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The noble pen shell Pinna nobilis is a Mediterranean endemic and emblematic giant bivalve. Already considered by the late 20th century to be an endangered species, it is facing a dramatic and rapidly expanding epizooty that has decimated populations since mid-2016. The ecological importance of P. nobilis has prompted important investigations for conservation purposes. Here, we report a first analysis of the whole genome sequencing of this animal. This was performed on an Illumina HiSeq X platform using a single paired-end library of short fragments (2 × 150 bp). The de novo contig assembly had a total size of 584 Mb (96,738 contigs, N50 = 7.6 kb, with 0.4% of ambiguous nucleotides), representing 77.5% of the predicted genome size of 754 Mb. The P. nobilis genome is highly AT-rich, with a GC content of 35.6%. At 1%, heterozygosity was in the range of other bivalves with sequenced genomes. Over one-third (36.2%) of the genome consisted of repeated elements with a surprising larger number of short interspersed nuclear elements compared to other molluscan genomes. We were also able to reconstruct the full mitochondrial genome (c. 19 kb, with 12 protein-coding genes, 2 rRNA and 22 tRNA genes). In the context of the epizootic outbreak affecting P. nobilis , a first insight into the innate immune and stress-related genes found in the sequence is provided. [ABSTRACT FROM AUTHOR]

The antifouling (AF) properties of phytochemicals isolated from the Mediterranean brown seaweed Taonia atomaria have been assayed against several colonizing organisms. Eight compounds were isolated and their chemical structures characterized by spectroscopic techniques (NMR, MS) and comparison with literature data. The phytochemical composition of the extracts of T. atomaria was found to be mainly attributable to sesquiterpenes. Accordingly, a new germacrane, germacra-4(15),5,10(14)-trien-9-ol ( 1), is described together with five other sesquiterpenoids (-)-gleenol ( 2); α-cadinol methyl ether ( 3), (-)- trans-calamenene ( 4), (1 S, 5 E, 7 S) 1-acetoxygermacra-4(15),5,10(14)-triene ( 5), and 4-peroxymuurol-5-ene ( 6). Moreover, two lipidic compounds, a polyunsaturated fatty acid, (5 Z, 8 Z, 11 Z, 14 Z, 17 Z)-eicosa-5,8,11,14,17-pentaenoic acid ( 7), and a glycerol derivative, sn-3- O-(geranylgeranyl)glycerol ( 8), were also isolated. The known compounds 3, 6- 7 are described here for the first time from this alga. In comparison with commercial antifoulants [tributyltin oxide (TBTO), zineb, and copper pyrithione (CuPT)], compounds 1, 2, 4, 6, and 8 were evaluated for their toxicity and capacity to inhibit the settlement of five bacteria isolated from marine biofilms. Toxicity and anti-settlement activity of 1, 2, and 6 were also assessed against two barnacle species ( Amphibalanus amphitrite and Balanus perforatus). Regarding bacteria, a significant anti-adhesion effect was recorded for 8 (EC between 25 and 50 μM) with moderate associated toxicity. Considering the settlement inhibition of barnacle cyprids, (-)-gleenol ( 2) exhibited a strong AF efficacy (EC < 5 μM) with moderate toxicity. In addition, as B. perforatus showed similar responses to the reference A. amphitrite with all compounds, this species may be proposed as an alternative model for anti-macrofouling assessment, especially in temperate areas. Overall, these results suggest that a number of the tested compounds could, alone or in combination, play a critical role in limiting the fouling at the T. atomaria surface. [ABSTRACT FROM AUTHOR]

The phosphopantetheinyl transferases (PPTases) are responsible for the activation of the carrier protein domains of the polyketide synthases (PKS), non ribosomal peptide synthases (NRPS) and fatty acid synthases (FAS). The analysis of the Streptomyces ambofaciens ATCC23877 genome has revealed the presence of four putative PPTase encoding genes. One of these genes appears to be essential and is likely involved in fatty acid biosynthesis. Two other PPTase genes, samT0172 (alpN) and samL0372, are located within a type II PKS gene cluster responsible for the kinamycin production and an hybrid NRPS-PKS cluster involved in antimycin production, respectively, and their products were shown to be specifically involved in the biosynthesis of these secondary metabolites. Surprisingly, the fourth PPTase gene, which is not located within a secondary metabolite gene cluster, appears to play a pleiotropic role. Its product is likely involved in the activation of the acyl- and peptidyl-carrier protein domains within all the other PKS and NRPS complexes encoded by S. ambofaciens. Indeed, the deletion of this gene affects the production of the spiramycin and stambomycin macrolide antibiotics and of the grey spore pigment, all three being PKS-derived metabolites, as well as the production of the nonribosomally produced compounds, the hydroxamate siderophore coelichelin and the pyrrolamide antibiotic congocidine. In addition, this PPTase seems to act in concert with the product of samL0372 to activate the ACP and/or PCP domains of the antimycin biosynthesis cluster which is also responsible for the production of volatile lactones. [ABSTRACT FROM AUTHOR]

The genome sequence of Streptomyces ambofaciens, a species known to produce the congocidine and spiramycin antibiotics, has revealed the presence of numerous gene clusters predicted to be involved in the biosynthesis of secondary metabolites. Among them, the type II polyketide synthase-encoding alp cluster was shown to be responsible for the biosynthesis of a compound with antibacterial activity. Here, by means of a deregulation approach, we gained access to workable amounts of the antibiotics for structure elucidation. These compounds, previously designated as alpomycin, were shown to be known members of kinamycin family of antibiotics. Indeed, a mutant lacking AlpW, a member of the TetR regulator family, was shown to constitutively produce kinamycins. Comparative transcriptional analyses showed that expression of alpV, the essential regulator gene required for activation of the biosynthetic genes, is strongly maintained during the stationary growth phase in the alpW mutant, a stage at which alpV transcripts and thereby transcripts of the biosynthetic genes normally drop off. Recombinant AlpW displayed DNA binding activity toward specific motifs in the promoter region of its own gene and that of alpV and alpZ. These recognition sequences are also targets for AlpZ, the γ-butyrolactone-like receptor involved in the regulation of the alp cluster. However, unlike that of AlpZ, the AlpW DNA-binding ability seemed to be insensitive to the signaling molecules controlling antibiotic biosynthesis. Together, the results presented in this study reveal S. ambofaciens to be a new producer of kinamycins and AlpW to be a key late repressor of the cellular control of kinamycin biosynthesis. [ABSTRACT FROM AUTHOR]

The potential iron siderophore transporter genes have been determined from the genome sequence of Streptomyces coelicolor A3(2). One of these gene clusters, cdtABC, was disrupted and characterized to determine its role in the uptake of the siderophores produced by S. coelicolor. Resistance to the siderophore-like antibiotics, salmycin and albomycin, was tested in the parent and cdtABC mutant, showing that the parent, but not the mutant, was sensitive to salmycin, while both were resistant to albomycin. Ferrioxamine competition assays against salmycin suggest that the uptake of salmycin is via a ferrioxamine transport system. However, Fe-55 ferrioxamine B uptake experiments did not reveal any difference between the parent and mutant. This suggests that CdtABC specifically transports salmycin, while ferrioxamine uptake maybe substituted by another transport system. [ABSTRACT FROM AUTHOR]

The fan mussel Pinna nobilis is currently on the brink of extinction due to a multifactorial disease mainly caused to the highly pathogenic parasite Haplosporidium pinnae, meaning that the selection pressure outweighs the adaptive potential of the species. Hopefully, rare individuals have been observed somehow resistant to the parasite, stretching the need to identify the traits underlying this better fitness. Among the candidate to explore at first intention are fast‐evolving immune genes, of which toll‐like receptor (TLR). In this study, we examined the genetic diversity at 14 TLR loci across P. nobilis, Pinna rudis and P. nobilis × P. rudis hybrid genomes, collected at four physically distant regions, that were found to be either resistant or sensitive to the parasite H. pinnae. We report a high genetic diversity, mainly observed at cell surface TLRs compared with that of endosomal TLRs. However, the endosomal TLR‐7 exhibited unexpected level of diversity and haplotype phylogeny. The lack of population structure, associated with a high genetic diversity and elevated dN/dS ratio, was interpreted as balancing selection, though both directional and purifying selection were detected. Interestingly, roughly 40% of the P. nobilis identified as resistant to H. pinnae were introgressed with P. rudis TLR. Specifically, they all carried a TLR‐7 of P. rudis origin, whereas sensitive P. nobilis were not introgressed, at least at TLR loci. Small contributions of TLR‐6 and TLR‐4 single‐nucleotide polymorphisms to the clustering of resistant and susceptible individuals could be detected, but their specific role in resistance remains highly speculative. This study provides new information on the diversity of TLR genes within the P. nobilis species after MME and additional insights into adaptation to H. pinnae that should contribute to the conservation of this Mediterranean endemic species. [ABSTRACT FROM AUTHOR]

Streptomyces ambofaciens produces an orange pigment and the antibiotic alpomycin, both of which are products of a type II polyketide synthase gene cluster identified in each of the terminal inverted repeats of the linear chromosome. Five regulatory genes encoding Streptomyces antibiotic regulatory proteins (alpV, previously shown to be an essential activator gene; alpT; and alpU) and TetR family receptors (alpZ and alpW) were detected in this cluster. Here, we demonstrate that AlpZ, which shows high similarity to γ-butyrolactone receptors, is at the top of a pathway-specific regulatory hierarchy that prevents synthesis of the alp polyketide products. Deletion of the two copies of alpZ resulted in the precocious production of both alpomycin and the orange pigment, suggesting a repressor role for AlpZ. Consistent with this, expression of the five alp-located regulatory genes and of two representative biosynthetic structural genes (alpA and alpR) was induced earlier in the alpZ deletion strain. Furthermore, recombinant AlpZ was shown to bind to specific DNA sequences within the promoter regions of alpZ, alpV, and alpXW, suggesting direct transcriptional control of these genes by AlpZ. Analysis of solvent extracts of S. ambofaciens cultures identified the existence of a factor which induces precocious production of alpomycin and pigment in the wild-type strain and which can disrupt the binding of AlpZ to its DNA targets. This activity is reminiscent of γ-butyrolactone-type molecules. However, the AlpZ-interacting molecule(s) was shown to be resistant to an alkali treatment capable of inactivating γ-butyrolactones, suggesting that the AlpZ ligand(s) does not possess a lactone functional group. [ABSTRACT FROM AUTHOR]

With the intensification of maritime traffic, recently emerged infectious diseases have become major drivers in the decline and extinction of species. Since 2016, mass mortality events have decimated the endemic Mediterranean Sea bivalve Pinna nobilis, affecting ca. 100% of individuals. These events have largely been driven by Haplosporidium pinnae's infection, an invasive species which was likely introduced by shipping. While monitoring wild populations of P. nobilis, we observed individuals that survived such a mass mortality event during the summer of 2018 (France). We considered these individuals resistant, as they did not show any symptoms of the disease, while the rest of the population in the area was devastated. Furthermore, the parasite was not detected when we conducted a PCR amplification of a species-specific fragment of the small subunit ribosomal DNA. In parallel, the transcriptomic analysis showed evidence of some parasite RNA indicating that the resistant individuals had been exposed to the parasite without proliferating. To understand the underlying mechanisms of resistance in these individuals, we compared their gene expression with that of susceptible individuals. We performed de novo transcriptome assembly and annotated the expressed genes. A comparison of the transcriptomes in resistant and susceptible individuals highlighted a gene expression signature of the resistant phenotype. We found significant differential expressions of genes involved in immunity and cell architecture. This data provides the first insights into how individuals escape the pathogenicity associated with infection. [ABSTRACT FROM AUTHOR]

Background: The control of antibiotic production in Streptomyces coelicolor A3(2) involves complicated regulatory networks with multiple regulators controlling the expression of antibiotic biosynthetic pathways. One such regulatory network is that of the ?-butyrolactones, the so-called S. coelicolor butanolide (SCB) system. The γ-butyrolactones in this system serve as signalling molecules and bind to the receptor protein ScbR, releasing the repression of its target genes. The resulting expression changes affect the production of the two pigmented antibiotics Act and Red, as well as the transcription of the cpk antibiotic biosynthesis gene cluster and the synthesis of the γ-butyrolactones themselves.Results: We identified a natural variant of ScbR in S. coelicolor (ScbRM600) that differs from ScbR in the genome-sequenced strain M145 (ScbRM145) by a single amino acid change, R120S. ScbRM600 is impaired in its DNA binding ability and alters the expression of the pathway-specific regulatory genes of the red and cpk antibiotic biosynthesis gene clusters. Also, expression of the ?-butyrolactone biosynthesis gene scbA and production of the signalling molecules is slightly reduced.Conclusions: The γ-butyrolactone receptor, ScbR, plays a key role in the SCB regulatory cascade and in determining the onset of the expression of the antibiotic regulatory genes [ABSTRACT FROM AUTHOR]

The duplicated hasR and hasL genes of Streptomyces ambofaciens encode alternative sigma factors (named σBR and σBL) belonging to the σB general stress response family in Bacillus subtilis. The duplication appears to be the result of a recent event that occurred specifically in S. ambofaciens. The two genes are 98% identical, and their deduced protein products exhibit 97% identity at the amino acid level. In contrast with the coding sequences, their genetic environments and their transcriptional control are strongly divergent. While hasL is monocistronic, hasR is arranged in a polycistronic unit with two upstream open reading frames, arsR and prsR, that encode putative anti-anti-σ and anti-σ factors, respectively. Transcription of each has gene is initiated from two promoters. In each case, one promoter was shown to be developmentally controlled and to be similar to those recognized by the B. subtilis general stress response sigma factor σB. Expression from this type of promoter for each of the has genes dramatically increases during the course of growth in liquid or on solid media and following oxidative and osmotic stresses. Reverse transcription-PCR measurements indicate that hasR is 100 times more strongly expressed than hasL from the σB-like promoter. Transcription from the second promoter of each gene (located upstream of arsR in the case of the hasR locus) appears to be constitutive and weak. Quantitative transcriptional analysis in single and double has mutant strains revealed that σBR and σBL direct their own transcription as well as that of their duplicates. Only a slight sensitivity in response to oxidative conditions could be assigned to either single or double mutants, revealing the probable redundancy of the σ factors implied in stress response in Streptomyces. [ABSTRACT FROM AUTHOR]

Marine microalgae and cyanobacteria have largely been studied for their biotechnological potential and proved their ability to produce a wide array of bioactive molecules. We investigated the antifouling potential of unexplored benthic tropical microalgae using anti-adhesion and toxicity bioassays against two major micro- and ma crobiofoulers, namely bacteria and barnacles. Fifty strains belonging to six phyla [Cyanobacteria, Miozoa (Dinoflagellata), Bacillariophyta, Cryptophyta, Rhodophyta and Haptophyta] were isolated from southwestern Islands of the Indian Ocean. They were chosen in order to represent as much as possible the huge biodiversity of such a rich tropical ecosystem. The associated chemodiversity was highlighted by both NMR- and LC-MS-based metabolomics. The screening of 84 algal fractions revealed that the anti-adhesion activity was concentrated in methanolic ones (i.e. 93% of all active fractions). Our results confirmed that microalgae constitute a promising source of natural antimicrofoulants as 17 out of the 30 active fractions showed high or very high capacity to inhibit the adhesion of three biofilm-forming marine bacteria. Dinoflagellate-derived fractions were the most active, both in terms of number and intensity. However, dinoflagellates were also more toxic and may not be suitable as a source of environmentally friendly antifouling compounds, in contrast to diatoms, e.g. Navicula mollis. The latter and two dinoflagellates of the genus Amphidinium also had interesting anti-settlement activities while being moderately toxic to barnacle larvae. Our approach, combining the bioprospecting of a large number of tropical microalgae for their anti-settlement potential and metabolomics analyses, constituted a first step towards the discovery of alternative ecofriendly antifoulants. [ABSTRACT FROM AUTHOR]

Summary: γ-Butyrolactone bacterial hormones regulate antibiotic production and morphological differentiation in Streptomyces species. One γ-butyrolactone, SCB1, has been previously characterized in Streptomyces coelicolor. Here we report the characterization of two additional γ-butyrolactones, named SCB2 (2-[1′-hydroxyoctyl]-3-hydroxymethylbutanolide) and SCB3 (2-[1′-hydroxy-6′-methyloctyl]-3-hydroxymethylbutanolide), possessing an antibiotic stimulatory activity. To elucidate the specificity determinants of these ligands for the receptor protein, ScbR, 30 chemically synthesized γ-butyrolactone analogs were tested by utilizing the release of ScbR from DNA upon binding to a γ-butyrolactone, which can be detected by kanamycin resistance. The butyrolactone detection method developed here revealed that ScbR shows preference toward a ligand possessing a 7–10 carbon C-2 side chain, a C-1′-β-hydroxyl group, and a C-6′-methyl branch that coincides with SCB3. Moreover, this method was successfully used to screen for potential γ-butyrolactone producers from commercial-antibiotic-producing Streptomyces. [Copyright &y& Elsevier]

Fouling Release Coatings are marine antifouling coatings based on silicone elastomers. Contrary to commonly used biocide-based antifouling coatings, they do not release biocides into the marine environment, however, they suffer from poor antifouling efficacy during idle periods. To improve their antifouling performances in static conditions, various amounts of hydrolyzable polymers were incorporated within a silicone matrix. These hydrolyzable polymers were chosen for the well-known hydrolytic degradation mechanism of their main chain, e.g. poly(ε-caprolactone) (PCL), or of their ester pending groups, e.g. poly(bis(trimethylsilyloxy)methylsilyl methacrylate) (PMATM2). The degradation kinetics of such hydrolyzable silicone coatings were assessed by mass loss measurements during immersion in deionized water. Coatings containing PMATM2 exhibited a maximum mass loss after 12 weeks, whereas PCL-based coatings showed no significant mass loss after 24 weeks. Dynamic contact angle measurements revealed the modifications of the coatings surface chemistry with an amphiphilic behavior after water exposure. The attachment of macrofoulers on these coatings were evaluated by field tests in the Mediterranean Sea, demonstrating the short or long-term antifouling effect of these hydrolyzable polymers embedded in the silicone matrix. The settlement of A. amphitrite barnacles on the different coatings indicated inhospitable behaviors towards larval barnacles for coatings with at least 15 wt % of additives. [ABSTRACT FROM AUTHOR]