The genome sequence of Streptomyces lividans 66 reveals a novel tRNA-dependent peptide biosynthetic system within a metal-related genomic island

Microbial Biotechnolog

Actinomycete integrative and conjugative elements

This paper reviews current knowledge on actinomycete integrative and conjugative elements (AICEs). The best characterised AICEs, pSAM2 of Streptomyces ambofaciens (10.9 kb), SLP1 (17.3 kb) of Streptomyces coelicolor and pMEA300 of Amycolatopsis methanolica (13.3 kb), are present as integrative elements in specific tRNA genes, and are capable of conjugative transfer. These AICEs have a highly conserved structural organisation, with functional modules for excision/integration, replication, conjugative transfer, and regulation. Recently, it has been shown that pMEA300 and the related elements pMEA100 of Amycolatopsis mediterranei and pSE211 of Saccharopolyspora erythraea form a novel group of AICEs, the pMEA-elements, based on the unique characteristics of their replication initiator protein RepAM. Evaluation of a large collection of Amycolatopsis isolates has allowed identification of multiple pMEA-like elements. Our data show that, as AICEs, they mainly coevolved with their natural host in an integrated form, rather than being dispersed via horizontal gene transfer. The pMEA-like elements could be separated into two distinct populations from different geographical origins. One group was most closely related to pMEA300 and was found in isolates from Australia and Asia and pMEA100-related sequences were present in European isolates. Genome sequence data have enormously contributed to the recent insight that AICEs are present in many actinomycete genera. The sequence data also provide more insight into their evolutionary relationships, revealing their modular composition and their likely combined descent from bacterial plasmids and bacteriophages. Evidence is accumulating that AICEs act as modulators of host genome diversity and are also involved in the acquisition of secondary metabolite clusters and foreign DNA via horizontal gene transfer. Although still speculative, these AICEs may play a role in the spread of antibiotic resistance factors into pathogenic bacteria. The novel insights on AICE characteristics presented in this review may be used for the effective construction of new vectors that allows us to engineer and optimise strains for the production of commercially and medically interesting secondary metabolites, and bioactive proteins

Concentration-Dependent, Size-Independent Toxicity of Citrate Capped AuNPs in Drosophila melanogaster

The expected potential benefits promised by nanotechnology in various fields have led to a rapid increase of the presence of engineered nanomaterials in a high number of commercial goods. This is generating increasing questions about possible risks for human health and environment, due to the lack of an in-depth assessment of the physical/chemical factors responsible for their toxic effects. In this work, we evaluated the toxicity of monodisperse citrate-capped gold nanoparticles (AuNPs) of different sizes (5, 15, 40, and 80 nm) in the model organism Drosophila melanogaster, upon ingestion. To properly evaluate and distinguish the possible dose- and/or size-dependent toxicity of the AuNPs, we performed a thorough assessment of their biological effects, using two different dose-metrics. In the first approach, we kept constant the total surface area of the differently sized AuNPs (Total Exposed Surface area approach, TES), while, in the second approach, we used the same number concentration of the four different sizes of AuNPs (Total Number of Nanoparticles approach, TNN). We observed a significant AuNPs-induced toxicity in vivo, namely a strong reduction of Drosophila lifespan and fertility performance, presence of DNA fragmentation, as well as a significant modification in the expression levels of genes involved in stress responses, DNA damage recognition and apoptosis pathway. Interestingly, we found that, within the investigated experimental conditions, the toxic effects in the exposed organisms were directly related to the concentration of the AuNPs administered, irrespective of their size

Minimum Information about a Biosynthetic Gene cluster

A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit. To facilitate consistent and systematic deposition and retrieval of data on biosynthetic gene clusters, we propose the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard.Chemistry and Chemical Biolog

Marker-Free Genome Engineering in Amycolatopsis Using the pSAM2 Site-Specific Recombination System

Actinobacteria of the genus Amycolatopsis are important for antibiotic production and other valuable biotechnological applications such as bioconversion or bioremediation. Despite their importance, tools and methods for their genetic manipulation are less developed than in other actinobacteria such as Streptomyces. We report here the use of the pSAM2 site-specific recombination system to delete antibiotic resistance cassettes used in gene replacement experiments or to create large genomic deletions. For this purpose, we constructed a shuttle vector, replicating in Escherichia coli and Amycolatopsis, expressing the integrase and the excisionase from the Streptomyces integrative and conjugative element pSAM2. These proteins are sufficient for site-specific recombination between the attachment sites attL and attR. We also constructed two plasmids, replicative in E. coli but not in Amycolatopsis, for the integration of the attL and attR sites on each side of a large region targeted for deletion. We exemplified the use of these tools in Amycolatopsis mediterranei by obtaining with high efficiency a marker-free deletion of one single gene in the rifamycin biosynthetic gene cluster or of the entire 90-kb cluster. These robust and simple tools enrich the toolbox for genome engineering in Amycolatopsis

Evidence for a High Heterogeneity Between the Terminal Regions of the Chromosomes of Two Phylogenetically Close Species, Streptomyces ambofaciens and Streptomyces coelicolor by Genome Comparison

The chromosomal DNA of Streptomyces ambofaciens is a 8 Mb linear molecule that ends with large Terminal Inverted Repeats (TIRs) of about 200 kb. The size and the sequence of the TIRs are extremely variable between the different linear replicons of Streptomyces (e.g., 21,6 kb in Streptomyces coelicolor A3(2), Bentley et al., 2002; 174 bp in Streptomyces avermitilis, Omura et al., 2001). Previous works have shown that the terminal regions of the S. ambofaciens chromosome are strongly implied in genomic instability. Indeed, large rearrangements can frequently occur in those regions (Leblond et Decaris, 1999). Despite a close phylogenetic relationship between S. ambofaciens and the already sequenced species S. coelicolor (Bentley et al., 2002), we found out that the terminal regions appear highly variable compared to the central region. A similar situation has been recently described by comparing S. avermitilis and S. coelicolor A3(2) (Ikeda et al., 2003). However, those two species are phylogenetically distant whereas S. ambofaciens is extremely close to S. coelicolor. Therefore, comparative genomics between S. ambofaciens and S. coelicolor will increase our understanding of Streptomyces genome evolution.In the prospect of studying variability and comparative genomics, we have been undertaking the sequence analysis of the terminal regions (about 2.5 Mb) of the S. ambofaciens linear chromosome, sequencing being done using a set of ordered cosmids and a BAC library. A PCR approach was also used to sequence the last kilobases at the extremities that were not cloned into the libraries.The sequence of the TIRs of S. ambofaciens ATCC23877 is now nearly completed and the analysis has confirmed that the subtelomeric regions are highly variable compared to S. coelicolor for the gene organization and the encoded functions. In the 200 kb already available, we have identified 187 potential genes. About 24% of them are completely absent in the S. coelicolor genome and moreover, the other 76% show similarities with genes scattered all along the chromosome of S. coelicolor. These results reveal a low level of conservation of gene organization in the terminal regions. Preliminary results suggested a higher conservation of the central region. A library of 8457 sequences of the whole BAC ends, covering approximately 3.5 Mb of chromosomal DNA, has been analysed in order to confirm the conservation of the central chromosomal region. This library was used to carry out a statistical analysis about the conservation and the distribution of DNA homologies in the different regions of the chromosome.The complete genome sequence of S. avermitilis has recently been published and the comparative genomics with this less close species will be presented. Moreover, approximately 1000 kb are now sequenced apart from the TIRs and preliminary analyses revealed the presence of several gene clusters involved in secondary metabolite biosynthesis