1. Genome Architecture

Ralph Kirby and Carton W. Chen

Linear replicons are relatively uncommon among bacteria and their preponderance among the Actinomycetales, and within the Streptomyces in particular, poses some interesting questions. These novel bacterial replicons are capped by terminal proteins that are covalently bound to the 5' ends of the linear DNA and these terminal structures are directly involved in replicating and protecting the ends of the linear genome. In addition and perhaps related to their linear nature, these genomes are among the largest bacterial chromosomes. As far as can be ascertained at present, these large genomes have a specific organizational structure in terms of their genes. The genome structure can be divided into a core region that is present syntenously in most Actinomycetales, two terminal regions that are highly variable throughout the explored Streptomyces and two regions to the left and right of the core region that contain many syntenous genes specific to the Streptomyces and not found in other Actinomycetales. Genome dynamics seems to be important to the Streptomyces with plasmid-chromosome interactions, horizontal gene transfer and interspecific recombination probably playing important roles in how these genomes to adapt to the diverse environment they reside in. Exploring the genome architecture of the Streptomyces helps our understanding of how and why the genus Streptomyces has a unique place in the evolution of the bacteria.

2. Streptomyces Conjugative Genetic Elements

Jutta Vogelmann, Wolfgang Wohlleben and Günther Muth

Antibiotic producing actinomycetes contain a huge variety of different plasmids, distinguished in size, topology, replication mechanism and copy number. Some are able to integrate into the chromosome by site specific recombination. With the exception of the huge linear plasmids, Streptomyces plasmids encode only functions involved in replication, stable maintenance and conjugative transfer. The Streptomyces conjugation system is unique, requiring a single plasmid-encoded protein, TraB. TraB is a hexameric ring ATPase with similarity to the septal DNA translocator proteins FtsK/SpoIIIE which are involved in chromosome segregation during cell division and sporulation. TraB binds non-covalently to 8bp TRS repeats present in the clt locus and transfers double stranded plasmid DNA from the donor to the recipient. Presence of clt-like sequences in the chromosome of S. coelicolor suggests that chromosomal genes are mobilized independently from the plasmid. Following primary transfer from the donor into the recipient, the plasmid is translocated via septal crosswalls resulting in intramycelial plasmid spreading. Plasmid spreading involves five to seven plasmid-encoded Spd-proteins. Protein-protein interaction studies with Spd-proteins of the conjugative plasmid pSVH1 suggest formation of a large DNA-translocation apparatus. One component, the integral membrane protein SpdB2 was shown to form pore structures in lipid bilayers.

3. Differentiation in Streptomyces: The Properties and Programming of Diverse Cell-types

Keith F. Chater

Streptomyces colonies are complex differentiated organisms, generated from a single ovoid spore by filamentous growth and branching. Eventually, much of this biomass is converted to large numbers of spores in long chains on specialised aerial hyphae. During colony development, different cellular compartments have different physiology and metabolism, and exoskeletal and cytoskeletal elements bring about different morphological changes. These cellular differentiating processes are underpinned by a large number of regulatory genes, often operating in cascades. During the transition from biomass accumulation to reproductive development, antibiotics are made, sometimes under the control of developmental regulators.

4. Protein Secretion in Streptomyces

Tracy Palmer and Matthew I. Hutchings

The saprophytic lifestyle of Streptomyces requires them to secrete prolific numbers of proteins. For example, inspection of the genome sequence of Streptomyces coelicolor indicates it encodes some 819 proteins with predicted signal peptides. This represents more than 10% of the protein coding genes and is most likely an underestimate. Many secreted proteins are required for nutrient capture, and there is an abundance of secreted hydrolases for the breakdown of complex carbohydrates (including cellulose and chitin), peptides and phospho-compounds. In addition to proteins that are secreted into the milieu, many proteins are covalently anchored to the cell surface by means of either a lipid anchor to the membrane or by covalent attachment to the cell wall through the sortase system. Here we summarise what is known about the different protein secretion systems utilised by Streptomyces, and the mechanisms by which proteins are anchored to the extracellular surface.

5. Central Carbon Metabolic Pathways in Streptomyces

Geertje van Keulen, Jeroen Siebring and Lubbert Dijkhuizen

Streptomyces and other actinomycetes are fascinating soil bacteria of major economic importance. They produce 70% of antibiotics known to man and numerous other pharmaceuticals for treatment of, e.g. cancer, a range of infections, high cholesterol, or have immunosuppressive activity. It is not surprising that the multitude of gene clusters encoding for the biosynthesis of known and unknown secondary metabolites in genome sequences of a wide range of actinomycetes have received much attention in the last few years. In contrast, there is much less understanding of primary metabolism and its control in actinomycetes, despite its importance as supply pathways of precursors for secondary metabolite production. This review describes current information on the central carbon metabolic pathways in streptomycetes, focussing on glycolysis, pentose phosphate pathway, Entner-Doudoroff pathway, gluconeogenesis, and the source of phosphate for phosphorylation reactions. In addition, recent developments providing a greater insight into links with secondary metabolism in Streptomyces are reviewed.

6. Regulation of Nitrogen Assimilation in Streptomycetes and Other Actinobacteria

Wolfgang Wohlleben, Yvonne Mast and Jens Reuther

Streptomycetes, as most bacteria, possess two ways to assimilate ammonium: Whereas the glutamate dehydrogenase is active under high nitrogen supply, the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway is required under nitrogen limiting conditions. The major GS activity is mediated by the typical prokaryotic-type GSI enzyme. A second GS activity is conveyed by the GSII enzyme, belonging to the eukaryotic-type GS. This enzyme contributes to the overall GS activity mainly under N-limitations and during stationary growth. Three further genes (glnA1, glnA2, glnA3) encoding GSI-like enzymes to which no function could yet be assigned are located in all genomes of Streptomyces sequenced so far. The activity of GSI is post-translationally regulated by an adenylyltransferase (GlnE), which modifies the enzyme in response to nitrogen availability. In contrast to enteric bacteria, the PII protein and its modifying enzyme, the adenylyltransferase GlnD, are not involved in the control of GlnE. The central transcriptional regulator GlnR which can act as activator and repressor, respectively, controls the expression of various genes involved in nitrogen supply, such as genes encoding a urease and an ammonium transporter, as well as genes of the ammonium assimilation pathways. GlnR itself can be multiply modified probably reflecting different nitrogen conditions.

7. Network Mechanisms of Phosphate Control of Primary and Secondary Metabolism

Juan F. Martín, Alberto Sola-Landa, Fernando Santos-Beneit and Antonio Rodríguez-García

The biosynthesis of hundreds of antibiotics and other secondary metabolites is repressed by inorganic phosphate, in spite of the fact that those metabolites are synthesized by quite different pathways. Derepression of many antibiotic biosynthesis genes occurs when the concentration of inorganic phosphate becomes limiting for growth. Phosphate control of primary and secondary metabolism in Streptomyces species is mediated by the two component system PhoR-PhoP. The genes under direct control of PhoP (the pho regulon) have been identified in S. coelicolor. They are involved in a variety of cellular functions, most notably in phosphate scavenging and phosphate transport. Phosphate control of secondary metabolism genes is not exerted by direct binding of PhoP to the promoters of those genes. Rather, PhoP controls directly other intermediate regulators in a regulatory cascade that in turn modulate expression of pathway-specific regulators. Indeed PhoP regulates expression of afsS in S. coelicolor that in turn triggers expression of actII-ORF4 and redD controlling actinorhodin and undecylprodigiosin biosynthesis. PhoP binds to a PHO box in the afsS promoter that overlaps with the AfsR binding sequence. Phosphate control also interacts with nitrogen regulation. PhoP binds to the promoters of glnR, glnA and other key genes of the nitrogen metabolism. This article provides new insights into the mechanisms underlying systems biology in Streptomyces.

8. Gamma-Butyrolactones and their Role in Antibiotic Regulation

Marco Gottelt, Stefan Kol and Eriko Takano

γ-Butyrolactones are small signalling molecules regulating antibiotic production and sometimes also morphological development in streptomycetes. The related regulatory systems are complex, and some are species specific. Focusing on well-studied examples in S. griseus, S. virginiae and S. coelicolor, we depict common basic features and differences between the γ-butyrolactone systems. Biosynthesis and structural diversity of the γ-butyrolactones are described, as well as the mode of action and structure-function relationship of the bacterial "hormone receptors". We also report the recent discovery of a previously undescribed antibiotic compound in S. coelicolor by deletion of a γ-butyrolactone homologue, and the identification and characterisation of a second form of the major γ-butyrolactone receptor in the model streptomycete S. coelicolor.

9. Clavulanic Acid and Clavams Biosynthesis and Regulation

Paloma Liras, Irene Santamarta and Rosario Pérez-Redondo

The (3R,5R) clavulanic acid and (3S,5S) clavam molecules share a structure formed by a four member β-lactam and a five member oxaxolidine ring and have several initial common steps in their biosynthesis pathways. The precursors of the molecules are glyceraldehyde-3-phosphate and arginine, condensed by the carboxyethylarginine synthetase (CeaS). The next steps in the pathway occur by the subsequent action of the β-lactam synthase (Bls) forming the β-lactam ring, a proclavaminic acid guanidine hydrolase (PAH) and the clavaminate synthase (Csa2), which forms the two rings clavam structure of clavaminic acid. Modifications of this compound result in late step intermediates for clavulanic acid biosynthesis, N-glycylclavaminic acid or clavaldehyde, and in the clavams structures. In addition to the clavulanic acid gene cluster, two additional clusters containing paralogous genes for clavulanic acid biosynthesis and clavam biosynthesis have been located in S. clavuligerus. Biochemical characterization of the clavam non producer mutants will clarify the biosynthetic pathway of these compounds.

10. Genome-guided Exploration of Streptomyces ambofaciens Secondary Metabolism

Bertrand Aigle, R. Bunet, C. Corre, A. Garenaux, S. Huang, L. Laureti, S. Lautru, M.V. Mendes, S. Nezbedová, H.C. Nguyen, L. Song, J. Weiser, G.L. Challis, P. Leblond and J.-L. Pernodet

Members of the Streptomyces genus are among the most prolific microorganisms producing secondary metabolites with wide uses in medicine and in agriculture. Sequencing of the genome of the model Streptomyces, Streptomyces coelicolor, has highlighted an unexpected feature, i.e. that the potential of these organisms to synthesise secondary metabolites has been largely underestimated. They indeed possess many more gene clusters encoding natural product-like biosynthetic pathways than there are known natural products. Similar observations have since been made for other bacterial or fungal genomes. Thus, it became clear that microbial secondary metabolism had been seriously underestimated and that genome-based approaches were very promising for the search of new bioactive compounds. Here, we present an overview of the secondary metabolite biosynthetic potential of Streptomyces ambofaciens, a species known for decades as producer of the macrolide spiramycin and the pyrrolamide congocidine. Interestingly, genome analysis has revealed that despite of the close phylogenetic relatedness between S. coelicolor and S. ambofaciens, most of its secondary metabolite gene clusters are species-specific.

11. Gene Clusters for Bioactive Natural Products in Actinomycetes and their Use in Combinatorial Biosynthesis

Carlos Olano, Carmen Méndez and José A. Salas

During the last twenty five years the isolation and characterization of gene clusters involved in the biosynthesis of actinomycete secondary metabolites has permitted the elucidation of the biochemical steps involved in the production of different structural classes of bioactive compounds. The characterization of these clusters has represented a great source of genes for the generation of novel "unnatural natural" compounds by using combinatorial biosynthesis. The development of more effective methods for DNA sequencing, the improvement of targeted inactivation and heterologous host expression systems has strengthened the effectiveness of combinatorial biosynthesis. For these reasons combinatorial DNA technology has become during the last decade one of the most important approaches for generating chemical structural diversity and for increasing the number of potential useful compounds.

12. Genomics of Streptomyces Cytochromes P450

David C. Lamb, Bin Zhao, F. Peter Guengerich, Steven L. Kelly and Michael R. Waterman

In the present chapter, a review of the current status regarding the molecular biology and biochemistry of streptomycete cytochromes P450 (CYPs) will be presented. Emphasis will be placed upon CYPs with novel functions and identifiying the roles of CYPs in the four sequenced and publically available streptomycete genomes: Streptomyces coelicolor, Streptomyces avermitilis, Streptomyces griseus, and Streptomyces scabies. Additionally, the first detailed analysis of the CYP complement of a Streptomyces genome is being undertaken in the laboratories of the authors and colleagues. A summary of these findings will be presented. Finally, discussion on how new drugs might be generated from Streptomyces by modifying the structure of specific CYPs, as well as thoughts regarding actinomycete P450 research in the future, will be outlined.