Isolation and genome sequencing of 14 Spongia sp. bacterial associates expands the taxonomic and functional breadth of the cultivatable marine sponge microbiome

Abstract

19Marine sponges live with complex microbial consortia, which have been considered as 20 potential sources of novel natural products. However, the usual recalcitrance of host-21 associated microorganisms to cultivation makes studying sponge symbionts challenging. To 22 tackle this complexity, exploration of cultivated sponge-associated bacteria and their coding 23 potential is unavoidable. In this study, we isolate and report the draft genome sequences of 14 24 bacterial strains from the marine sponge Spongia sp. using R2A and VXA media. The strains 25 belong to the classes Actinobacteria, Gammaproteobacteria, Alphaproteobacteria, and 26 Cytophagia spanning 11 formally described genera plus two potentially novel genera in the 27Rhodobacteraceae family and one potentially novel family in the Cytophagales order. 28Functional genomics revealed presumed symbiosis factors typical of specific taxonomic 29 groups (i.e. taurine metabolism genes among the Alphaproteobacteria, chitinase encoding 30 genes and eukaryotic-like proteins in the Cytophagia genome) while multidrug efflux pumps, 31 1 Current affiliation: Sorbonne Universités/CNRS, Station Biologique de Roscoff, UMR 8227, Integrative Biology of Marine Models, CS 90074, Roscoff, France also important in host-microbe interactions, were common across all genomes. Moreover, we 32 detected 76 secondary-metabolite biosynthetic gene clusters putatively involved in the 33 production of drug-like compounds or signalling molecules across all genomes, warranting 34 future biotechnologically driven research into their coding potential. 35 Keywords 36Porifera, genomics, host-microbe interactions, symbiosis, marine microbiology, secondary 37 metabolite 38

Marine Sponge and Octocoral-Associated Bacteria Show Versatile Secondary Metabolite Biosynthesis Potential and Antimicrobial Activities against Human Pathogens

“…This study was embedded in the national research project “SymbioReactor”, supported by the ‘Portuguese Ministry of the Sea’, and aimed to exploit the diversity, antimicrobial activities, and natural product biosynthesis capacities of two comprehensive culture collections, totaling 919 marine host-associated bacteria. The first “MicroEcoEvo” collection comprised 800 isolates and was established between 2010 and 2020 during multiple sampling events of the temperate marine sponges Sarcotragus spinosulus , Icrcina variabilis and Spongia officinalis [ 24 , 25 , 26 ], the octocorals Eunicella labiata [ 27 ] and Eunicella verrucosa [ 36 ] and fish larvae of Sparus aurata [ 28 , 29 , 88 ] from Portugal. Sampling and sample processing were previously described [ 24 , 25 , 26 , 27 , 28 , 29 , 88 ].…”

“…The first “MicroEcoEvo” collection comprised 800 isolates and was established between 2010 and 2020 during multiple sampling events of the temperate marine sponges Sarcotragus spinosulus , Icrcina variabilis and Spongia officinalis [ 24 , 25 , 26 ], the octocorals Eunicella labiata [ 27 ] and Eunicella verrucosa [ 36 ] and fish larvae of Sparus aurata [ 28 , 29 , 88 ] from Portugal. Sampling and sample processing were previously described [ 24 , 25 , 26 , 27 , 28 , 29 , 88 ]. Animal tissue was washed with artificial seawater to remove external bacterial colonizers after which the soft tissue was homogenized in sterile Ca 2 + and Mg 2 + -free artificial seawater (CMFASW: 27 g L −1 NaCl, 1 g L −1 Na 2 SO 4 , 0.8 g L −1 KCl and 0.18 g L −1 NaHCO 3 , 1 g of soft tissue per 9 mL CMFASW w/v) using a sterile mortar and pestle.…”

“…One milliliter of each homogenate was then serially diluted with CMFASW, and various dilutions were plated for cultivation. Several marine culture media and conditions were employed, including full strength Marine Agar [ 24 , 28 , 29 , 36 , 88 ] and half strength (1:2 diluted) Marine Agar (MA) [ 27 ], 50× diluted Marine Gellan Gum (MG50) [ 25 ] as well as R2A and VXA [ 26 ] media. Incubation temperatures ranged from 18 °C [ 27 ] to 25 °C [ 24 ] and incubation time from three days [ 24 ] to up to eight weeks [ 25 ] in the different studies (see Table S1 for more details on the isolates and culture conditions of the “MicroEcoEvo” collection).…”

“…Genomic DNA was extracted from the resulting cell pellets using the Wizard® Genomic DNA Purification Kit (Promega, Madison, USA) according to the manufacturer’s instructions. The Purified DNA from all isolates was subjected to 16S rRNA gene amplification by PCR and Sanger sequencing for identification as described in our previous studies [ 24 , 25 , 26 , 27 ]. The 16S rRNA gene sequences are deposited at NCBI GenBank, and sequences and accession numbers are available in Tables S1 and S2 .…”

“…Finally, we tested whether the bacterial isolates inhibit the growth of human pathogenic bacteria and fungi to select promising antimicrobial strains worth further investigation. The first collection, hereafter called the “MicroEcoEvo” collection, was established between 2010 and 2020 during multiple sampling events of temperate marine sponges [ 24 , 25 , 26 ], octocorals [ 27 ] and fish larvae [ 28 , 29 , 30 ] in Portugal, by employing various marine culture media and conditions. The second collection, hereafter called the “EcoTech-SPONGE” collection, comprises bacterial isolates from various tropical marine sponges collected off the coast of Taiwan in 2017 [ 31 , 32 ].…”