Impact of hydrodynamics on community structure and metabolic production of marine biofouling formed in a highly energetic estuary.

Biofouling is a specific lifestyle including both marine prokaryotic and eukaryotic communities. Hydrodynamics are poorly studied parameters affecting biofouling formation. This study aimed to investigate how water dynamics in the Etel Estuary (Northwest Atlantic coasts of France) influences the colonization of artificial substrates. Hydrodynamic conditions, mainly identified as shear stress, were characterized by measuring current velocity, turbulence intensity and energy using Acoustic Doppler Current Profiler (ADCP). One-month biofouling was analyzed by coupling metabarcoding (16S rRNA, 18S rRNA and COI genes), untargeted metabolomics (liquid chromatography coupled with high-resolution mass spectrometry, LC-HRMS) and characterization of the main biochemical components of the microbial exopolymeric matrix. A higher richness was observed for biofouling communities (prokaryotes and eukaryotes) exposed to the strongest currents. Ectopleura (Cnidaria) and its putative symbionts Endozoicomonas (Gammaproteobacteria) were dominant in the less dynamic conditions. Eukaryotes assemblages were specifically shaped by shear stress, leading to drastic changes in metabolite profiles. Under high hydrodynamic conditions, the exopolymeric matrix increased and was composed of 6 times more polysaccharides than proteins, these latter playing a crucial role in the adhesion and cohesion properties of biofilms. This original multidisciplinary approach demonstrated the importance of shear stress on both the structure of marine biofouling and the metabolic response of these complex communities.

Impact of phosphate concentration on the metabolome of biofilms of the marine bacterium Pseudoalteromonas lipolytica.

INTRODUCTION: Marine biofilms are the most widely distributed mode of life on Earth and drive biogeochemical cycling processes of most elements. Phosphorus (P) is essential for many biological processes such as energy transfer mechanisms, biological information storage and membrane integrity. OBJECTIVES: Our aim was to analyze the effect of a gradient of ecologically relevant phosphate concentrations on the biofilm-forming capacity and the metabolome of the marine bacterium Pseudoalteromonas lipolytica TC8. METHODS: In addition to the evaluation of the effect of different phosphate concentration on the biomass, structure and gross biochemical composition of biofilms of P. lipolytica TC8, untargeted metabolomics based on liquid chromatography-mass spectrometry (LC-MS) analysis was used to determine the main metabolites impacted by P-limiting conditions. Annotation of the most discriminating and statistically robust metabolites was performed through the concomitant use of molecular networking and MS/MS fragmentation pattern interpretation. RESULTS: At the lowest phosphate concentration, biomass, carbohydrate content and three-dimensional structures of biofilms tended to decrease. Furthermore, untargeted metabolomics allowed for the discrimination of the biofilm samples obtained at the five phosphate concentrations and the highlighting of a panel of metabolites mainly implied in such a discrimination. A large part of the metabolites of the resulting dataset were then putatively annotated. Ornithine lipids were found in increasing quantity when the phosphate concentration decreased, while the opposite trend was observed for oxidized phosphatidylethanolamines (PEs). CONCLUSION: This study demonstrated the suitability of LC-MS-based untargeted metabolomics for evaluating the effect of culture conditions on marine bacterial biofilms. More precisely, these results supported the high plasticity of the membrane of P. lipolytica TC8, while the role of the oxidized PEs remains to be clarified.

Disrupting quorum sensing alters social interactions in Chromobacterium violaceum.

Quorum sensing (QS) is a communication system used by bacteria to coordinate a wide panel of biological functions in a cell density-dependent manner. The Gram-negative Chromobacterium violaceum has previously been shown to use an acyl-homoserine lactone (AHL)-based QS to regulate various behaviors, including the production of proteases, hydrogen cyanide, or antimicrobial compounds such as violacein. By using combined metabolomic and proteomic approaches, we demonstrated that QS modulates the production of antimicrobial and toxic compounds in C. violaceum ATCC 12472. We provided the first evidence of anisomycin antibiotic production by this strain as well as evidence of its regulation by QS and identified new AHLs produced by C. violaceum ATCC 12472. Furthermore, we demonstrated that targeting AHLs with lactonase leads to major QS disruption yielding significant molecular and phenotypic changes. These modifications resulted in drastic changes in social interactions between C. violaceum and a Gram-positive bacterium (Bacillus cereus), a yeast (Saccharomyces cerevisiae), immune cells (murine macrophages), and an animal model (planarian Schmidtea mediterranea). These results underscored that AHL-based QS plays a key role in the capacity of C. violaceum to interact with micro- and macroorganisms and that quorum quenching can affect microbial population dynamics beyond AHL-producing bacteria and Gram-negative bacteria.

Metabolomic and proteomic changes induced by growth inhibitory concentrations of copper in the biofilm-forming marine bacterium Pseudoalteromonas lipolytica.

Copper is an essential element for living cells but this metal is present in some marine environments at such high concentrations that it can be toxic for numerous organisms. In polluted areas, marine organisms may develop specific adaptive responses to prevent cell damage. To investigate the influence of copper on the metabolism of a single organism, a dual approach combining metabolomics and proteomics was undertaken on the biofilm-forming bacterial strain Pseudoalteromonas lipolytica TC8. In order to highlight differential adaptation according to the phenotype, the response of P. lipolytica TC8 to copper stress was studied in planktonic and biofilm culture modes under growth inhibitory copper concentrations. As expected, copper exposure led to the induction of defense and detoxification mechanisms. Specific metabolite and protein profiles were thus observed in each condition (planktonic vs. biofilm and control vs. copper-treated cultures). Copper exposure seems to induce drastic changes in the lipid composition of the bacterial cell membrane and to modulate the abundance of proteins functionally known to be involved in copper cell homeostasis in both planktonic and biofilm culture modes. Much more proteins differentially expressed after copper treatment were observed in biofilms than in planktonic cells, which could indicate a more heterogeneous response of biofilm cells to this metallic stress.

Metabolome and proteome changes between biofilm and planktonic phenotypes of the marine bacterium Pseudoalteromonas lipolytica TC8.

A number of bacteria adopt various lifestyles such as planktonic free-living or sessile biofilm stages. This enables their survival and development in a wide range of contrasting environments. With the aim of highlighting specific metabolic shifts between these phenotypes and to improve the overall understanding of marine bacterial adhesion, a dual metabolomics/proteomics approach was applied to planktonic and biofilm cultures of the marine bacterium Pseudoalteromonas lipolytica TC8. The liquid chromatography mass spectrometry (LC-MS) based metabolomics study indicated that membrane lipid composition was highly affected by the culture mode: phosphatidylethanolamine (PEs) derivatives were over-produced in sessile cultures while ornithine lipids (OLs) were more specifically synthesized in planktonic samples. In parallel, differences between proteomes revealed that peptidases, oxidases, transcription factors, membrane proteins and the enzymes involved in histidine biosynthesis were over-expressed in biofilms while proteins involved in heme production, nutrient assimilation, cell division and arginine/ornithine biosynthesis were specifically up-regulated in free-living cells.

Discrimination of Four Marine Biofilm-Forming Bacteria by LC-MS Metabolomics and Influence of Culture Parameters.

Most marine bacteria can form biofilms, and they are the main components of biofilms observed on marine surfaces. Biofilms constitute a widespread life strategy, as growing in such structures offers many important biological benefits. The molecular compounds expressed in biofilms and, more generally, the metabolomes of marine bacteria remain poorly studied. In this context, a nontargeted LC-MS metabolomics approach of marine biofilm-forming bacterial strains was developed. Four marine bacteria, Persicivirga (Nonlabens) mediterranea TC4 and TC7, Pseudoalteromonas lipolytica TC8, and Shewanella sp. TC11, were used as model organisms. The main objective was to search for some strain-specific bacterial metabolites and to determine how culture parameters (culture medium, growth phase, and mode of culture) may affect the cellular metabolism of each strain and thus the global interstrain metabolic discrimination. LC-MS profiling and statistical partial least-squares discriminant analyses showed that the four strains could be differentiated at the species level whatever the medium, the growth phase, or the mode of culture (planktonic vs biofilm). A MS/MS molecular network was subsequently built and allowed the identification of putative bacterial biomarkers. TC8 was discriminated by a series of ornithine lipids, while the P. mediterranea strains produced hydroxylated ornithine and glycine lipids. Among the P. mediterranea strains, TC7 extracts were distinguished by the occurrence of diamine derivatives, such as putrescine amides.

Characterization and anti-biofilm activity of extracellular polymeric substances produced by the marine biofilm-forming bacterium Pseudoalteromonas ulvae strain TC14.

This study investigated soluble (Sol-EPS), loosely bound (LB-EPS), and tightly bound extracellular polymeric substances (TB-EPS) harvested from biofilm and planktonic cultures of the marine bacterium Pseudoalteromonas ulvae TC14. The aim of the characterization (colorimetric methods, FTIR, GC-MS, NMR, HPGPC, and AFM analyses) was to identify new anti-biofilm compounds; activity was assessed using the BioFilm Ring Test®. A step-wise separation of EPS was designed, based on differences in water-solubility and acidity. An acidic fraction was isolated from TB-EPS, which strongly inhibited biofilm formation by marine bacterial strains in a concentration-dependent manner. The main constituents of this fraction were characterized as two glucan-like polysaccharides. An active poly(glutamyl-glutamate) fraction was also recovered from TB-EPS. The distribution of these key EPS components in Sol-EPS, LB-EPS, and TB-EPS was distinct and differed quantitatively in biofilm vs planktonic cultures. The anti-biofilm potential of the fractions emphasizes the putative antifouling role of EPS in the environment.

Modulation of violacein production and phenotypes associated with biofilm by exogenous quorum sensing N-acylhomoserine lactones in the marine bacterium Pseudoalteromonas ulvae TC14.

Various phenotypes ranging from biofilm formation to pigment production have been shown to be regulated by quorum sensing (QS) in many bacteria. However, studies of the regulation of pigments produced by marine bacteria in saline conditions and of biofilm-associated phenotypes are scarcer. This study focuses on the demonstration of the existence of a QS communication system involving N-acylhomoserine lactones (AHLs) in the Mediterranean Sea strain Pseudoalteromonas ulvae TC14. We have investigated whether TC14 produces the violacein pigment, and whether intrinsic or exogenous AHLs could influence its production and modulate biofilm-associated phenotypes. Here, we demonstrate that the purple pigment produced by TC14 is violacein. The study shows that in planktonic conditions, TC14 produces more pigment in the medium in which it grows less. Using different approaches, the results also show that TC14 does not produce intrinsic AHLs in our conditions. When exogenous AHLs are added in planktonic conditions, the production of violacein is upregulated by C6-, C12-, 3-oxo-C8 and 3-oxo-C12-HSLs (homoserine lactones), and downregulated by 3-oxo-C6-HSL. In sessile conditions, 3-oxo-C8-HSL upregulates the production of violacein. The study of the biofilm-associated phenotypes shows that oxo-derived-HSLs decrease adhesion, swimming and biofilm formation. While 3-oxo-C8 and 3-oxo-C12-HSLs decrease both swimming and adhesion, 3-oxo-C6-HSLs decrease not only violacein production in planktonic conditions but also swimming, adhesion and more subtly biofilm formation. Therefore, TC14 may possess a functional LuxR-type QS receptor capable of sensing extrinsic AHLs, which controls violacein production, motility, adhesion and biofilm formation.

Cystophloroketals A-E, Unusual Phloroglucinol-Meroterpenoid Hybrids from the Brown Alga Cystoseira tamariscifolia.

Cystophloroketals A-E (1-5), five new phloroglucinol-meroditerpenoid hybrids, have been isolated together with their putative biosynthetic precursor, the monocyclic meroditerpenoid 6, from the Mediterranean brown alga Cystoseira tamariscifolia. They represent the first examples of meroditerpenoids linked to a phloroglucinol through a 2,7-dioxabicyclo[3.2.1]octane moiety. The chemical structures, including absolute configurations, were elucidated on the basis of extensive spectroscopic analysis (HR-ESIMS, 1D and 2D NMR, and ECD) and TDDFT ECD calculations. Compounds 1-6 were tested for their antifouling activity against several marine colonizing species (bacteria, fungi, invertebrates, micro- and macroalgae). Compound 6 showed high potency for the inhibition of macrofoulers (invertebrates and macroalgae), while cystophloroketals B (2) and D (4) displayed strong inhibition of the germination of the two macroalgae tested and moderate antimicrobial activities (bacteria, microalgae, and fungi).

Extraction, Purification, and NMR Analysis of Terpenes from Brown Algae.

Algal terpenes constitute a wide and well-documented group of marine natural products with structures differing from their terrestrial plant biosynthetic analogues. Amongst macroalgae, brown seaweeds are considered as one of the richest source of biologically and ecologically relevant terpenoids. These metabolites, mostly encountered in algae of the class Phaeophyceae, are mainly diterpenes and meroditerpenes (metabolites of mixed biogenesis characterized by a toluquinol or a toluquinone nucleus linked to a diterpene moiety).In this chapter, we describe analytical processes commonly employed for the isolation and structural characterization of the main terpenoid constituents obtained from organic extracts of brown algae. The successive steps include (1) extraction of lipidic content from algal samples; (2) purification of terpenes by column chromatography and semi-preparative high-performance liquid chromatography; and (3) structure elucidation of the isolated terpenes by means of 1D and 2D nuclear magnetic resonance (NMR). More precisely, we propose a representative methodology which allows the isolation and structural determination of the monocyclic meroditerpene methoxybifurcarenone (MBFC) from the Mediterranean brown alga Cystoseira amentacea var. stricta. This methodology has a large field of applications and can then be extended to terpenes isolated from other species of the family Sargassaceae.

Identification of bacterial strains isolated from the Mediterranean Sea exhibiting different abilities of biofilm formation.

The Mediterranean Sea has rarely been investigated for the characterization of marine bacteria as compared to other marine environments such as the Atlantic or Pacific Ocean. Bacteria recovered from inert surfaces are poorly studied in these environments, when it has been shown that the community structure of attached bacteria can be dissimilar from that of planktonic bacteria present in the water column. The objectives of this study were to identify and characterize marine bacteria isolated from biofilms developed on inert surfaces immersed in the Mediterranean Sea and to evaluate their capacity to form a biofilm in vitro. Here, 13 marine bacterial strains have been isolated from different supports immersed in seawater in the Bay of Toulon (France). Phylogenetic analysis and different biological and physico-chemical properties have been investigated. Among the 13 strains recovered, 8 different genera and 12 different species were identified including 2 isolates of a novel bacterial species that we named Persicivirga mediterranea and whose genus had never been isolated from the Mediterranean Sea. Shewanella sp. and Pseudoalteromonas sp. were the most preponderant genera recovered in our conditions. The phenotypical characterization revealed that one isolate belonging to the Polaribacter genus differed from all the other ones by its hydrophobic properties and poor ability to form biofilms in vitro. Identifying and characterizing species isolated from seawater including from Mediterranean ecosystems could be helpful for example, to understand some aspects of bacterial biodiversity and to further study the mechanisms of biofilm (and biofouling) development in conditions approaching those of the marine environment.

Correlation between synthesis variation of 2-alkylquinolones and the antifungal activity of a Burkholderia cepacia strain collection.

Twenty epiphytic and rhizospheric bacterial strains harbouring strong antifungal activities were isolated from the Tunisian environment. This group of bacteria was identified as Burkholderia cepacia genomovar I using 16S rDNA and recA fragment gene sequence analyses for two selected strains and RFLP technique for the eighteen other ones. This identification did not show variability between isolates despite the significant differences in the antifungal activities of their culture supernatant and the organic crude extract against Aspergillus niger and other phytopathogenic fungi. Chromatographic and mass spectrometric analyses of these extracts allowed us to confirm the difference between strains of the group. Their metabolic production showed differences in term of contents and quantities of secreted molecules, particularly those which were identified to be involved in the antifungal activities. Two metabolites, named Bc-255 and Bc-257 secreted by the entire group at different amounts, have been purified and tested separately against A. niger. Bc-255 showed an activity twice as high as those shown by Bc-257. The structural characterization of these two compounds by mass spectrometry and nuclear magnetic resonance spectroscopy allowed their identification as two analogous 2-alkylquinolones with only one difference at the alkyl chain.

Environmental Burkholderia cepacia strain Cs5 acting by two analogous alkyl-quinolones and a didecyl-phthalate against a broad spectrum of phytopathogens fungi.

An environmental Burkholderia cepacia strain named Cs5 was isolated and identified first using API biochemical identification system and then with 16S rDNA and recA sequence homology search. This bacterium exhibited a broad spectrum of fungicidal activities against Alternaria alternata, Aspergillus niger, Fusarium culmorum, F. graminearum, F. oxysporum and Rhizoctonia solani. In the liquid conditions, the MIC of A. niger and R. solani were reached with, respectively, 1.25-2% of the Cs5 liquid culture supernatant. However, in the solid conditions, the same inhibition was caused in the presence of 3% of the Cs5 supernatant. The exhibition of these two fungi at low concentrations of supernatant Cs5 caused various morphological changes of their mycelia which were observed by confocal microscopy. Three antifungal compounds, named Cs5-255, Cs5-257 and Cs5-446, were purified from the Cs5 culture. The structural analysis of these molecules showed that Cs5-255 and Cs5-257 are analogous and belonged to the alkyl-quinolone family, while Cs5-446 was a didecyl-phthalate, isolated for the first time from a bacterium.

Antifungal activities of an endophytic Pseudomonas fluorescens strain Pf1TZ harbouring genes from pyoluteorin and phenazine clusters.

Pseudomonas fluorescens Pf1TZ was inhibitory in vitro to a number of phytopathogenic fungi and could protect vine plantlets against Botrytis cinerea. Total protection was reached after 3 weeks of bacterial inoculation. The endophytism of Pf1TZ was confirmed by confocal microscopy using its inherent fluorescence. The molecular characterization of Pf1TZ indicated the presence of genes from clusters encoding pyoluteorin and phenazine. The chromatographic purification and LC-MS(n) analysis revealed that the most active one has a molecular mass of 504 Da.

Diterpenoids from the Mediterranean brown alga Dictyota sp. evaluated as antifouling substances against a marine bacterial biofilm.

Four new cyclized diterpenes, one xenicane (1) and three dolabellanes (2-4), were isolated, along with seven previously reported metabolites [3beta-hydroxydilophol (5), dictyols E (6) and C (7), hydroxycrenulide (8), 9-acetoxy-15-hydroxy-1,6-dollabelladiene (9), hydroxyacetyldictyolal (10), and fucoxanthin], from a Mediterranean species of Dictyota sp. collected in Le Brusc Lagoon (French Mediterranean coast). Their structures, as well as their relative configurations, were determined through extensive spectrometric (IR, HRESIMS, 1D and 2D NMR) data analysis and molecular modeling studies and by comparison with those reported in literature. Some of the isolated metabolites were evaluated for their antiadhesion activity against a marine bacterial biofilm (Pseudoalteromonas sp. D41).

Meroditerpenoids and derivatives from the brown alga Cystoseira baccata and their antifouling properties.

The brown alga Cystoseira baccata harvested along the Atlantic coasts of Morocco yielded seven new meroditerpenoids (1-4) and derivatives (5-7), whose chemical structures were elucidated mainly by 2D NMR and mass spectrometry. Surprisingly, for all these compounds, which possess a bicyclo[4.3.0]nonane ring system, a trans fusion of the bicyclic system was deduced by stereochemical studies even though such compounds isolated from Cystoseira species are known to have a typical cis orientation for the bridgehead methyls. The antifouling and antibacterial activities of compounds 1-5 and 7 were evaluated, as well as their toxicity toward nontarget species. Compounds 4, 5, and 7 showed antifouling activities against growth of microalgae, macroalgal settlement, and mussel phenoloxidase activity, while being nontoxic to larvae of sea urchins and oysters.

Antifouling activity of meroditerpenoids from the marine brown alga Halidrys siliquosa.

Nine tetraprenyltoluquinol-related metabolites (1-9) have been isolated from the organic extract of the temperate brown alga Halidrys siliquosa that exhibits antifouling properties. The planar structure of compounds 1-9 was elucidated on the basis of extensive spectroscopic analysis and by comparison of the data with those of related metabolites. Antifouling and toxicity tests were conducted on these compounds: the most active (compounds 2, 6, and 9) inhibited both the growth of four strains of bacteria (MICs < 2.5 microg/mL) and settlement of cyprids of Balanus amphitrite (EC50 < 5 microg/mL), the latter at nontoxic concentrations (LC50 > 5 microg/mL).

Polar acyclic diterpenoids from Bifurcaria bifurcata (Fucales, Phaeophyta).

From the lipophilic extract of the brown alga Bifurcaria bifurcata collected off the Atlantic coast of Southern Brittany (Quiberon, France), five polar linear diterpenoids have been isolated. These metabolites have been identified as hydroxylated derivatives of 13-oxo- and 13-hydroxygeranylgeraniol. Their structures were characterized on the basis of chemical and spectral evidence including two-dimensional NMR experiments and mass spectrometric techniques. The absolute configuration of the 13-position has been determined, for the 13-hydroxygeranylgeraniol derivatives, to be R by means of a modified Mosher's method and therefore that of 13-hydroxygeranylgeraniol (eleganediol) has been revised. Along with these compounds, three related known geranylgeraniol derivatives were also identified, and these data were used for chemotaxonomical purposes.

Trihydroxylated linear diterpenes from the brown alga Bifurcaria bifurcata.

Two novel polar diterpenes were isolated from the brown alga Bifurcaria bifurcata collected off the Atlantic coast of Morocco, and their structures established by spectral methods. Both compounds are trihydroxylated acyclic diterpenes derived from 12-hydroxygeranylgeraniol. They were tested in vitro for their cytotoxicity and proved to be active against the NSCLC-N6 cell line. Their absolute configuration at the C-12 position has been determined with a modified Mosher's method [J. Am. Chem. Soc. 113 (1991) 4092] and that of the 12-hydroxygeranylgeraniol (bifurcadiol) has been revised.

Extracellular and surface-exposed polysaccharides of non-tuberculous mycobacteria.

We studied the outermost constituents of the cell envelopes, which are involved in the interaction between the bacilli and the host cells, of five pathogenic and non-pathogenic mycobacterial species for comparison with those we have previously characterized from M. tuberculosis. The extracellular materials (ECMs) were isolated by ethanol precipitation and compared to the surface-exposed materials (SXMs) extracted by mechanical means. The materials from both sources were composed almost exclusively of polysaccharides and proteins. Two groups of mycobacteria were clearly distinguishable. The first group comprised the pathogenic species M. kansasii which produced large amounts of ECM, the glycosyl composition of which was similar to that of the SXM. The second group comprised M. avium and the non-pathogenic strains of M. gastri, M. phlei and M. smegmatis which produced small amounts of ECK This latter group could be subdivided into those which produced carbohydrate-rich ECM (M. avium and M. gastri) and those forming protein-rich ECM (M. phlei and M. smegmatis), a classification that correlated with the difference in the growth rate of the two subgroups. The glycosyl composition of the ECM of a given species was qualitatively similar to that of the SXM, except for M. avium and M. phlei whose SXM were devoid of arabinose. In addition to glucose, mannose and arabinose, xylose was detected in the hydrolysis products of the ECM and SXM of M. smegmatis, the SXM of M. phlei and the ECM of some batches of M. avium. The polysaccharide constituents of the ECM and SXM of the different mycobacteria were purified by anion-exchange and gel-filtration chromatography; all were found to be neutral compounds devoid of acyl substituents. The extracellular polysaccharides consisted of high-molecular-mass glycogen-like glucans, arabinomannans and mannans, structurally similar to the corresponding substances previously characterized from the capsule of M. tuberculosis. The same types of polysaccharides were characterized from the SXM of all the strains, except M. avium and M. phlei which were devoid of arabinomannans. This study questions the unique and universal representation of the mycobacterial cell envelope and the existence of the so-called acidic polysaccharide-rich outer layer.