Exploring the Exopolysaccharide Production Potential of Bacterial Strains Isolated from Tunisian Blue Crab Portunus segnis Microbiota.

The blue crab (BC) Portunus segnis is considered an invasive species colonizing Tunisian coasts since 2014. This work aims to explore its associated bacteria potential to produce anionic exopolysaccharides (EPSs) in order to open up new ways of valorization. In this study, different BC samples were collected from the coastal area of Sfax, Tunisia. First, bacterial DNA was extracted from seven different fractions (flesh, gills, viscera, carapace scraping water, and three wastewaters from the production plant) and then sequenced using the metabarcoding approach targeting the V3-V4 region of the 16S rDNA to describe their microbiota composition. Metabarcoding data showed that the dominant bacterial genera were mainly Psychrobacter, Vagococcus, and Vibrio. In parallel, plate counting assays were performed on different culture media, and about 250 bacterial strains were isolated and identified by sequencing the 16S rDNA. EPS production by this new bacterial diversity was assessed to identify new compounds of biotechnological interest. The identification of the bacterial strains in the collection confirmed the dominance of Psychrobacter spp. strains. Among them, 43 were identified as EPS producers, as revealed by Stains-all dye in agarose gel electrophoresis. A Buttiauxella strain produced an EPS rich in both neutral sugars including rare sugars such as rhamnose and fucose and uronic acids. This original composition allows us to assume its potential for biotechnological applications and, more particularly, for developing innovative therapeutics. This study highlights bacterial strains associated with BC; they are a new untapped source for discovering innovative bioactive compounds for health and cosmetic applications, such as anionic EPS.

Screening of marine lactic acid bacteria for Vibrio parahaemolyticus inhibition and application to depuration in Pacific oysters (Crassostrea gigas).

AIMS: This study aims to assess the use of marine lactic acid bacteria (LAB) to reduce Vibrio parahaemolyticus levels during oyster depuration process. METHODS AND RESULTS: The inhibitory effect of 30 marine LAB strains against V. parahaemolyticus strains was evaluated by in vitro assays. A total of three positive strains (Latilactobacillus sakei SF1583, Lactococcus lactis SF1945, and Vagococcus fluvialis CD264) were selected for V. parahaemolyticus levels reduction during oyster depuration. Pacific oysters Crassostrea gigas were artificially and independently contaminated by four GFP-labelled V. parahaemolyticus strains (IFVp201, IFVp69, IFVp195, and LMG2850T) at 105 CFU ml-1 and then exposed by balneation to 106 CFU ml-1 of each LAB strains during 24 h, at 19°C. Quantification of V. parahaemolyticus in haemolymph by flow cytometry revealed variations in natural depuration of the different V. parahaemolyticus strains alone. Furthermore, the addition of LABs improved up to 1-log bacteria ml-1 the reduction of IFVp201 concentration in comparison to the control condition. CONCLUSIONS: Although further optimizations of procedure are needed, addition of marine LABs during oyster depuration may be an interesting strategy to reduce V. parahaemolyticus levels in Crassostrea gigas.

Marine versus Non-Marine Bacterial Exopolysaccharides and Their Skincare Applications.

Bacteria are well-known to synthesize high molecular weight polysaccharides excreted in extracellular domain, which constitute their protective microenvironment. Several bacterial exopolysaccharides (EPS) are commercially available for skincare applications in cosmetic products due to their unique structural features, conferring valuable biological and/or textural properties. This review aims to give an overview of bacterial EPS, an important group of macromolecules used in cosmetics as actives and functional ingredients. For this purpose, the main chemical characteristics of EPS are firstly described, followed by the basics of the development of cosmetic ingredients. Then, a focus on EPS production, including upstream and downstream processes, is provided. The diversity of EPS used in the cosmetic industry, and more specifically of marine-derived EPS is highlighted. Marine bacteria isolated from extreme environments are known to produce EPS. However, their production processes are highly challenging due to high or low temperatures; yield must be improved to reach economically viable ingredients. The biological properties of marine-derived EPS are then reviewed, resulting in the highlight of the challenges in this field.

NMR Analyses of the Enzymatic Degradation End-Products of Diabolican: The Secreted EPS of Vibrio diabolicus CNCM I-1629.

Diabolican, or HE800, is an exopolysaccharide secreted by the non-pathogenic Gram-negative marine bacterium Vibrio diabolicus (CNCM I-1629). This polysaccharide was enzymatically degraded by the Bacteroides cellulosilyticus WH2 hyaluronan lyase. The end products were purified by size-exclusion chromatography and their structures were analyzed in depth by nuclear magnetic resonance (NMR). The oligosaccharide structures confirmed the possible site of cleavage of the enzyme showing plasticity in the substrate recognitions. The production of glycosaminoglycan-mimetic oligosaccharides of defined molecular weight and structure opens new perspectives in the valorization of the marine polysaccharide diabolican.

Deep-sea Hydrothermal Vent Bacteria as a Source of Glycosaminoglycan-Mimetic Exopolysaccharides.

Bacteria have developed a unique strategy to survive in extreme environmental conditions through the synthesis of an extracellular polymeric matrix conferring upon the cells a protective microenvironment. The main structural component of this complex network constitutes high-molecular weight hydrophilic macromolecules, namely exopolysaccharides (EPS). EPS composition with the presence of particular chemical features may closely be related to the specific conditions in which bacteria evolve. Deep-sea hydrothermal vent bacteria have already been shown to produce EPS rich in hexosamines and uronic acids, frequently bearing some sulfate groups. Such a particular composition ensures interesting functional properties, including biological activities mimicking those known for glycosaminoglycans (GAG). The aim of the present study was to go further into the exploration of the deep-sea hydrothermal vent IFREMER (French Research Institute for Exploitation of the Sea) collection of bacteria to discover new strains able to excrete EPS endowed with GAG-like structural features. After the screening of our whole collection containing 692 strains, 38 bacteria have been selected for EPS production at the laboratory scale. EPS-producing strains were identified according to 16S rDNA phylogeny. Chemical characterization of the obtained EPS highlighted their high chemical diversity with the presence of atypical compositional patterns. These EPS constitute potential bioactives for a number of biomedical applications, including regenerative medicines and cancer treatment.

Characterization of New Oligosaccharides Obtained by An Enzymatic Cleavage of the Exopolysaccharide Produced by the Deep-Sea Bacterium Alteromonas infernus Using its Cell Extract.

Bacteria from deep-sea hydrothermal vents constitute an attractive source of bioactive molecules. In particular, exopolysaccharides (EPS) produced by these bacteria become a renewable source of both biocompatible and biodegradable molecules. The low molecular weight (LMW) derivatives of the GY785 EPS produced by the deep-sea hydrothermal vent strain Alteromonas infernus have previously displayed some biological properties, similar to those of glycosaminoglycans (GAG), explored in cancer and tissue engineering. These GAG-mimetic derivatives are obtained through a free radical depolymerization process, which could, however, affect their structural integrity. In a previous study, we have shown that A. infernus produces depolymerizing enzymes active on its own EPS. In the present study, an enzymatic reaction was optimized to generate LMW derivatives of the GY785 EPS, which could advantageously replace the present bioactive derivatives obtained by a chemical process. Analysis by mass spectrometry of the oligosaccharide fractions released after enzymatic treatment revealed that mainly a lyase activity was responsible for the polysaccharide depolymerization. The repeating unit of the GY785 EPS produced by enzyme cleavage was then fully characterized.

Purification of the exopolysaccharide produced by Alteromonas infernus: identification of endotoxins and effective process to remove them.

Alteromonas infernus bacterium isolated from deep-sea hydrothermal vents can produce by fermentation a high molecular weight exopolysaccharide (EPS) called GY785. This EPS described as a new source of glycosaminoglycan-like molecule presents a great potential for pharmaceutical and biotechnological applications. However, this unusual EPS is secreted by a Gram-negative bacterium and can be therefore contaminated by endotoxins, in particular the lipopolysaccharides (LPS). Biochemical and chemical analyses of the LPS extracted from A. infernus membranes have shown the lack of the typical LPS architecture since 3-deoxy-D-manno-oct-2-ulopyranosonic acid (Kdo), glucosamine (GlcN), and phosphorylated monosaccharides were not present. Unlike for other Gram-negative bacteria, the results revealed that the outer membrane of A. infernus bacterium is most likely composed of peculiar glycolipids. Furthermore, the presence of these glycolipids was also detected in the EPS batches produced by fermentation. Different purification and chemical detoxification methods were evaluated to efficiently purify the EPS. Only the method based on a differential solubility of EPS and glycolipids in deoxycholate detergent showed the highest decrease in the endotoxin content. In contrast to the other tested methods, this new protocol can provide an effective method for obtaining endotoxin-free EPS without any important modification of its molecular weight, monosaccharide composition, and sulfate content.

Genome sequence of Vibrio diabolicus and identification of the exopolysaccharide HE800 biosynthesis locus.

Vibrio diabolicus, a marine bacterium originating from deep-sea hydrothermal vents, produces the HE800 exopolysaccharide with high value for biotechnological purposes, especially for human health. Its genome was sequenced and analyzed; phylogenetic analysis using the core genome revealed V. diabolicus is close to another deep-sea Vibrio sp. (Ex25) within the Harveyi clade and Alginolyticus group. A genetic locus homologous to the syp cluster from Vibrio fischeri was demonstrated to be involved in the HE800 production. However, few genetic particularities suggest that the regulation of syp expression may be different in V. diabolicus. The presence of several types of glycosyltransferases within the locus indicates a capacity to generate diversity in the glycosidic structure, which may confer an adaptability to environmental conditions. These results contribute to better understanding exopolysaccharide biosynthesis and for developing new efficient processes to produce this molecule for biotechnological applications.

Virulence phenotypes differ between toxigenic Vibrio parahaemolyticus isolated from western coasts of Europe.

Vibrio parahaemolyticus is the leading bacterial cause of gastroenteritis associated with seafood consumption worldwide. Not all members of the species are thought to be pathogenic, thus identification of virulent organisms is essential to protect public health and the seafood industry. Correlations of human disease and known genetic markers (e.g. thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH)) appear complex. Some isolates recovered from patients lack these factors, while their presence has become increasingly noted in isolates recovered from the environment. Here, we used whole-genome sequencing in combination with mammalian and insect models of infection to assess the pathogenic potential of V. parahaemolyticus isolated from European Atlantic shellfish production areas. We found environmental V. parahaemolyticus isolates harboured multiple virulence-associated genes, including TDH and/or TRH. However, carriage of these factors did not necessarily reflect virulence in the mammalian intestine, as an isolate containing TDH and the genes coding for a type 3 secretion system (T3SS) 2α virulence determinant, appeared avirulent. Moreover, environmental V. parahaemolyticus lacking TDH or TRH could be assigned to groups causing low and high levels of mortality in insect larvae, with experiments using defined bacterial mutants showing that a functional T3SS1 contributed to larval death. When taken together, our findings highlight the genetic diversity of V. parahaemolyticus isolates found in the environment, their potential to cause disease and the need for a more systematic evaluation of virulence in diverse V. parahaemolyticus to allow better genetic markers.

Screening of enzymatic activities for the depolymerisation of the marine bacterial exopolysaccharide HE800.

The exopolysaccharide (EPS) HE800 is a marine-derived polysaccharide (from 8 × 10(5) to 1.5 × 10(6) g mol(-1)) produced by Vibrio diabolicus and displaying original structural features close to those of glycosaminoglycans. In order to confer new biological activities to the EPS HE800 or to improve them, structural modifications need to be performed. In particular, depolymerisation is required to generate low-molecular-weight derivatives. To circumvent the use of chemical methods that lack specificity and reproducibility, enzymes able to perform such reaction are sought. This study reports the screening for enzymes capable of depolymerising the EPS HE800. A large diversity of enzyme sources has been studied: commercially available glycoside hydrolases with broad substrate specificity, lyases, and proteases as well as growing microorganisms. Interestingly, we found that the genus Enterococcus and, more particularly, the strain Enterococcus faecalis were able to depolymerise the EPS HE800. Partial characterization of the enzymatic activity gives evidence for a random and incomplete depolymerisation pattern that yields low-molecular-weight products of 40,000 g mol(-1). Genomic analysis and activity assays allowed the identification of a relevant open reading frame (ORF) which encodes an endo-N-acetyl-galactosaminidase. This study establishes the foundation for the development of an enzymatic depolymerisation process.

Influence of the Carbon and Nitrogen Sources on Diabolican Production by the Marine Vibrio diabolicus Strain CNCM I-1629.

Recent advances in glycobiotechnology show that bacterial exopolysaccharides (EPS) presenting glycosaminoglycan (GAG)-like properties can provide a valuable source of bio-active macromolecules for industrial applications. The HE800 EPS, named diabolican, is a marine-derived anionic high-molecular-weight polysaccharide produced by Vibrio diabolicus CNCM I-1629 which displays original structural features close to those of hyaluronic acid. We investigated the impact of carbon and nitrogen substrates on both Vibrio diabolicus growth and diabolican production. Both substrates were screened by a one-factor-at-a-time method, and experimental designs were used to study the effect of glucose, mannitol, and ammonium acetate various concentrations. Results showed that the medium composition affected not only the bacterium growth and EPS yield, but also the EPS molecular weight (MW). EPS yields of 563 and 330 mg L-1 were obtained in the presence of 69.3 g L-1 glucose and 24.6 g L-1 mannitol, respectively, both for 116.6 mM ammonium acetate. MW was the highest, with 69.3 g L-1 glucose and 101.9 mM ammonium acetate (2.3 × 106 g mol-1). In parallel, the bacterial maximum specific growth rate was higher when both carbon and nitrogen substrate concentrations were low. This work paves the way for the optimization of marine exopolysaccharide production of great interest in the fields of human health and cosmetics.

Marine polysaccharides: a source of bioactive molecules for cell therapy and tissue engineering.

The therapeutic potential of natural bioactive compounds such as polysaccharides, especially glycosaminoglycans, is now well documented, and this activity combined with natural biodiversity will allow the development of a new generation of therapeutics. Advances in our understanding of the biosynthesis, structure and function of complex glycans from mammalian origin have shown the crucial role of this class of molecules to modulate disease processes and the importance of a deeper knowledge of structure-activity relationships. Marine environment offers a tremendous biodiversity and original polysaccharides have been discovered presenting a great chemical diversity that is largely species specific. The study of the biological properties of the polysaccharides from marine eukaryotes and marine prokaryotes revealed that the polysaccharides from the marine environment could provide a valid alternative to traditional polysaccharides such as glycosaminoglycans. Marine polysaccharides present a real potential for natural product drug discovery and for the delivery of new marine derived products for therapeutic applications.

Genomic diversity of Serratia proteamaculans and Serratia liquefaciens predominant in seafood products and spoilage potential analyses.

Serratia sp. cause food losses and waste due to spoilage; it is noteworthy that they represent a dominant population in seafood. The main spoilage associated species comprise S. liquefaciens, S. grimesii, S. proteamaculans and S. quinivorans, also known as S. liquefaciens-like strains. These species are difficult to discriminate since classical 16S rRNA gene-based sequences do not possess sufficient resolution. In this study, a phylogeny based on the short-length luxS gene was able to speciate 47 Serratia isolates from seafood, with S. proteamaculans being the main species from fresh salmon and tuna, cold-smoked salmon, and cooked shrimp while S. liquefaciens was only found in cold-smoked salmon. The genome of the first S. proteamaculans strain isolated from the seafood matrix (CD3406 strain) was sequenced. Pangenome analyses of S. proteamaculans and S. liquefaciens indicated high adaptation potential. Biosynthetic pathways involved in antimicrobial compounds production and in the main seafood spoilage compounds were also identified. The genetic equipment highlighted in this study contributed to gain further insights into the predominance of Serratia in seafood products and their capacity to spoil.

Bacteria enhance the production of extracellular polymeric substances by the green dinoflagellate Lepidodinium chlorophorum.

High biomasses of the marine dinoflagellate Lepidodinium chlorophorum cause green seawater discolorations along Southern Brittany (NE Atlantic, France). The viscosity associated to these phenomena has been related to problems in oyster cultivation. The harmful effect of L. chlorophorum might originate from the secretion of Extracellular Polymeric Substances (EPS). To understand whether the EPS are produced by L. chlorophorum or its associated bacteria, or if they are a product of their interaction, batch cultures were performed under non-axenic and pseudo-axenic conditions for three strains. Maximum dinoflagellate cell abundances were observed in pseudo-axenic cultures. The non-sinking fraction of polymers (Soluble Extracellular Polymers, SEP), mainly composed of proteins and the exopolysaccharide sulphated galactan, slightly increased in pseudo-axenic cultures. The amount of Transparent Exopolymer Particles (TEP) per cell increased under non-axenic conditions. Despite the high concentrations of Particulate Organic Carbon (POC) measured, viscosity did not vary. These results suggest that the L. chlorophorum-bacteria interaction could have a detrimental consequence on the dinoflagellate, translating in a negative effect on L. chlorophorum growth, as well as EPS overproduction by the dinoflagellate, at concentrations that should not affect seawater viscosity.

Draft-genome sequence data and phylogenomic comparison of two marine-sourced bacterial strains Pseudoalteromonas sp. MIP2626 and Psychrobacter sp. BI730.

Halophilic and psychrophilic marine bacteria are source of interesting bioactive molecules for biotechnology. We report here the whole-genome sequences of two of them, Pseudoalteromonas sp. MIP2626 isolated from tropical peeled shrimps and Psychrobacter sp. BI730 isolated from deep-sea hydrothermal vent. Sequencing of both genomes was performed by Illumina HiSeq platform (2 × 150 pb). De novo assemblies using Spades v3.9 generated 136 contigs for Pseudoalteromonas MIP2626 and 42 contigs for Psychrobacter BI730, representing a genome size of 3.9 Mb and 3.2 Mb, respectively. Phylogenetic based on 16S rRNA gene sequence and phylogenomic analyses were reported to compare the new sequences with Pseudoalteromonas and Psychrobacter representative strains available in the public databases. The genome sequences have been deposited at GenBank under the accession numbers JAATTW000000000 for Pseudoalteromonas sp. MIP2626 and JAATTV000000000 for Psychrobacter sp. BI730.

New Insight into Antimicrobial Compounds from Food and Marine-Sourced Carnobacterium Species through Phenotype and Genome Analyses.

Carnobacterium maltaromaticum and Carnobacterium divergens, isolated from food products, are lactic acid bacteria known to produce active and efficient bacteriocins. Other species, particularly those originating from marine sources, are less studied. The aim of the study is to select promising strains with antimicrobial potential by combining genomic and phenotypic approaches on large datasets comprising 12 Carnobacterium species. The biosynthetic gene cluster (BGCs) diversity of 39 publicly available Carnobacterium spp. genomes revealed 67 BGCs, distributed according to the species and ecological niches. From zero to six BGCs were predicted per strain and classified into four classes: terpene, NRPS (non-ribosomal peptide synthetase), NRPS-PKS (hybrid non-ribosomal peptide synthetase-polyketide synthase), RiPP (ribosomally synthesized and post-translationally modified peptide). In parallel, the antimicrobial activity of 260 strains from seafood products was evaluated. Among the 60% of active strains, three genomes were sequenced and submitted to a dereplication process. C. inhibens MIP2551 produced a high amountof H2O2, probably thanks to the presence of four oxidase-encoding genes. C. maltaromaticum EBP3019 and SF668 strains were highly efficient against Listeria monocytogenes. A new extracellular 16 kDa unmodified bacteriocin in the EBP3019 strain and five different bacteriocins in SF668 were highlighted. In this study, the overview of antimicrobial BGC and inhibitory activities of Carnobacterium spp. allowed the prediction of potential innovative natural products that could be relevant for biotechnological applications.

Comparative genomics reveals a widespread distribution of an exopolysaccharide biosynthesis gene cluster among Vibrionaceae.

OBJECTIVES: The eps locus in Vibrio diabolicus is involved in the production of the biotechnologically valuable HE800 EPS. In this study, the distribution and diversity of similar eps gene clusters across Vibrionaceae and its variability in relation to phylogenetic relationship were investigated. The aim was to provide a better knowledge of the eps gene cluster importance and to facilitate discovery of new EPS with potent interesting bioactivities. RESULTS: Seventy percent of the 103 genome sequences examined display such an eps locus with a high level of synteny. However, genetic divergence was found inside some monophyletic clades or even between some strains of the same species. It includes gene insertions, truncations, and deletions. Comparative analysis also reveals some variations in glycosyltransferase and export systems genes. Phylogenetic analysis of the Vibrionaceae eps gene clusters within Vibrionaceae suggests a vertical transfer by speciation but also pinpoints rearrangement events independent of the speciation.

Enzymatic depolymerization of the GY785 exopolysaccharide produced by the deep-sea hydrothermal bacterium Alteromonas infernus: Structural study and enzyme activity assessment.

Polysaccharides have attracted much attention due to their interesting physico-chemical and also biological properties that are explored in food, cosmetic and pharmaceutical industries. GY785 exopolysaccharide (EPS) presenting an unusual structure is secreted by the deep-sea hydrothermal bacterium, Alteromonas infernus. Low-molecular weight (LMW) derivatives obtained by chemical depolymerization of the native high molecular weight (HMW) EPS were previously shown to exhibit biological properties similar to glycosaminoglycans (GAG). In the present study, in order to generate well defined derivatives with a better control of the depolymerization, an enzymatic approach was applied for the first time. Various commercially available enzymes were firstly screened for their depolymerizing activities, however none of them was able to degrade the polysaccharide. Enzymatic assays performed with A. infernus protein extracts have shown that the bacterium produces by itself endogenous enzymes able to depolymerize its own EPS. The oligosaccharides released by the enzymes were analyzed and their structures allowed to assess that the protein extract contains several depolymerizing activities.

Bioprospecting for Exopolysaccharides from Deep-Sea Hydrothermal Vent Bacteria: Relationship between Bacterial Diversity and Chemical Diversity.

Many bacteria biosynthesize structurally diverse exopolysaccharides (EPS) and excrete them into their surrounding environment. The EPS functional features have found many applications in industries such as cosmetics and pharmaceutics. In particular, some EPS produced by marine bacteria are composed of uronic acids, neutral sugars, and N-acetylhexosamines, and may also bear some functional sulfate groups. This suggests that they can share common structural features with glycosaminoglycans (GAG) like the two EPS (HE800 and GY785) originating from the deep sea. In an attempt to discover new EPS that may be promising candidates as GAG-mimetics, fifty-one marine bacterial strains originating from deep-sea hydrothermal vents were screened. The analysis of the EPS chemical structure in relation to bacterial species showed that Vibrio, Alteromonas, and Pseudoalteromonas strains were the main producers. Moreover, they produced EPS with distinct structural features, which might be useful for targeting marine bacteria that could possibly produce structurally GAG-mimetic EPS.

Complete Genome Sequence of Lactococcus piscium CNCM I-4031, a Bioprotective Strain for Seafood Products.

Lactococcus piscium CNCM I-4031 is a psychotrophic foodborne lactic acid bacterium showing potential interest for the biopreservation of seafood products due to its inhibition properties toward pathogenic and spoilage bacteria. The analysis of its genome will provide a better understanding of the mechanisms of interaction between these bacteria.