Gregory L. Challis

Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2)

Streptomyces coelicolor is a representative of the group of soil-dwelling, filamentous bacteria responsible for producing most natural antibiotics used in human and veterinary medicine. Here we report the 8,667,507 base pair linear chromosome of this organism, containing the largest number of genes so far discovered in a bacterium. The 7,825 predicted genes include more than 20 clusters coding for known or predicted secondary metabolites. The genome contains an unprecedented proportion of regulatory genes, predominantly those likely to be involved in responses to external stimuli and stresses, and many duplicated gene sets that may represent ‘tissue-specific’ isoforms operating in different phases of colonial development, a unique situation for a bacterium. An ancient synteny was revealed between the central ‘core’ of the chromosome and the whole chromosome of pathogens Mycobacterium tuberculosis and Corynebacterium diphtheriae. The genome sequence will greatly increase our understanding of microbial life in the soil as well as aiding the generation of new drug candidates by genetic engineering.

Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature

This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.

Predictive, structure-based model of amino acid recognition by nonribosomal peptide synthetase adenylation domains

BACKGROUND Nonribosomal peptide synthetases (NRPSs) are large modular proteins that selectively bind, activate and condense amino acids in an ordered manner. Substrate recognition and activation occurs by reaction with ATP within the adenylation (A) domain of each module. Recently, the crystal structure of the A domain from the gramicidin synthetase (GrsA) with L-phenylalanine and adenosine monophosphate bound has been determined. RESULTS Critical residues in all known NRPS A domains have been identified that align with eight binding-pocket residues in the GrsA A domain and define sets of remarkably conserved recognition templates. Phylogenetic relationships among these sets and the likely specificity determinants for polar and nonpolar amino acids were determined in light of extensive published biochemical data for these enzymes. The binding specificity of greater than 80% of the known NRPS A domains has been correlated with more than 30 amino acid substrates. CONCLUSIONS The analysis presented allows the specificity of A domains of unknown function (e.g. from polymerase chain reaction amplification or genome sequencing) to be predicted. Furthermore, it provides a rational framework for altering of A domain specificity by site-directed mutagenesis, which has significant potential for engineering the biosynthesis of novel natural products.

Discovery of microbial natural products by activation of silent biosynthetic gene clusters

Microorganisms produce a wealth of structurally diverse specialized metabolites with a remarkable range of biological activities and a wide variety of applications in medicine and agriculture, such as the treatment of infectious diseases and cancer, and the prevention of crop damage. Genomics has revealed that many microorganisms have far greater potential to produce specialized metabolites than was thought from classic bioactivity screens; however, realizing this potential has been hampered by the fact that many specialized metabolite biosynthetic gene clusters (BGCs) are not expressed in laboratory cultures. In this Review, we discuss the strategies that have been developed in bacteria and fungi to identify and induce the expression of such silent BGCs, and we briefly summarize methods for the isolation and structural characterization of their metabolic products.

Strategies for the discovery of new natural products by genome mining

New drugs from silent gene clusters: Analysis of genome sequence data has identified numerous “cryptic” gene clusters encoding novel natural product biosynthetic assembly lines; this suggests that many new bioactive metabolites remain to be discovered, even in extensively investigated organisms. Several related and complementary strategies for identifying the products of these clusters have emerged recently and revitalized the search for novel bioactive natural products.

A Widely Distributed Bacterial Pathway for Siderophore Biosynthesis Independent of Nonribosomal Peptide Synthetases

Iron is an essential nutrient for virtually all microorganisms because it is a cofactor for several electron-transport proteins involved in vital life processes like aerobic and anaerobic ATP biosynthesis. However, the bioavailability of iron, which exists predominantly in its ferric form in aerobic environments, such as soil, is very low despite the fact that iron is the fourth most abundant element in the Earth’s crust. This is because, at neutral and alkaline pH, ferric iron forms insoluble, polymeric oxyhydroxide complexes that cannot be assimilated by microorganisms. Consequently, iron acquisition from the environment poses a significant challenge to saprophytic microorganisms. Similar bioavailability problems exist in the intercellular matrices of higher eucaryotes, where ferric iron is tightly bound to solubilising transport and storage glycoproteins, such as transferrin and lactoferrin. Thus, iron assimilation by invading pathogens, which is considered essential for establishing infection, also poses a significant challenge. A common strategy used by many pathogenic and saprophytic microorganisms to tackle the problem of low iron bioavailability is the biosynthesis and excretion of high-affinity iron chelators known as siderophores. 2] Once an excreted siderophore has scavenged ferric iron from the environment or host, the resulting iron–siderophore complex is readsorbed by bacterial cells by a membrane-associated ATP-dependent transport system that often exhibits high substrate selectivity. In fungi, the readsorption of iron–siderophore complexes is mediated by the siderophore iron transport (SIT) family of the major facilitator protein superfamily. Several different mechanisms have been proposed for the recovery of ferric iron from the siderophore complex and reduction to the ferrous form for storage and utilisation. 5] Many siderophores are polypeptides that are biosynthesised by members of the nonribosomal peptide synthetase (NRPS) multienzyme family, which is also responsible for the biosynthesis of the majority of microbial peptide antibiotics. The enzymology of NRPS-catalysed siderophore biosynthesis has been intensively studied over the last decade, and the biosynthetic mechanisms for several types of structurally diverse peptide siderophore are now well understood. On the other hand, several bacterial siderophores are not polypetides, but are assembled instead from alternating dicarboxylic acid and diamine or amino alcohol building blocks (which are nevertheless derived from amino acids) linked by amide or ester bonds. Examples include aerobactin (1), rhizobactin 1021 (2), achromobactin (3), vibrioferrin (4), 11] alcaligin (5), and desferrioxamine E (6). Pioneering biochemical genetic studies in the 1980s by Neilands and co-workers established that aero-

Identification of a bioactive 51-membered macrolide complex by activation of a silent polyketide synthase in Streptomyces ambofaciens

There is a constant need for new and improved drugs to combat infectious diseases, cancer, and other major life-threatening conditions. The recent development of genomics-guided approaches for novel natural product discovery has stimulated renewed interest in the search for natural product-based drugs. Genome sequence analysis of Streptomyces ambofaciens ATCC23877 has revealed numerous secondary metabolite biosynthetic gene clusters, including a giant type I modular polyketide synthase (PKS) gene cluster, which is composed of 25 genes (nine of which encode PKSs) and spans almost 150 kb, making it one of the largest polyketide biosynthetic gene clusters described to date. The metabolic product(s) of this gene cluster are unknown, and transcriptional analyses showed that it is not expressed under laboratory growth conditions. The constitutive expression of a regulatory gene within the cluster, encoding a protein that is similar to Large ATP binding of the LuxR (LAL) family proteins, triggered the expression of the biosynthetic genes. This led to the identification of four 51-membered glycosylated macrolides, named stambomycins A–D as metabolic products of the gene cluster. The structures of these compounds imply several interesting biosynthetic features, including incorporation of unusual extender units into the polyketide chain and in trans hydroxylation of the growing polyketide chain to provide the hydroxyl group for macrolide formation. Interestingly, the stambomycins possess promising antiproliferative activity against human cancer cell lines. Database searches identify genes encoding LAL regulators within numerous cryptic biosynthetic gene clusters in actinomycete genomes, suggesting that constitutive expression of such pathway-specific activators represents a powerful approach for novel bioactive natural product discovery.

The Complete Genome Sequence and Comparative Genome Analysis of the High Pathogenicity Yersinia enterocolitica Strain 8081

The human enteropathogen, Yersinia enterocolitica, is a significant link in the range of Yersinia pathologies extending from mild gastroenteritis to bubonic plague. Comparison at the genomic level is a key step in our understanding of the genetic basis for this pathogenicity spectrum. Here we report the genome of Y. enterocolitica strain 8081 (serotype 0:8; biotype 1B) and extensive microarray data relating to the genetic diversity of the Y. enterocolitica species. Our analysis reveals that the genome of Y. enterocolitica strain 8081 is a patchwork of horizontally acquired genetic loci, including a plasticity zone of 199 kb containing an extraordinarily high density of virulence genes. Microarray analysis has provided insights into species-specific Y. enterocolitica gene functions and the intraspecies differences between the high, low, and nonpathogenic Y. enterocolitica biotypes. Through comparative genome sequence analysis we provide new information on the evolution of the Yersinia. We identify numerous loci that represent ancestral clusters of genes potentially important in enteric survival and pathogenesis, which have been lost or are in the process of being lost, in the other sequenced Yersinia lineages. Our analysis also highlights large metabolic operons in Y. enterocolitica that are absent in the related enteropathogen, Yersinia pseudotuberculosis, indicating major differences in niche and nutrients used within the mammalian gut. These include clusters directing, the production of hydrogenases, tetrathionate respiration, cobalamin synthesis, and propanediol utilisation. Along with ancestral gene clusters, the genome of Y. enterocolitica has revealed species-specific and enteropathogen-specific loci. This has provided important insights into the pathology of this bacterium and, more broadly, into the evolution of the genus. Moreover, wider investigations looking at the patterns of gene loss and gain in the Yersinia have highlighted common themes in the genome evolution of other human enteropathogens.

Genome Mining for Novel Natural Product Discovery

Genomics has resulted in the deposition of a huge quantity of DNA sequence data from a wide variety of organisms in publicly accessible databases. Such data can be exploited to generate new knowledge in several areas relevant to medicinal chemistry including the characterization of human physiological processes, the identification and validation of new drug targets in human pathogens, and the discovery of new chemical entities (NCEsa) from natural sources, which may form the basis for new drug leads. The term “genome mining” has been used in various fields to describe the exploitation of genomic information for the discovery of new processes, targets, and products. This Miniperspective will focus on the development of genome mining approaches for the discovery of new natural products. It will also discuss future prospects for the application of genome mining technology to NCE discovery and lead generation.

The dynamic architecture of the metabolic switch in Streptomyces coelicolor

BackgroundDuring the lifetime of a fermenter culture, the soil bacterium S. coelicolor undergoes a major metabolic switch from exponential growth to antibiotic production. We have studied gene expression patterns during this switch, using a specifically designed Affymetrix genechip and a high-resolution time-series of fermenter-grown samples.ResultsSurprisingly, we find that the metabolic switch actually consists of multiple finely orchestrated switching events. Strongly coherent clusters of genes show drastic changes in gene expression already many hours before the classically defined transition phase where the switch from primary to secondary metabolism was expected. The main switch in gene expression takes only 2 hours, and changes in antibiotic biosynthesis genes are delayed relative to the metabolic rearrangements. Furthermore, global variation in morphogenesis genes indicates an involvement of cell differentiation pathways in the decision phase leading up to the commitment to antibiotic biosynthesis.ConclusionsOur study provides the first detailed insights into the complex sequence of early regulatory events during and preceding the major metabolic switch in S. coelicolor, which will form the starting point for future attempts at engineering antibiotic production in a biotechnological setting.