Molecular richness and biotechnological potential of bacteria cultured from Irciniidae sponges in the north-east Atlantic

Several bioactive compounds originally isolated from marine sponges have been later ascribed or suggested to be synthesized by their symbionts. The cultivation of sponge-associated bacteria provides one possible route to the discovery of these metabolites. Here, we determine the bacterial richness cultured from two irciniid sponge species, Sarcotragus spinosulus and Ircinia variabilis, and ascertain their biotechnological potential. A total of 279 isolates were identified from 13 sponge specimens. These were classified into 17 genera - with Pseudovibrio, Ruegeria and Vibrio as the most dominant - and 3 to 10 putatively new bacterial species. While 16S rRNA gene sequencing identified 29 bacterial phylotypes at the 'species' level (97% sequence similarity), whole-genome BOX-PCR fingerprinting uncovered 155 genotypes, unveiling patterns of specimen-dependent occurrence of prevailing bacterial genomes across sponge individuals. Among the BOX-PCR genotypes recovered, 34% were active against clinically relevant strains, with Vibrio isolates producing the most active antagonistic effect. Several Pseudovibrio genotypes showed the presence of polyketide synthase (PKS) genes, and these were for the first time detected in isolates of the genus Aquimarina (Bacteroidetes). Our results highlight great biotechnological potential and interest for the Irciniidae sponge family and their diversified bacterial genomes.Portuguese Foundation for Science and Technology (FCT) [PTDC/MAR/101431/2008]; FCT [SFRH/BD/60873/2009, SFRH/BPD/62946/2009

The Natural Product Domain Seeker NaPDoS: A Phylogeny Based Bioinformatic Tool to Classify Secondary Metabolite Gene Diversity

New bioinformatic tools are needed to analyze the growing volume of DNA sequence data. This is especially true in the case of secondary metabolite biosynthesis, where the highly repetitive nature of the associated genes creates major challenges for accurate sequence assembly and analysis. Here we introduce the web tool Natural Product Domain Seeker (NaPDoS), which provides an automated method to assess the secondary metabolite biosynthetic gene diversity and novelty of strains or environments. NaPDoS analyses are based on the phylogenetic relationships of sequence tags derived from polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes, respectively. The sequence tags correspond to PKS-derived ketosynthase domains and NRPS-derived condensation domains and are compared to an internal database of experimentally characterized biosynthetic genes. NaPDoS provides a rapid mechanism to extract and classify ketosynthase and condensation domains from PCR products, genomes, and metagenomic datasets. Close database matches provide a mechanism to infer the generalized structures of secondary metabolites while new phylogenetic lineages provide targets for the discovery of new enzyme architectures or mechanisms of secondary metabolite assembly. Here we outline the main features of NaPDoS and test it on four draft genome sequences and two metagenomic datasets. The results provide a rapid method to assess secondary metabolite biosynthetic gene diversity and richness in organisms or environments and a mechanism to identify genes that may be associated with uncharacterized biochemistry

Comparative genome structure, secondary metabolite, and effector coding capacity across Cochliobolus pathogens.

The genomes of five Cochliobolus heterostrophus strains, two Cochliobolus sativus strains, three additional Cochliobolus species (Cochliobolus victoriae, Cochliobolus carbonum, Cochliobolus miyabeanus), and closely related Setosphaeria turcica were sequenced at the Joint Genome Institute (JGI). The datasets were used to identify SNPs between strains and species, unique genomic regions, core secondary metabolism genes, and small secreted protein (SSP) candidate effector encoding genes with a view towards pinpointing structural elements and gene content associated with specificity of these closely related fungi to different cereal hosts. Whole-genome alignment shows that three to five percent of each genome differs between strains of the same species, while a quarter of each genome differs between species. On average, SNP counts among field isolates of the same C. heterostrophus species are more than 25× higher than those between inbred lines and 50× lower than SNPs between Cochliobolus species. The suites of nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS), and SSP-encoding genes are astoundingly diverse among species but remarkably conserved among isolates of the same species, whether inbred or field strains, except for defining examples that map to unique genomic regions. Functional analysis of several strain-unique PKSs and NRPSs reveal a strong correlation with a role in virulence

Genome-scale diversity and niche adaptation analysis of Lactococcus lactis by comparative genome hybridization using multi-strain arrays

Contains fulltext : 95929.pdf (publisher's version ) (Open Access)Lactococcus lactis produces lactic acid and is widely used in the manufacturing of various fermented dairy products. However, the species is also frequently isolated from non-dairy niches, such as fermented plant material. Recently, these non-dairy strains have gained increasing interest, as they have been described to possess flavour-forming activities that are rarely found in dairy isolates and have diverse metabolic properties. We performed an extensive whole-genome diversity analysis on 39 L. lactis strains, isolated from dairy and plant sources. Comparative genome hybridization analysis with multi-strain microarrays was used to assess presence or absence of genes and gene clusters in these strains, relative to all L. lactis sequences in public databases, whereby chromosomal and plasmid-encoded genes were computationally analysed separately. Nearly 3900 chromosomal orthologous groups (chrOGs) were defined on basis of four sequenced chromosomes of L. lactis strains (IL1403, KF147, SK11, MG1363). Of these, 1268 chrOGs are present in at least 35 strains and represent the presently known core genome of L. lactis, and 72 chrOGs appear to be unique for L. lactis. Nearly 600 and 400 chrOGs were found to be specific for either the subspecies lactis or subspecies cremoris respectively. Strain variability was found in presence or absence of gene clusters related to growth on plant substrates, such as genes involved in the consumption of arabinose, xylan, alpha-galactosides and galacturonate. Further niche-specific differences were found in gene clusters for exopolysaccharides biosynthesis, stress response (iron transport, osmotolerance) and bacterial defence mechanisms (nisin biosynthesis). Strain variability of functions encoded on known plasmids included proteolysis, lactose fermentation, citrate uptake, metal ion resistance and exopolysaccharides biosynthesis. The present study supports the view of L. lactis as a species with a very flexible genome

Gene family encoding the major toxins of lethal Amanita mushrooms

Amatoxins, the lethal constituents of poisonous mushrooms in the genus Amanita, are bicyclic octapeptides. Two genes in A. bisporigera, AMA1 and PHA1, directly encode α-amanitin, an amatoxin, and the related bicyclic heptapeptide phallacidin, a phallotoxin, indicating that these compounds are synthesized on ribosomes and not by nonribosomal peptide synthetases. α-Amanitin and phallacidin are synthesized as proproteins of 35 and 34 amino acids, respectively, from which they are predicted to be cleaved by a prolyl oligopeptidase. AMA1 and PHA1 are present in other toxic species of Amanita section Phalloidae but are absent from nontoxic species in other sections. The genomes of A. bisporigera and A. phalloides contain multiple sequences related to AMA1 and PHA1. The predicted protein products of this family of genes are characterized by a hypervariable “toxin” region capable of encoding a wide variety of peptides of 7–10 amino acids flanked by conserved sequences. Our results suggest that these fungi have a broad capacity to synthesize cyclic peptides on ribosomes

Direct structural insight into the substrate-shuttling mechanism of yeast fatty acid synthase by electron cryomicroscopy

Yeast fatty acid synthase (FAS) is a 2.6-MDa barrel-shaped multienzyme complex, which carries out cyclic synthesis of fatty acids. By electron cryomicroscopy of single particles we obtained a three-dimensional map of yeast FAS at 5.9-Å resolution. Compared to the crystal structures of fungal FAS, the EM map reveals major differences and new features that indicate a considerably different arrangement of the complex in solution compared to the crystal structures, as well as a high degree of variance inside the barrel. Distinct density regions in the reaction chambers next to each of the catalytic domains fitted the substrate-binding acyl carrier protein (ACP) domain. In each case, this resulted in the expected distance of ∼18 Å from the ACP substrate-binding site to the active site of the catalytic domains. The multiple, partially occupied positions of the ACP within the reaction chamber provide direct structural insight into the substrate-shuttling mechanism of fatty acid synthesis in this large cellular machine

Combinatorial biosynthesis of novel antibiotics related to daptomycin

Daptomycin, a cyclic lipopeptide produced by Streptomyces roseosporus, is the active ingredient of Cubicin (daptomycin-for-injection), a first-in-class antibiotic approved for treatment of skin and skin-structure infections caused by Gram-positive pathogens and bacteremia and endocarditis caused by Staphylococcus aureus, including methicillin-resistant strains. Genetic engineering of the nonribosomal peptide synthetase (NRPS) in the daptomycin biosynthetic pathway was exploited for the biosynthesis of novel active antibiotics. λ-Red-mediated recombination was used to exchange single or multiple modules in the DptBC subunit of the NRPS to modify the daptomycin cyclic peptide core. We combined module exchanges, NRPS subunit exchanges, inactivation of the tailoring enzyme glutamic acid 3-methyltransferase, and natural variations of the lipid tail to generate a library of novel lipopeptides, some of which were as active as daptomycin against Gram-positive bacteria. One compound was more potent against an Escherichia coli imp mutant that has increased outer membrane permeability. This study established a robust combinatorial biosynthesis platform to produce novel peptide antibiotics in sufficient quantities for antimicrobial screening and drug development