Paul F. Long

A chain initiation factor common to both modular and aromatic polyketide synthases

Antibiotic-producing polyketide synthases (PKSs) are enzymes responsible for the biosynthesis in Streptomyces and related filamentous bacteria of a remarkably broad range of bioactive metabolites, including antitumour aromatic compounds such as mithramycin and macrolide antibiotics such as erythromycin. The molecular basis for the selection of the starter unit on aromatic PKSs is unknown. Here we show that a component of aromatic PKS, previously named ‘chain-length factor’, is a factor required for polyketide chain initiation and that this factor has decarboxylase activity towards malonyl-ACP (acyl carrier protein). We have re-examined the mechanism of initiation on modular PKSs and have identified as a specific initiation factor a domain of previously unknown function named KSQ, which operates like chain-length factor. Both KSQ and chain-length factor are similar to the ketosynthase domains that catalyse polyketide chain extension in modular multifunctional PKSs and in aromatic PKSs, respectively, except that the ketosynthase domain active-site cysteine residue is replaced by a highly conserved glutamine in KSQ and in chain-length factor. The glutamine residue is important both for decarboxylase activity and for polyketide synthesis.

Shotgun Cloning and Heterologous Expression of the Patellamide Gene Cluster as a Strategy to Achieving Sustained Metabolite Production

Shotgun cloning into E. coli of genomic DNA from Prochloron sp., symbiont of the seasquirt Lissoclinum patella, resulted in the heterologous expression of the patellamide gene cluster and subsequent production of patellamide D (1) and ascidiacyclamide (2) at levels of 80?100 ng?mL?1.

ClustScan: an integrated program package for the semi-automatic annotation of modular biosynthetic gene clusters and in silico prediction of novel chemical structures

The program package ‘ClustScan’ (Cluster Scanner) is designed for rapid, semi-automatic, annotation of DNA sequences encoding modular biosynthetic enzymes including polyketide synthases (PKS), non-ribosomal peptide synthetases (NRPS) and hybrid (PKS/NRPS) enzymes. The program displays the predicted chemical structures of products as well as allowing export of the structures in a standard format for analyses with other programs. Recent advances in understanding of enzyme function are incorporated to make knowledge-based predictions about the stereochemistry of products. The program structure allows easy incorporation of additional knowledge about domain specificities and function. The results of analyses are presented to the user in a graphical interface, which also allows easy editing of the predictions to incorporate user experience. The versatility of this program package has been demonstrated by annotating biochemical pathways in microbial, invertebrate animal and metagenomic datasets. The speed and convenience of the package allows the annotation of all PKS and NRPS clusters in a complete Actinobacteria genome in 2–3 man hours. The open architecture of ClustScan allows easy integration with other programs, facilitating further analyses of results, which is useful for a broad range of researchers in the chemical and biological sciences.

Enzymes of the shikimic acid pathway encoded in the genome of a basal metazoan, Nematostella vectensis, have microbial origins.

The shikimic acid pathway is responsible for the biosynthesis of many aromatic compounds by a broad range of organisms, including bacteria, fungi, plants, and some protozoans. Animals are considered to lack this pathway, as evinced by their dietary requirement for shikimate-derived aromatic amino acids. We challenge the universality of this traditional view in this report of genes encoding enzymes for the shikimate pathway in an animal, the starlet sea anemone Nematostella vectensis. Molecular evidence establishes horizontal transfer of ancestral genes of the shikimic acid pathway into the N. vectensis genome from both bacterial and eukaryotic (dinoflagellate) donors. Bioinformatic analysis also reveals four genes that are closely related to those of Tenacibaculum sp. MED152, raising speculation for the existence of a previously unsuspected bacterial symbiont. Indeed, the genome of the holobiont (i.e., the entity consisting of the host and its symbionts) comprises a high content of Tenacibaculum-like gene orthologs, including a 16S rRNA sequence that establishes the phylogenetic position of this associate to be within the family Flavobacteriaceae. These results provide a complementary view for the biogenesis of shikimate-related metabolites in marine Cnidaria as a “shared metabolic adaptation” between the partners.

Effect of Antibiotics for Otitis Media on Mastoiditis in Children: A Retrospective Cohort Study Using the United Kingdom General Practice Research Database

BACKGROUND. Information is needed on whether mastoiditis has increased in association with the decline in antibiotics prescribed to children by primary care physicians in the United Kingdom. OBJECTIVE. To determine time trends in mastoiditis incidence, the frequency of antecedent otitis media, and the effect of antibiotics for otitis media on the risk of mastoiditis in children. PATIENTS AND METHODS. We conducted a retrospective cohort study by using the UK General Practice Research Database. Children aged 3 months to 15 years between 1990 and 2006 were included. Risk of mastoiditis within 3 months after otitis media diagnosis and the protective effect of antibiotics were determined. RESULTS. There were 2 622 348 children within the General Practice Research Database; 854 had mastoiditis, only one third of whom (35.7%) had antecedent otitis media. Mastoiditis incidence remained stable between 1990 and 2006 (∼1.2 per 10 000 child-years). Risk of mastoiditis, after otitis media, was 1.8 per 10 000 episodes (139 of 792 623) after antibiotics compared with 3.8 per 10 000 (149 of 389 649) without antibiotics, and increased with age. Antibiotics halved the risk of mastoiditis. General practitioners would need to treat 4831 otitis media episodes with antibiotics to prevent 1 child from developing mastoiditis. If antibiotics were no longer prescribed for otitis media, an extra 255 cases of childhood mastoiditis would occur, but there would be 738 775 fewer antibiotic prescriptions per year in the United Kingdom. CONCLUSIONS. Most children with mastoiditis have not seen their general practitioner for otitis media. Antibiotics halve the risk of mastoiditis, but the high number of episodes needing treatment to prevent 1 case precludes the treatment of otitis media as a strategy for preventing mastoiditis. Although mastoiditis is a serious disease, most children make an uncomplicated recovery after mastoidectomy or intravenous antibiotics. Treating these additional otitis media episodes could pose a larger public health problem in terms of antibiotic resistance.

Changes in clinical indications for community antibiotic prescribing for children in the UK from 1996 to 2006: will the new NICE prescribing guidance on upper respiratory tract infections just be ignored?

Objective: To analyse changes in clinical indications for community antibiotic prescribing for children in the UK between 1996 and 2006 and relate these findings to the new NICE guidelines for the treatment of upper respiratory tract infections in children. Study design: Retrospective cohort study. Method: The IMS Health Mediplus database was used to obtain annual antibiotic prescribing rates and associated clinical indications in 0–18-year-old patients between 1 January 1996 and 31 December 2006 in the UK. Results: Antibiotic prescribing declined by 24% between 1996 and 2000 but increased again by 10% during 2003–2006. Respiratory tract infection was the most common indication for which an antibiotic was prescribed, followed by “abnormal signs and symptoms”, ear and skin infections. Antibiotic prescriptions for respiratory tract infections have decreased by 31% (p<0.01) mainly because of reduced prescribing for lower respiratory tract infections (56% decline, p<0.001) and specific upper respiratory tract infections including tonsillitis/pharyngitis (48% decline, p<0.001) and otitis (46% decline, p<0.001). Prescribing for non-specific upper respiratory tract infection increased fourfold (p<0.001). Prescribing for “abnormal signs and symptoms” increased significantly since 2001 (40% increase, p<0.001). Conclusion: There has been a marked decrease in community antibiotic prescribing linked to lower respiratory tract infection, tonsillitis, pharyngitis and otitis. Overall prescribing is now increasing again but is associated with non-specific upper respiratory tract infection diagnoses. General practitioners may be avoiding using diagnoses where formal guidance suggests antibiotic prescribing is not indicated. The new NICE guidance on upper respiratory tract infections is at risk of being ignored.

Engineering specificity of starter unit selection by the erythromycin‐producing polyketide synthase

Chain initiation on many modular polyketide synthases is mediated by acyl transfer from the CoA ester of a dicarboxylic acid, followed by decarboxylation in situ by KSQ, a ketosynthase‐like decarboxylase domain. Consistent with this, the acyltransferase (AT) domains of all KSQ‐containing loading modules are shown here to contain a key arginine residue at their active site. Site‐specific replacement of this arginine residue in the oleandomycin (ole) loading AT domain effectively abolished AT activity, consistent with its importance for catalysis. Substitution of the ole PKS loading module, or of the tylosin PKS loading module, for the erythromycin (ery) loading module gave polyketide products almost wholly either acetate derived or propionate derived, respectively, instead of the mixture found normally. An authentic extension module AT domain, rap AT2 from the rapamycin PKS, functioned appropriately when engineered in the place of the ole loading AT domain, and gave rise to substantial amounts of C13‐methylerythromycins, as predicted. The role of direct acylation of the ketosynthase domain of ex‐tension module 1 in chain initiation was confirmed by demonstrating that a mutant of the triketide synthase DEBS1‐TE, in which the 4′‐phosphopante‐theine attachment site for starter acyl groups was specifically removed, produced triketide lactone pro‐ducts in detectable amounts.

Engineering of complex polyketide biosynthesis — insights from sequencing of the monensin biosynthetic gene cluster

The biosynthesis of complex reduced polyketides is catalysed in actinomycetes by large multifunctional enzymes, the modular Type I polyketide synthases (PKSs). Most of our current knowledge of such systems stems from the study of a restricted number of macrolide-synthesising enzymes. The sequencing of the genes for the biosynthesis of monensin A, a typical polyether ionophore polyketide, provided the first genetic evidence for the mechanism of oxidative cyclisation through which polyethers such as monensin are formed from the uncyclised products of the PKS. Two intriguing genes associated with the monensin PKS cluster code for proteins, which show strong homology with enzymes that trigger double bond migrations in steroid biosynthesis by generation of an extended enolate of an unsaturated ketone residue. A similar mechanism operating at the stage of an enoyl ester intermediate during chain extension on a PKS could allow isomerisation of an E double bond to the Z isomer. This process, together with epoxidations and cyclisations, form the basis of a revised proposal for monensin formation. The monensin PKS has also provided fresh insight into general features of catalysis by modular PKSs, in particular into the mechanism of chain initiation. Journal of Industrial Microbiology & Biotechnology (2001) 27, 360–367.

Genetics of Streptomyces rimosus, the Oxytetracycline Producer

SUMMARY From a genetic standpoint, Streptomyces rimosus is arguably the best-characterized industrial streptomycete as the producer of oxytetracycline and other tetracycline antibiotics. Although resistance to these antibiotics has reduced their clinical use in recent years, tetracyclines have an increasing role in the treatment of emerging infections and noninfective diseases. Procedures for in vivo and in vitro genetic manipulations in S. rimosus have been developed since the 1950s and applied to study the genetic instability of S. rimosus strains and for the molecular cloning and characterization of genes involved in oxytetracycline biosynthesis. Recent advances in the methodology of genome sequencing bring the realistic prospect of obtaining the genome sequence of S. rimosus in the near term.

Proteomic characterisation of toxins isolated from nematocysts of the South Atlantic jellyfish Olindias sambaquiensis

Surprisingly little is known of the toxic arsenal of cnidarian nematocysts compared to other venomous animals. Here we investigate the toxins of nematocysts isolated from the jellyfish Olindias sambaquiensis. A total of 29 unique ms/ms events were annotated as potential toxins homologous to the toxic proteins from diverse animal phyla, including cone-snails, snakes, spiders, scorpions, wasp, bee, parasitic worm and other Cnidaria. Biological activities of these potential toxins include cytolysins, neurotoxins, phospholipases and toxic peptidases. The presence of several toxic enzymes is intriguing, such as sphingomyelin phosphodiesterase B (SMase B) that has only been described in certain spider venoms, and a prepro-haystatin P-IIId snake venom metalloproteinase (SVMP) that activates coagulation factor X, which is very rare even in snake venoms. Our annotation reveals sequence orthologs to many representatives of the most important superfamilies of peptide venoms suggesting that their origins in higher organisms arise from deep eumetazoan innovations. Accordingly, cnidarian venoms may possess unique biological properties that might generate new leads in the discovery of novel pharmacologically active drugs.