpene synthase from farnesyl pyrophosphate.

Genomics of Actinobacteria: Tracing the Evolutionary History of an Ancient Phylum

SUMMARY Actinobacteria constitute one of the largest phyla among Bacteria and represent gram-positive bacteria with a high G+C content in their DNA. This bacterial group includes microorganisms exhibiting a wide spectrum of morphologies, from coccoid to fragmenting hyphal forms, as well as possessing highly variable physiological and metabolic properties. Furthermore, Actinobacteria members have adopted different lifestyles, and can be pathogens (e.g., Corynebacterium, Mycobacterium, Nocardia, Tropheryma, and Propionibacterium), soil inhabitants (Streptomyces), plant commensals (Leifsonia), or gastrointestinal commensals (Bifidobacterium). The divergence of Actinobacteria from other bacteria is ancient, making it impossible to identify the phylogenetically closest bacterial group to Actinobacteria. Genome sequence analysis has revolutionized every aspect of bacterial biology by enhancing the understanding of the genetics, physiology, and evolutionary development of bacteria. Various actinobacterial genomes have been sequenced, revealing a wide genomic heterogeneity probably as a reflection of their biodiversity. This review provides an account of the recent explosion of actinobacterial genomics data and an attempt to place this in a biological and evolutionary context.

Molecular Regulation of Antibiotic Biosynthesis in Streptomyces

SUMMARY Streptomycetes are the most abundant source of antibiotics. Typically, each species produces several antibiotics, with the profile being species specific. Streptomyces coelicolor, the model species, produces at least five different antibiotics. We review the regulation of antibiotic biosynthesis in S. coelicolor and other, nonmodel streptomycetes in the light of recent studies. The biosynthesis of each antibiotic is specified by a large gene cluster, usually including regulatory genes (cluster-situated regulators [CSRs]). These are the main point of connection with a plethora of generally conserved regulatory systems that monitor the organisms physiology, developmental state, population density, and environment to determine the onset and level of production of each antibiotic. Some CSRs may also be sensitive to the levels of different kinds of ligands, including products of the pathway itself, products of other antibiotic pathways in the same organism, and specialized regulatory small molecules such as gamma-butyrolactones. These interactions can result in self-reinforcing feed-forward circuitry and complex cross talk between pathways. The physiological signals and regulatory mechanisms may be of practical importance for the activation of the many cryptic secondary metabolic gene cluster pathways revealed by recent sequencing of numerous Streptomyces genomes.

The complex extracellular biology of Streptomyces

Streptomycetes, soil-dwelling mycelial bacteria that form sporulating aerial branches, have an exceptionally large number of predicted secreted proteins, including many exported via the twin-arginine transport system. Their use of noncatalytic substrate-binding proteins and hydrolytic enzymes to obtain soluble nutrients from carbohydrates such as chitin and cellulose enables them to interact with other organisms. Some of their numerous secreted proteases participate in developmentally significant extracellular cascades, regulated by inhibitors, which lead to cannibalization of the substrate mycelium biomass to support aerial growth and sporulation. They excrete many secondary metabolites, including important antibiotics. Some of these play roles in interactions with eukaryotes. Surprisingly, some antibiotic biosynthetic enzymes are extracellular. Antibiotic production is often regulated by extracellular signalling molecules, some of which also control morphological differentiation. Amphipathic proteins, assembled with the help of cellulose-like material, are required for both hyphal attachment to surfaces and aerial reproductive growth. Comparative genomic analysis suggests that the acquisition of genes for extracellular processes has played a huge part in speciation. The rare codon TTA, which is present in the key pleiotropic regulatory gene adpA and many pathway-specific regulatory genes for antibiotic production, has a particular influence on extracellular biology.

Regulation of sporulation in Streptomyces coelicolor A3(2): a checkpoint multiplex?

By complementing developmental mutants of Streptomyces coelicolor A3(2), at least 15 regulatory genes for sporulation have been identified and studied at the molecular level, and some of their intracellular interactions have been characterised. Extensive interplay of the regulatory cascade with metabolic, morphological, homeostatic and stress-related checkpoints is emerging.

λ Red-Mediated Genetic Manipulation of Antibiotic-Producing Streptomyces

Publisher Summary This chapter explores the importance of λ Red recombination to be used in Streptomyces . This rapid and highly efficient method has made the generation of gene disruptions more precise and allows the construction of in-frame deletions. So far, more than 100 segments of the S. coelicolor genome ranging in size between 4 bp and over 7 kb have been replaced by PCR-targeting. The technique has also succeeded in other Streptomyces species. The chapter discusses the use of this technology for various other DNA modifications such as introducing point mutations, promoter replacements, and gene fusions. Combining the different approaches helps manipulate Streptomyces DNA more rapidly and precisely than using traditional techniques. The facile integration of whole antibiotic gene clusters into Streptomyces chromosomes makes high-throughput manipulation of the clusters possible. The genes for synthesis of any one antibiotic in streptomycetes are invariably clustered together on the chromosome (or sometimes on a plasmid). The availability of plasmid vectors, which can efficiently carry stable large inserts into different Streptomyces spp. has been exploited in a number of laboratories to allow production in a heterologous host.

Taking a genetic scalpel to the Streptomyces colony

Summary: 1997 Fred Griffith Review Lecture (Delivered at the 138th Meeting of the Society for General Microbiology, 2 September 1977)

Spore colour in Streptomyces coelicolor A3(2) involves the developmentally regulated synthesis of a compound biosynthetically related to polyketide antibiotics

Streptomyces coelicolor produces spores whose development of a grey colour requires the activity of the whiE locus. The cloned whiE locus was identified after mobilization into a whiE mutant of a library of S. coelicolor DNA inserted into a transmissible plasmid vector. The whiE region of the cloned DNA was localized both by subcloning and by mutagenesis of the cloned DNA with the Streptomyces transposon Tn4560. Nucleotide sequencing of this region revealed seven open reading frames, of which six show homo‐logy at the level of deduced gene products with genes involved in the synthesis of polyketide antibiotics. A previously described S. coelicolor DNA segment encoding biosynthesis of a brown pigment (Horinouchi and Beppu, 1985) corresponds to the cloned whiE DNA. It is proposed that whiE is normally expressed only in the aerial hyphae, where the biosynthetic product is responsible for spore colour.

Multiple paralogous genes related to the Streptomyces coelicolor developmental regulatory gene whiB are present in Streptomyces and other actinomycetes

The whiB sporulation gene of Streptomyces coelicolor was shown [Davis, N. K. & Chater, K. F. (1992). Mol Gen Genet 232, 351-358] to encode a small, cysteine-rich putative transcription factor unlike any that had been described previously. The large database of DNA sequences of mycobacteria (like Streptomyces, members of the Actinomycetales) has revealed a family of genes encoding proteins related to WhiB. Mycobacterium tuberculosis contains at least six such genes (whiB homologues in mycobacteria: whmA-F) and a likely seventh, whmG. Using conserved features of Whm proteins, a PCR-based approach led to the discovery that S. coelicolor A3(2) contains several similar genes. Cloning and sequencing of these whiB-like (wbI) genes revealed likely orthologues of four of the whm genes of M. tuberculosis. In all, S. coelicolor contains at least five wbI genes in addition to whiB itself. All five were shown by RT-PCR to be transcribed. A Southern blotting survey using each wbI gene as a probe showed that nearly all of a series of representatives of ten actinomycete genera (including morphologically simple organisms) contain close homologues of several wbI genes, suggesting that the ancient progenitor of all these organisms already contained a family of such genes, which have not been found in any other organisms.

A bacterial hormone (the SCB1) directly controls the expression of a pathway‐specific regulatory gene in the cryptic type I polyketide biosynthetic gene cluster of Streptomyces coelicolor

Gamma‐butyrolactone signalling molecules are produced by many Streptomyces species, and several have been shown to regulate antibiotic production. In Streptomyces coelicolor A3(2) at least one γ‐butyrolactone (SCB1) has been shown to stimulate antibiotic production, and genes encoding proteins that are involved in its synthesis (scbA) and binding (scbR) have been characterized. Expression of these genes is autoregulated by a complex mechanism involving the γ‐butyrolactone. In this study, additional genes influenced by ScbR were identified by DNA microarray analysis, and included a cryptic cluster of genes for a hypothetical type I polyketide. Further analysis of this gene cluster revealed that the pathway‐specific regulatory gene, kasO, is a direct target for regulation by ScbR. Gel retardation and DNase I footprinting analyses identified two potential binding sites for ScbR, one at −3 to −35 nt and the other at −222 to −244 nt upstream of the kasO transcriptional start site. Addition of SCB1 eliminated the DNA binding activity of ScbR at both sites. The expression of kasO was growth phase regulated in the parent (maximal during transition phase), undetectable in a scbA null mutant, and constitutively expressed in a scbR null mutant. Addition of SCB1 to the scbA mutant restored the expression of kasO, indicating that ScbR represses kasO until transition phase, when presumably SCB1 accumulates in sufficient quantity to relieve kasO repression. Expression of the cryptic antibiotic gene cluster was undetectable in a kasO deletion mutant. This is the first report with comprehensive in vivo and in vitro data to show that a γ‐butyrolactone‐binding protein directly regulates a secondary metabolite pathway‐specific regulatory gene in Streptomyces.