Watasemycin biosynthesis in Streptomyces venezuelae : thiazoline C-methylation by a type B radical-SAM methylase homologue

2-Hydroxyphenylthiazolines are a family of iron-chelating nonribosomal peptide natural products that function as virulence-conferring siderophores in various Gram-negative bacteria. They have also been reported as metabolites of Gram-positive Streptomyces species. Transcriptional analyses of Streptomyces venezuelae ATCC 10712 revealed that its genome contains a putative 2-hydroxyphenylthiazoline biosynthetic gene cluster. Heterologous expression of the gene cluster in Streptomyces coelicolor M1152 showed that the mono- and dimethylated derivatives, thiazostatin and watasemycin, respectively, of the 2-hydroxyphenylthiazoline enantiopyochelin are two of its metabolic products. In addition, isopyochelin, a novel isomer of pyochelin containing a C-methylated thiazolidine, was identified as a third metabolic product of the cluster. Metabolites with molecular formulae corresponding to aerugine and pulicatins A/B were also detected. The structure and stereochemistry of isopyochelin were confirmed by comparison with synthetic standards. The role of two genes in the cluster encoding homologues of PchK, which is proposed to catalyse thiazoline reduction in the biosynthesis of enantiopyochelin in Pseudomonas protegens, was investigated. One was required for the production of all the metabolic products of the cluster, whereas the other appears not to be involved in the biosynthesis of any of them. Deletion of a gene in the cluster encoding a type B radical-SAM methylase homologue abolished the production of watasemycin, but not thiazostatin or isopyochelin. Feeding of thiazostatin to the mutant lacking the functional PchK homologue resulted in complete conversion to watasemycin, demonstrating that thiazoline C-methylation by the type B radical-SAM methylase homologue is the final step in watasemycin biosynthesis

New Insights into Chloramphenicol Biosynthesis in Streptomyces venezuelae ATCC 10712

Comparative genome analysis revealed seven uncharacterized genes, sven0909 to sven0915, adjacent to the previously identified chloramphenicol biosynthetic gene cluster (sven0916–sven0928) of Streptomyces venezuelae strain ATCC 10712 that was absent in a closely related Streptomyces strain that does not produce chloramphenicol. Transcriptional analysis suggested that three of these genes might be involved in chloramphenicol production, a prediction confirmed by the construction of deletion mutants. These three genes encode a cluster-associated transcriptional activator (Sven0913), a phosphopantetheinyl transferase (Sven0914), and a Na(+)/H(+) antiporter (Sven0915). Bioinformatic analysis also revealed the presence of a previously undetected gene, sven0925, embedded within the chloramphenicol biosynthetic gene cluster that appears to encode an acyl carrier protein, bringing the number of new genes likely to be involved in chloramphenicol production to four. Microarray experiments and synteny comparisons also suggest that sven0929 is part of the biosynthetic gene cluster. This has allowed us to propose an updated and revised version of the chloramphenicol biosynthetic pathway

Analysis of the tunicamycin biosynthetic gene cluster of streptomyces chartreusis reveals new insights into tunicamycin production and immunity

The tunicamycin biosynthetic gene cluster of Streptomyces chartreusis consists of 14 genes (tunA to tunN) with a high degree of apparent translational coupling. Transcriptional analysis revealed that all of these genes are likely to be transcribed as a single operon from two promoters, tunp1 and tunp2. In-frame deletion analysis revealed that just six of these genes (tunABCDEH) are essential for tunicamycin production in the heterologous host Streptomyces coelicolor, while five (tunFGKLN) with likely counterparts in primary metabolism are not necessary, but presumably ensure efficient production of the antibiotic at the onset of tunicamycin biosynthesis. Three genes are implicated in immunity, namely, tunI and tunJ, which encode a two-component ABC transporter presumably required for export of the antibiotic, and tunM, which encodes a putative S-adenosylmethionine (SAM)-dependent methyltransferase. Expression of tunIJ or tunM in S. coelicolor conferred resistance to exogenous tunicamycin. The results presented here provide new insights into tunicamycin biosynthesis and immunity

Orion Crew Module Aerodynamic Testing

The Apollo-derived Orion Crew Exploration Vehicle (CEV), part of NASA s now-cancelled Constellation Program, has become the reference design for the new Multi-Purpose Crew Vehicle (MPCV). The MPCV will serve as the exploration vehicle for all near-term human space missions. A strategic wind-tunnel test program has been executed at numerous facilities throughout the country to support several phases of aerodynamic database development for the Orion spacecraft. This paper presents a summary of the experimental static aerodynamic data collected to-date for the Orion Crew Module (CM) capsule. The test program described herein involved personnel and resources from NASA Langley Research Center, NASA Ames Research Center, NASA Johnson Space Flight Center, Arnold Engineering and Development Center, Lockheed Martin Space Sciences, and Orbital Sciences. Data has been compiled from eight different wind tunnel tests in the CEV Aerosciences Program. Comparisons are made as appropriate to highlight effects of angle of attack, Mach number, Reynolds number, and model support system effects

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 \u3e20 distinct compound classes is also reviewed, and commonalities are discussed

Cdk5 controls lymphatic vessel development and function by phosphorylation of Foxc2.

The lymphatic system maintains tissue fluid balance, and dysfunction of lymphatic vessels and valves causes human lymphedema syndromes. Yet, our knowledge of the molecular mechanisms underlying lymphatic vessel development is still limited. Here, we show that cyclin-dependent kinase 5 (Cdk5) is an essential regulator of lymphatic vessel development. Endothelial-specific Cdk5 knockdown causes congenital lymphatic dysfunction and lymphedema due to defective lymphatic vessel patterning and valve formation. We identify the transcription factor Foxc2 as a key substrate of Cdk5 in the lymphatic vasculature, mechanistically linking Cdk5 to lymphatic development and valve morphogenesis. Collectively, our findings show that Cdk5-Foxc2 interaction represents a critical regulator of lymphatic vessel development and the transcriptional network underlying lymphatic vascular remodeling

Synthetic RNA Silencing of Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2)

We demonstrate the first application of synthetic RNA gene silencers in Streptomyces coelicolor A3(2). Peptide nucleic acid and expressed antisense RNA silencers successfully inhibited actinorhodin production. Synthetic RNA silencing was target-specific and is a new tool for gene regulation and metabolic engineering studies in Streptomyces.Peer reviewe

Developmentally regulated volatiles geosmin and 2-methylisoborneol attract a soil arthropod to Streptomyces bacteria promoting spore dispersal

Volatile compounds emitted by bacteria are often sensed by other organisms as odours, but their ecological roles are poorly understood1,2. Well-known examples are the soil-smelling terpenoids geosmin and 2-methylisoborneol (2-MIB)3,4, which humans and various animals sense at extremely low concentrations5,6. The conservation of geosmin biosynthesis genes among virtually all species of Streptomyces bacteria (and genes for the biosynthesis of 2-MIB in about 50%)7,8, suggests that the volatiles provide a selective advantage for these soil microbes. We show, in the present study, that these volatiles mediate interactions of apparent mutual benefit between streptomycetes and springtails (Collembola). In field experiments, springtails were attracted to odours emitted by Streptomyces colonies. Geosmin and 2-MIB in these odours induce electrophysiological responses in the antennae of the model springtail Folsomia candida, which is also attracted to both compounds. Moreover, the genes for geosmin and 2-MIB synthases are under the direct control of sporulation-specific transcription factors, constraining emission of the odorants to sporulating colonies. F. candida feeds on the Streptomyces colonies and disseminates spores both via faecal pellets and through adherence to its hydrophobic cuticle. The results indicate that geosmin and 2-MIB production is an integral part of the sporulation process, completing the Streptomyces life cycle by facilitating dispersal of spores by soil arthropods