Genomic blueprints of sponge-prokaryote symbiosis are shared by low abundant and cultivatable Alphaproteobacteria

Marine sponges are early-branching, filter-feeding metazoans that usually host complex microbiomes comprised of several, currently uncultivatable symbiotic lineages. Here, we use a low-carbon based strategy to cultivate low-abundance bacteria from Spongia officinalis. This approach favoured the growth of Alphaproteobacteria strains in the genera Anderseniella, Erythrobacter, Labrenzia, Loktanella, Ruegeria, Sphingorhabdus, Tateyamaria and Pseudovibrio, besides two likely new genera in the Rhodobacteraceae family. Mapping of complete genomes against the metagenomes of S. officinalis, seawater, and sediments confirmed the rare status of all the above-mentioned lineages in the marine realm. Remarkably, this community of low-abundance Alphaproteobacteria possesses several genomic attributes common to dominant, presently uncultivatable sponge symbionts, potentially contributing to host fitness through detoxification mechanisms (e.g. heavy metal and metabolic waste removal, degradation of aromatic compounds), provision of essential vitamins (e.g. B6 and B12 biosynthesis), nutritional exchange (especially regarding the processing of organic sulphur and nitrogen) and chemical defence (e.g. polyketide and terpenoid biosynthesis). None of the studied taxa displayed signs of genome reduction, indicative of obligate mutualism. Instead, versatile nutrient metabolisms along with motility, chemotaxis, and tight-adherence capacities - also known to confer environmental hardiness - were inferred, underlying dual host-associated and free-living life strategies adopted by these diverse sponge-associated Alphaproteobacteria.PTDC/MAR-BIO/1547/2014; full PhD scholarship from the Erasmus Mundus Programme/SALAM EMA2 lot7/SALA1206422info:eu-repo/semantics/publishedVersio

Metagenomics and functional genomics of bacterial symbionts of Spongia (Porifera, Dictyoceratida) specimens from the Algarvian shore (South Portugal)

Sponges are early-branched, filter-feeding metazoans that usually harbor complex microbial communities comprised of diverse “uncultivable” symbiotic bacteria. In this thesis, the functional and taxonomic features of the marine sponge microbiome are determined, using Spongia officinalis as model host organism. Emphasis is given to adaptive and functional traits of the profuse and biotechnologically-relevant alphaproteobacterial symbionts of sponges. A metagenomics-centred approach was employed to reveal microbial taxa and genomic signatures enriched in the Spongia officinalis endosymbiotic consortium, and thus likely to play pivotal roles in holobiont functioning. Further, a comparative genomics study is presented unveiling the common and specific traits of ten Alphaproteobacteria genera isolated from S. officinalis with alternative symbiont cultivation methodology. Finally, a sequence composition-dependent binning approach is employed to assemble, from metagenomic sequences, the genome of an uncultured alphaproteobacterial symbiont of S. officinalis belonging to the family Rhodospirillaceae. High abundance of polyketide and terpene synthase-, eukaryotic-like protein- (ELPs), type IV secretion system-, plasmid- and ABC transporter-encoding genes, among others, characterized the sponge microbial metagenomes. In contrast, motility and chemotaxis genes were abundant in seawater and sediment microbiomes, but nearly absent in the S. officinalis symbiotic consortium. Much higher frequencies of anti-viral CRISPR-Cas and restrictionmodification systems, along with much lower viral abundances, were observed in the spongeassociated metagenomes than in the environment and interpreted as true hallmarks of this symbiotic consortium. In line with outcomes retrieved for the whole symbiotic community, alphaproteobacterial symbionts of marine sponges likely contribute the most to host fitness through nutritional exchange, cell detoxification processes and chemical defense, the latter being theoretically promoted by both polyketide and terpenoid biosynthesis. The several alphaproteobacterial cultures retrieved in this thesis, displaying high natural product biosynthesis capacities, can now be explored in studies aiming at revealing novel biological activities and chemical structures from these symbionts.As esponjas marinhas (filo Porifera) são consideradas um dos mais simples grupos entre os metazoários em função de sua falta de organização em tecidos e órgãos verdadeiros. Porém, estes animais relativamente simples em termos de plano corporal normalmente abrigam comunidades muito complexas de microorganismos. Em função de seu surgimento basal na história evolutiva do planeta, o conhecimento a respeito deste “holobionte”, isto é, o consórcio de organismos formado pela esponja marinha hospedeira e todos os seus simbiontes microbianos, possui grande relavância ao avanço da nossa compreensão sobre as interações hospedeiro-microorganismos. Nesta tese de doutoramento, tive como objetivo a determinação das características funcionais e taxonómicas do microbioma das esponjas marinhas no contexto de seu ambiente circundante (água e sedimentos marinhos, e suas respecticvas microbiotas), dando ênfase aos traços adaptativos e funcionais de alfaproteobactérias associadas ao organismo modelo Spongia officinalis (“bath sponge”)

Bacterial isolates from the bryozoan Membranipora membranacea: influence of culture media on isolation and antimicrobioal activity

From specimens of the bryozoan Membranipora membranacea collected in the Baltic Sea, bacteria were isolated on four different media, which significantly increased the diversity of the isolated groups. All isolates were classifiedaccording to 16S rRNA gene sequence analysis and tested for antimicrobial properties using a panel of five indicator strainsand six different media. Each medium featured a unique set of isolated phylotypes, and a phylogenetically diverse collection of isolates was obtained. A total of 96 isolates were assigned to 49 phylotypes and 29 genera. Only one-third of the members of these genera had been isolated previously from comparable sources. The isolates were affiliated with Alpha- and Gammaproteobacteria, Bacilli, and Actinobacteria. A comparable large portion of up to 22 isolates, i.e., 15 phylotypes, probably represent new species. Likewise, 47 isolates (approximately 50%) displayed antibiotic activities, mostly against grampositive indicator strains. Of the active strains, 63.8 % had antibiotic traits only on one or two of the growth media, whereas only 12.7 % inhibited growth on five or all six media. The application of six different media for antimicrobial testing resulted in twice the number of positive hits as obtained with only a single medium. The use of different media for the isolation of bacteria as well as the variation of media considered suitable for the production of antibiotic substances significantly enhanced both the number of isolates obtained and the proportion of antibiotic active cultures. Thus the approach described herein offers an improved strategy in the search for new antibiotic compound

Bacterial isolates from the bryozoan Membranipora membranacea: influence of culture media on isolation and antimicrobial activity

From specimens of the bryozoan Membranipora membranacea collected in the Baltic Sea, bacteria were isolated on four different media, which significantly increased the diversity of the isolated groups. All isolates were classifiedaccording to 16S rRNA gene sequence analysis and tested for antimicrobial properties using a panel of five indicator strainsand six different media. Each medium featured a unique set of isolated phylotypes, and a phylogenetically diverse collection of isolates was obtained. A total of 96 isolates were assigned to 49 phylotypes and 29 genera. Only one-third of the members of these genera had been isolated previously from comparable sources. The isolates were affiliated with Alpha- and Gammaproteobacteria, Bacilli, and Actinobacteria. A comparable large portion of up to 22 isolates, i.e., 15 phylotypes, probably represent new species. Likewise, 47 isolates (approximately 50%) displayed antibiotic activities, mostly against grampositive indicator strains. Of the active strains, 63.8 % had antibiotic traits only on one or two of the growth media, whereas only 12.7 % inhibited growth on five or all six media. The application of six different media for antimicrobial testing resulted in twice the number of positive hits as obtained with only a single medium. The use of different media for the isolation of bacteria as well as the variation of media considered suitable for the production of antibiotic substances significantly enhanced both the number of isolates obtained and the proportion of antibiotic active cultures. Thus the approach described herein offers an improved strategy in the search for new antibiotic compounds. [Int Microbiol 2012; 15(1):17-32

Marine sponge and octocoral-associated bacteria show versatile secondary metabolite biosynthesis potential and antimicrobial activities against human pathogens

Marine microbiomes are prolific sources of bioactive natural products of potential pharmaceutical value. This study inspected two culture collections comprising 919 host-associated marine bacteria belonging to 55 genera and several thus-far unclassified lineages to identify isolates with potentially rich secondary metabolism and antimicrobial activities. Seventy representative isolates had their genomes mined for secondary metabolite biosynthetic gene clusters (SM-BGCs) and were screened for antimicrobial activities against four pathogenic bacteria and five pathogenic Candida strains. In total, 466 SM-BGCs were identified, with antimicrobial peptide- and polyketide synthase-related SM-BGCs being frequently detected. Only 38 SM-BGCs had similarities greater than 70% to SM-BGCs encoding known compounds, highlighting the potential biosynthetic novelty encoded by these genomes. Cross-streak assays showed that 33 of the 70 genome-sequenced isolates were active against at least one Candida species, while 44 isolates showed activity against at least one bacterial pathogen. Taxon-specific differences in antimicrobial activity among isolates suggested distinct molecules involved in antagonism against bacterial versus Candida pathogens. The here reported culture collections and genome-sequenced isolates constitute a valuable resource of understudied marine bacteria displaying antimicrobial activities and potential for the biosynthesis of novel secondary metabolites, holding promise for a future sustainable production of marine drug leads.info:eu-repo/semantics/publishedVersio

Biodegradation of Buried MC252 Oil in Coastal Beach Sands by PAH Degraders in Response to Oxygen Biostimulation

Six years following the Deepwater Horizon oil spill, buried MC252 oil persists on a beach segment in Fourchon, LA due to the natural anaerobic conditions of tidal groundwater. A field trial of in situ aerobic bioremediation of buried oil began on July 2015. Oxygen was added to the subsurface using Waterloo emitters which deliver O2 through diffusion via pressurized tubing in fixed groundwater wells. The multi-well injection system provided sufficient oxygen concentration in the groundwater in the immediate area of the emitters, resulting in a shift in the composition of the diverse, halophilic, hydrocarbon-degrading microbial population. Stable isotopic and radiocarbon data from dissolved inorganic carbon provided evidence of crude oil mineralization post-aeration, even in areas that had the highest level of contamination. Weathering ratios for 3-ring PAHs were reduced post-aeration, indicating increased rates of degradation of phenanthrenes and dibenzothiophenes. Serum bottle studies were conducted to analyze biodegradation of PAHs and alkanes as a response to adding varying levels of O2 over time. Oxygen amendments were found to stimulate biodegradation of recalcitrant PAHs more effectively in less oily sediments than in sediments with higher oil concentrations. The minimum oxygen amendments in this study were sufficient in shifting the microbial population to a more effectual hydrocarbon degrading community structure

Bacterial Diversity in a Dynamic and Extreme Sub-Arctic Watercourse (Pasvik River, Norwegian Arctic)

Microbial communities promptly respond to the environmental perturbations, especially in the Arctic and sub-Arctic systems that are highly impacted by climate change, and fluctuations in the diversity level of microbial assemblages could give insights on their expected response. 16S rRNA gene amplicon sequencing was applied to describe the bacterial community composition in water and sediment through the sub-Arctic Pasvik River. Our results showed that river water and sediment harbored distinct communities in terms of diversity and composition at genus level. The distribution of the bacterial communities was mainly affected by both salinity and temperature in sediment samples, and by oxygen in water samples. Glacial meltwaters and runoff waters from melting ice probably influenced the composition of the bacterial community at upper and middle river sites. Interestingly, marine-derived bacteria consistently accounted for a small proportion of the total sequences and were also more prominent in the inner part of the river. Results evidenced that particular conditions occurring at sampling sites (such as algal blooms, heavy metal contamination and anaerobiosis) may select species at local scale from a shared bacterial pool, thus favoring certain bacterial taxa. Conversely, the few phylotypes specifically detected in some sites are probably due to localized external inputs introducing allochthonous microbial groups

Complete genome sequence of Parvibaculum lavamentivorans type strain (DS-1T)

Parvibaculum lavamentivorans DS-1T is the type species of the novel genus Parvibaculum in the novel family Rhodobiaceae (formerly Phyllobacteriaceae) of the order Rhizobiales of Alphaproteobacteria. Strain DS-1T is a non-pigmented, aerobic, heterotrophic bacterium and represents the first tier member of environmentally important bacterial communities that catalyze the complete degradation of synthetic laundry surfactants. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,914,745 bp long genome with its predicted 3,654 protein coding genes is the first completed genome sequence of the genus Parvibaculum, and the first genome sequence of a representative of the family Rhodobiaceae

A path to biodiscovery: isolating Antarctic ascidian-associated bacteria and screening genomes for secondary metabolite-encoded biosynthetic gene clusters and signatures of cold adaptation

Natural products synthesized by host-associated microbes from polar regions pose tremendous potential for addressing human health challenges. One prominent example is the ascidian Synoicum adareanum, found on the seafloor of coastal regions of Antarctica, of interest in part due to one compound, palmerolide A. Palmerolide A is a cytotoxic macrolide with specific activity towards cancer cells which occurs in high concentration in its tissues. It has since been determined that one core bacterial member of the moderately diverse Synoicum adareanum microbiome is responsible for the biosynthesis of palmerolide A. This finding reinforces the significance of investigating natural product potential in host-associated microbes from high-latitude ecosystems. One goal of the Synoicum adareanum research program is establishing a sustainable biological source of palmerolide A either through cultivation or bioengineering solutions. Secondly, exploring the microbiome for additional cold-water biosynthesized natural products is of interest given recent metagenome results that suggest substantial natural product biosynthetic potential. To advance this research requires investigating cultivation strategies for the Verrucomicrobia-affiliated palmerolide A producer, establishing a more comprehensive understanding of the biosynthetic potential of other microbes in the Synoicum adareanum microbiome, and ascertaining the cold-adaptive characteristics of the genome and encoded proteome evolution of host-associated high latitude marine microorganisms. Here, we implemented a minimal media cultivation and dereplication strategy to isolate bacteria, rapidly screen for palmerolide A-encoding biosynthetic gene clusters using PCR and determine the identity of the novel isolates with long read 16S rRNA phylogenetic analyses. This work added phylogenetic breadth to previously cultivated bacterial classes Gammaproteobacteria and Alphaproteobacteria from the Synoicum adareanum microbiome, to now include Bacilli, Acidimicrobiia, Actinobacteria and Bacteroidia. Through sequencing representative genomes of 22 isolates, 135 biosynthetic gene cluster types across eight compound categories were identified within this expanded culture collection using bioinformatic approaches. Comparative genomic analyses between orthologous proteins predicted between our high-latitude, Antarctic isolates and representative low/mid latitude marine microorganisms of the same genera revealed signatures of cold adaptation in the Antarctic genomes. We found an overall decrease in proline residues that form rigid kinks in protein sequences. Likewise, charged amino acids and arginine/(arginine+lysine) content, which form protein-stabilizing salt bridges believed to hinder protein function at low temperatures were reduced. The predicted Antarctic proteins also followed the established trend of increased glycine and serine content in cold adapted proteins, understood to increase protein dynamics in cold through their small size and reduced ionic charge. Interestingly, significant numbers of coding sequences with lower GC content were found in the Antarctic genomes even if genome-wide GC contents were similar, or even higher, for the Antarctic organism. Also surprising, more predicted proteins with higher intrinsic disorder were observed in the low/mid latitude genomes, perhaps indicative of a possible environmental temperature threshold for disorder which balances protein function and cold denaturation resistance. These cultivation, phylogenomic and bioinformatic results provide a deeper view into this Antarctic marine ascidian, the diversity and potential for secondary metabolite production within its microbial community, and trends observed in protein and genome sequences of cold adapted organisms