Microbial community drivers of PK/NRP gene diversity in selected global soils

Background The emergence of antibiotic-resistant pathogens has created an urgent need for novel antimicrobial treatments. Advances in next-generation sequencing have opened new frontiers for discovery programmes for natural products allowing the exploitation of a larger fraction of the microbial community. Polyketide (PK) and non-ribosomal pepetide (NRP) natural products have been reported to be related to compounds with antimicrobial and anticancer activities. We report here a new culture-independent approach to explore bacterial biosynthetic diversity and determine bacterial phyla in the microbial community associated with PK and NRP diversity in selected soils. Results Through amplicon sequencing, we explored the microbial diversity (16S rRNA gene) of 13 soils from Antarctica, Africa, Europe and a Caribbean island and correlated this with the amplicon diversity of the adenylation (A) and ketosynthase (KS) domains within functional genes coding for non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), which are involved in the production of NRP and PK, respectively. Mantel and Procrustes correlation analyses with microbial taxonomic data identified not only the well-studied phyla Actinobacteria and Proteobacteria, but also, interestingly, the less biotechnologically exploited phyla Verrucomicrobia and Bacteroidetes, as potential sources harbouring diverse A and KS domains. Some soils, notably that from Antarctica, provided evidence of endemic diversity, whilst others, such as those from Europe, clustered together. In particular, the majority of the domain reads from Antarctica remained unmatched to known sequences suggesting they could encode enzymes for potentially novel PK and NRP. Conclusions The approach presented here highlights potential sources of metabolic novelty in the environment which will be a useful precursor to metagenomic biosynthetic gene cluster mining for PKs and NRPs which could provide leads for new antimicrobial metabolites

Designing and implementing an assay for the detection of rare and divergent NRPS and PKS clones in European, Antarctic and Cuban soils

The ever increasing microbial resistome means there is an urgent need for new antibiotics. Metagenomics is an underexploited tool in the field of drug discovery. In this study we aimed to produce a new updated assay for the discovery of biosynthetic gene clusters encoding bioactive secondary metabolites. PCR assays targeting the polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) were developed. A range of European soils were tested for their biosynthetic potential using clone libraries developed from metagenomic DNA. Results revealed a surprising number of NRPS and PKS clones with similarity to rare Actinomycetes. Many of the clones tested were phylogenetically divergent suggesting they were fragments from novel NRPS and PKS gene clusters. Soils did not appear to cluster by location but did represent NRPS and PKS clones of diverse taxonomic origin. Fosmid libraries were constructed from Cuban and Antarctic soil samples; 17 fosmids were positive for NRPS domains suggesting a hit rate of less than 1 in 10 genomes. NRPS hits had low similarities to both rare Actinobacteria and Proteobacteria; they also clustered with known antibiotic producers suggesting they may encode for pathways producing novel bioactive compounds. In conclusion we designed an assay capable of detecting divergent NRPS and PKS gene clusters from the rare biosphere; when tested on soil samples results suggest the majority of NRPS and PKS pathways and hence bioactive metabolites are yet to be discovere

Exploring the functional soil-microbe interface and exoenzymes through soil metaexoproteomics

Functionally important proteins at the interface of cell and soil are of potentially low abundance when compared with commonly recovered intracellular proteins. A novel approach was developed and used to extract the metaexoproteome, the subset of proteins found outside the cell, in the context of a soil enriched with the nitrogen-containing recalcitrant polymer chitin. The majority of proteins recovered was of bacterial origin and localized to the outer membrane or extracellular milieu. A wide variety of transporter proteins were identified, particularly those associated with amino-acid and phosphate uptake. The metaexoproteome extract retained chitinolytic activity and we were successful in detecting Nocardiopsis-like chitinases that correlated with the glycoside hydrolase family 18 (GH18) chi gene data and metataxonomic analysis. Nocardiopsis-like chitinases appeared to be solely responsible for chitinolytic activity in soil. This is the first study to detect and sequence bacterial exoenzymes with proven activity in the soil enzyme pool

Uso de estreptotricinas B y F, fungicidas naturales para el control de fitopatógenos en bananos y plátanos

Fecha de solicitud:16.02.2000.- Titular: Consejo Superior de Investigaciones Científicas (CSIC).- Centro de Química Farmacéutica[EN]To prevent the development of these diseases, streptothricins B and F may be applied both in aqueous solution and supplemented with detergents and/or mineral oil. With a view to its application in the field, preparation of the product from the fermented broth is characterized in that use is made of an ion exchange resin (IRC-50), where the product is fixed, it is washed with distilled water and it is eluted from the column with a solution of acetic acid between 4 and 8 M. The active fractions containing the product are rotoevaporated until a saturated solution of sodium acetate is obtained, at a final concentration of between 100-120 grams of streptothricin/litre.[ES]Para la prevención del desarrollo de estas enfermedades, la estreptotricinas B y F pueden aplicarse tanto en solución acuosa como suplementadas con detergentes y/o aceite mineral. La elaboración del producto, con vista a su aplicación en campo, a partir del caldo fermentado, caracterizado porque se utiliza una resina de intercambio iónico (IRC-50), donde se fija el producto, se lava con agua destilada y se eluye de la columna con una solución de ácido acético entre 4 y 8 M. Las fracciones activas conteniendo el producto se rotoevaporan hasta obtener una soluciób saturada de acetato de sodio quedando a un concentración final entre 100-120 gramos de estreptotricina/litrosPeer reviewe

Comparison of primer sets on genomic DNA of different actinomycetes.

<p>The positive PCR hits are reported with the + symbol. Examples of known biosynthetic products related to NRPS and PKS clusters present in the strains are reported in the “Antibiotic pathways” column (Source: database ClusterMine360).</p

Neighbour joining tree demonstrating relationship between NRPS clones recovered from fosmid libraries constructed from Cuban and Antarctic sample sites.

<p>Reference sequences from Genbank were included and are indicated by named species.</p