Co-Culturing Microalgae with Roseobacter Clade Bacteria as a Strategy for Vibrionaceae Control in Microalgae-Enriched Artemia.

Bacterial communities associated with fish larvae are highly influenced by the microbiota of live prey used as feed (rotifers or Artemia), generally dominated by bacterial strains with a low degree of specialization and high growth rates, (e.g., Vibrionaceae), which can be detrimental to larvae. Co-cultivation of microalgae used in the enrichment of Artemia (e.g., Phaeodactylum tricornutum, or Chlorella minutissima) with Vibrio-antagonistic probiotics belonging to the Roseobacter clade bacteria (e.g., Phaeobacter spp. or Ruegeria spp.) was studied. The introduction of the probiotics did not affect microalgae growth or significantly modify the composition of bacterial communities associated with both microalgae, as revealed by DGGE analysis. The inoculation of P. tricornutum with Ruegeria ALR6 allowed the maintenance of the probiotic in the scale-up of the microalgae cultures, both in axenic and non-axenic conditions. Using Ruegeria-inoculated P. tricornutum cultures in the enrichment of Artemia reduced the total Vibrionaceae count in Artemia by 2 Log units, therefore preventing the introduction of opportunistic or pathogenic bacteria to fish larvae fed with them.

Tropodithietic Acid, a Multifunctional Antimicrobial, Facilitates Adaption and Colonization of the Producer, Phaeobacter piscinae.

In the marine environment, surface-associated bacteria often produce an array of antimicrobial secondary metabolites, which have predominantly been perceived as competition molecules. However, they may also affect other hallmarks of surface-associated living, such as motility and biofilm formation. Here, we investigate the ecological significance of an antibiotic secondary metabolite, tropodithietic acid (TDA), in the producing bacterium, Phaeobacter piscinae S26. We constructed a markerless in-frame deletion mutant deficient in TDA biosynthesis, S26ΔtdaB. Molecular networking demonstrated that other chemical sulfur-containing features, likely related to TDA, were also altered in the secondary metabolome. We found several changes in the physiology of the TDA-deficient mutant, ΔtdaB, compared to the wild type. Growth of the two strains was similar; however, ΔtdaB cells were shorter and more motile. Transcriptome and proteome profiling revealed an increase in gene expression and protein abundance related to a type IV secretion system, and to a prophage, and a gene transfer agent in ΔtdaB. All these systems may contribute to horizontal gene transfer (HGT), which may facilitate adaptation to novel niches. We speculate that once a TDA-producing population has been established in a new niche, the accumulation of TDA acts as a signal of successful colonization, prompting a switch to a sessile lifestyle. This would lead to a decrease in motility and the rate of HGT, while filamentous cells could form the base of a biofilm. In addition, the antibiotic properties of TDA may inhibit invading competing microorganisms. This points to a role of TDA in coordinating colonization and adaptation. IMPORTANCE Despite the broad clinical usage of microbial secondary metabolites with antibiotic activity, little is known about their role in natural microbiomes. Here, we studied the effect of production of the antibiotic tropodithietic acid (TDA) on the producing strain, Phaeobacter piscinae S26, a member of the Roseobacter group. We show that TDA affects several phenotypes of the producing strain, including motility, cell morphology, metal metabolism, and three horizontal gene transfer systems: a prophage, a type IV secretion system, and a gene transfer agent. Together, this indicates that TDA participates in coordinating the colonization process of the producer. TDA is thus an example of a multifunctional secondary metabolite that can mediate complex interactions in microbial communities. This work broadens our understanding of the ecological role that secondary metabolites have in microbial community dynamics.

Simultaneous quantification of all B vitamins and selected biosynthetic precursors in seawater and bacteria by means of different mass spectrometric approaches.

B vitamins have high microbiological relevance in the marine environment, but their very low concentrations and the chemical heterogeneity of the individual vitamins make their analysis challenging. Mass spectrometric analysis of B vitamins in environmental samples at trace levels has mainly been performed using triple quadrupole mass spectrometers operated in targeted analysis mode. The development of such a method can be laborious and error prone. Additionally, high-resolution mass spectrometers can be used to measure a sample in full scan mode and subsequently search the total ion current chromatogram for extracted ion chromatograms of targeted vitamins. Three different analytical approaches for trace analysis of all B vitamins and some of their biosynthetic precursors were optimized and compared on two different mass spectrometers. A triple quadrupole mass spectrometer in selected reaction monitoring mode, and a high-resolution orbitrap mass spectrometer in parallel reaction monitoring, as well as in full scan mode were employed. Detection limits down to 10 ng/L were achieved with all three techniques. The methods were applied to a marine water sample from the North Sea and to the cell extract of a bacterial culture of Phaeobacter inhibens. Most vitamins and precursors were found in the bacterial cell extract and the seawater sample with all three measuring methods. The results of this study emphasize that, in addition to tandem mass spectrometry, high-resolution full scan mass spectrometry is a promising technique for the simultaneous detection of structurally diverse B vitamins in complex natural samples. This enables highly sensitive measurements without loss of detailed mass spectrometric information, which is inevitable when using a triple quadrupole system in MS/MS mode.

Impact of Quorum Sensing and Tropodithietic Acid Production on the Exometabolome of Phaeobacter inhibens.

Microbial interactions shape ecosystem diversity and chemistry through production and exchange of organic compounds, but the impact of regulatory mechanisms on production and release of these exometabolites is largely unknown. We studied the extent and nature of impact of two signaling molecules, tropodithietic acid (TDA) and the quorum sensing molecule acyl homoserine lactone (AHL) on the exometabolome of the model bacterium Phaeobacter inhibens DSM 17395, a member of the ubiquitous marine Roseobacter group. Exometabolomes of the wild type, a TDA and a QS (AHL-regulator) negative mutant were analyzed via Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Based on a total of 996 reproducibly detected molecular masses, exometabolomes of the TDA and QS negative mutant were ∼70% dissimilar to each other, and ∼90 and ∼60% dissimilar, respectively, to that of the wild type. Moreover, at any sampled growth phase, 40-60% of masses detected in any individual exometabolome were unique to that strain, while only 10-12% constituted a shared "core exometabolome." Putative annotation revealed exometabolites of ecological relevance such as vitamins, amino acids, auxins, siderophore components and signaling compounds with different occurrence patterns in the exometabolomes of the three strains. Thus, this study demonstrates that signaling molecules, such as AHL and TDA, extensively impact the composition of bacterial exometabolomes with potential consequences for species interactions in microbial communities.

Nanomolar Responsiveness of Marine Phaeobacter inhibens DSM 17395 toward Carbohydrates and Amino Acids.

Phaeobacter inhibens DSM 17395 is a heterotrophic member of the ubiquitous, marine Roseobacter group and specializes in the aerobic utilization of carbohydrates and amino acids via pathways widespread among roseobacters. The in vivo responsiveness of P. inhibens DSM 17395 was studied with nonadapted cells (succinate-grown), which were exposed to a single pulse (100-0.01 µM) each of N-acetylglucosamine, mannitol, xylose, leucine, phenylalanine, or tryptophan (effectors). Responsiveness was then determined by time-resolved transcript analyses (quantitative reverse transcription-PCR) of "degradation" and "uptake" genes selected based on previously reported substrate-specific proteome profiles. The transcriptional response thresholds were: 50-100 nM for nagK (N-acetylglucosamine kinase), paaA (ring 1,2-phenylacetyl-CoA epoxidase), and kynA (tryptophan 2,3-dioxygenase), 10-50 nM for xylA (xylose isomerase), and around 10 nM for mtlK (mannitol 2-dehydrogenase). A threshold for leucine could not be determined due to the elevated intrinsic presence of leucine in the exometabolome of succinate-grown cells (no effector addition). Notably, the response thresholds for presumptive carbohydrate-binding proteins of ABC-transporters were in the same range or even lower: 0.1-1 µM for c27930 (N-acetylglucosamine) and even below 10 nM for c13210 (mannitol) and xylF (xylose). These results shed new light on the sensory/regulatory sensitivity of a well-studied roseobacter for recognizing potential substrates at low ambient concentrations and on the concentration threshold below which these might escape biodegradation ("emergent recalcitrance" concept of dissolved organic matter persistence).

Luciferase-Based Determination of ATP/NAD(H) Pools in a Marine (Environmental) Bacterium.

In all living organisms, adenosine triphosphate (ATP) and NAD(H) represent universal molecular currencies for energy and redox state, respectively, and are thus widely applicable molecular proxies for an organism's viability and activity. To this end, corresponding luciferase-based assays in combination with a microplate reader were established with the marine model bacterium Phaeobacter inhibens DSM 17395 (Escherichia coli K12 served as reference). Grey multiwell plates best balanced sensitivity and crosstalk, and optimal incubation times were 5 min and 30 min for the ATP and NAD(H) assay, respectively, together allowing limits of detection of 0.042, 0.470 and 0.710 nM for ATP, NAD+, and NADH, respectively. Quenching of bacterial cell samples involved Tris-EDTA-DTAB and bicarbonate base-DTAB for ATP and NAD(H) assays, respectively. The ATP and NAD(H) yields determined for P. inhibens DSM 17395 at » ODmax were found to reside well within the range previously reported for E. coli and other bacteria, e.g., 3.28 µmol ATP (g cellsdry)-1. Thus, the here described methods for luciferase-based determination of ATP/NAD(H) pools open a promising approach to investigate energy and redox states in marine (environmental) bacteria.

Roseobacter Group Probiotics Exhibit Differential Killing of Fish Pathogenic Tenacibaculum Species.

Fish-pathogenic bacteria of the Tenacibaculum genus are a serious emerging concern in modern aquaculture, causing tenacibaculosis in a broad selection of cultured finfish. Data describing their virulence mechanisms are scarce and few means, antibiotic treatment aside, are available to control their proliferation in aquaculture systems. We genome sequenced a collection of 19 putative Tenacibaculum isolates from outbreaks at two aquaculture facilities and tested their susceptibility to treatment with tropodithietic acid (TDA)-producing Roseobacter group probiotics. We found that local outbreaks of Tenacibaculum can involve heterogeneous assemblages of species and strains with the capacity to produce multiple different virulence factors related to host invasion and infection. The probiotic Phaeobacter piscinae S26 proved efficient in killing pathogenic Tenacibaculum species such as T. maritimum, T. soleae, and some T. discolor strains. However, the T. mesophilum and T. gallaicum species exhibit natural tolerance toward TDA and are hence not likely to be easily killed by TDA-producing probiotics. Tolerance toward TDA in Tenacibaculum is likely involving multiple inherent physiological features pertaining to electron and proton transport, iron sequestration, and potentially also drug efflux mechanisms, since genetic determinants encoding such features were significantly associated with TDA tolerance. Collectively, our results support the use of TDA producers to prevent tenacibaculosis; however, their efficacy is likely limited to some Tenacibaculum species. IMPORTANCE A productive and sustainable aquaculture sector is needed to meet the UN sustainable development goals and supply the growing world population with high-protein food sources. A sustainable way to prevent disease outbreaks in the industry is the application of probiotic bacteria that can antagonize fish pathogens in the aquaculture systems. TDA-producing Roseobacter group probiotics have proven efficient in killing important vibrio pathogens and protecting fish larvae against infection, and yet their efficacy against different fish pathogenic species of the Tenacibaculum genus has not been explored. Therefore, we tested the efficacy of such potential probiotics against a collection of different Tenacibaculum isolates and found the probiotic to efficiently kill a subset of relevant strains and species, supporting their use as sustainable disease control measure in aquaculture.

Global metabolome analysis of Dunaliella tertiolecta, Phaeobacter italicus R11 Co-cultures using thermal desorption - Comprehensive two-dimensional gas chromatography - Time-of-flight mass spectrometry (TD-GC×GC-TOFMS).

Dunaliella tertiolecta is a marine microalgae that has been studied extensively as a potential carbon-neutral biofuel source (Tang et al., 2011). Microalgae oil contains high quantities of energy-rich fatty acids and lipids, but is not yet commercially viable as an alternative fuel. Carefully optimised growth conditions, and more recently, algal-bacterial co-cultures have been explored as a way of improving the yield of D. tertiolecta microalgae oils. The relationship between the host microalgae and bacterial co-cultures is currently poorly understood. Here, a complete workflow is proposed to analyse the global metabolomic profile of co-cultured D. tertiolectra and Phaeobacter italicus R11, which will enable researchers to explore the chemical nature of this relationship in more detail. To the best of the authors' knowledge this study is one of the first of its kind, in which a pipeline for an entirely untargeted analysis of the algal metabolome is proposed using a practical sample preparation, introduction, and data analysis routine.

Genomic Evolution of the Marine Bacterium Phaeobacter inhibens during Biofilm Growth.

Phaeobacter inhibens 2.10 is an effective biofilm former on marine surfaces and has the ability to outcompete other microorganisms, possibly due to the production of the plasmid-encoded secondary metabolite tropodithietic acid (TDA). P. inhibens 2.10 biofilms produce phenotypic variants with reduced competitiveness compared to the wild type. In the present study, we used longitudinal, genome-wide deep sequencing to uncover the genetic foundation that contributes to the emergent phenotypic diversity in P. inhibens 2.10 biofilm dispersants. Our results show that phenotypic variation is not due to the loss of the plasmid that carries the genes for TDA synthesis but instead show that P. inhibens 2.10 biofilm populations become rapidly enriched in single nucleotide variations in genes involved in the synthesis of TDA. While variants in genes previously linked to other phenotypes, such as lipopolysaccharide production (i.e., rfbA) and cellular persistence (i.e., metG), also appear to be selected for during biofilm dispersal, the number and consistency of variations found for genes involved in TDA production suggest that this metabolite imposes a burden on P. inhibens 2.10 cells. Our results indicate a strong selection pressure for the loss of TDA in monospecies biofilm populations and provide insight into how competition (or a lack thereof) in biofilms might shape genome evolution in bacteria. IMPORTANCE Biofilm formation and dispersal are important survival strategies for environmental bacteria. During biofilm dispersal, cells often display stable and heritable variants from the parental biofilm. Phaeobacter inhibens is an effective colonizer of marine surfaces, in which a subpopulation of its biofilm dispersal cells displays a noncompetitive phenotype. This study aimed to elucidate the genetic basis of these phenotypic changes. Despite the progress made to date in characterizing the dispersal variants in P. inhibens, little is understood about the underlying genetic changes that result in the development of the specific variants. Here, P. inhibens phenotypic variation was linked to single nucleotide polymorphisms (SNPs), in particular in genes affecting the competitive ability of P. inhibens, including genes related to the production of the antibiotic tropodithietic acid (TDA) and bacterial cell-cell communication (e.g., quorum sensing). This work is significant as it reveals how the biofilm lifestyle might shape genome evolution in a cosmopolitan bacterium.

The Metano Modeling Toolbox MMTB: An Intuitive, Web-Based Toolbox Introduced by Two Use Cases.

Genome-scale metabolic models are of high interest in a number of different research fields. Flux balance analysis (FBA) and other mathematical methods allow the prediction of the steady-state behavior of metabolic networks under different environmental conditions. However, many existing applications for flux optimizations do not provide a metabolite-centric view on fluxes. Metano is a standalone, open-source toolbox for the analysis and refinement of metabolic models. While flux distributions in metabolic networks are predominantly analyzed from a reaction-centric point of view, the Metano methods of split-ratio analysis and metabolite flux minimization also allow a metabolite-centric view on flux distributions. In addition, we present MMTB (Metano Modeling Toolbox), a web-based toolbox for metabolic modeling including a user-friendly interface to Metano methods. MMTB assists during bottom-up construction of metabolic models by integrating reaction and enzymatic annotation data from different databases. Furthermore, MMTB is especially designed for non-experienced users by providing an intuitive interface to the most commonly used modeling methods and offering novel visualizations. Additionally, MMTB allows users to upload their models, which can in turn be explored and analyzed by the community. We introduce MMTB by two use cases, involving a published model of Corynebacterium glutamicum and a newly created model of Phaeobacter inhibens.

The Roseobacter-Group Bacterium Phaeobacter as a Safe Probiotic Solution for Aquaculture.

Phaeobacter inhibens has been assessed as a probiotic bacterium for application in aquaculture. Studies addressing the efficacy and safety indicate that P. inhibens maintains its antagonistic activity against pathogenic vibrios in aquaculture live cultures (live feed and fish egg/larvae) while having no or a positive effect on the host organisms and a minor impact on the host microbiomes. While P. inhibens produces antibacterial and algicidal compounds, no study has so far found a virulent phenotype of P. inhibens cells against higher organisms. Additionally, an in silico search for antibiotic resistance genes using published genomes of representative strains did not raise concerns regarding the risk for antimicrobial resistance. P. inhibens occurs naturally in aquaculture systems, supporting its safe usage in this environment. In conclusion, at the current state of knowledge, P. inhibens is a "safe-to-use" organism.

Evaluation of the Production of Dissolved Organic Matter by Three Marine Bacterial Strains.

A large part of marine dissolved organic matter (DOM) is considered to be recalcitrant DOM (RDOM) produced by marine bacteria. However, it is still unclear whether differences in bacterial species and/or physiology control the efficiency of RDOM production. Here, batch culture experiments with glucose as the sole carbon source were carried out using three model marine bacterial strains, namely, Alteromonas macleodii (Alt), Vibrio splendidus (Vib), and Phaeobacter gallaeciensis (Pha). Dissolved organic carbon (DOC) concentrations drastically decreased during the exponential growth phases of these bacteria due to the consumption of glucose. The efficiency of bacterial DOC production at the end of incubation was largely different among the strains and was higher for Vib (20%) than for the other two strains (Alt, 4%; Pha, 6%). All strains produced fluorescent DOM (FDOM), including humic-like FDOM which is considered as recalcitrant component in the ocean, even though the composition of bacterial FDOM was also different among the strains. The efficiency of humic-like FDOM production during the exponential growth phase was different among the bacterial strains; that is, Pha produced humic-like FDOM efficiently compared with the other two species. The efficiency of humic-like FDOM production with mineralization of organic matter was lower during the exponential growth phase than during the stationary phase of Alt and Pha. Four processes for the production of bacterially derived recalcitrant humic-like FDOM are suggested from this study: (1) production during active growing (in all strains), (2) production with the reutilization of bacterial DOM (Alt), (3) production with the consumption of cellular materials (Pha), and (4) release from lysis (Vib). Our results suggest that bacterial species and physiology can regulate RDOM production and accumulation in the ocean.

Contrasting Immunomodulatory Effects of Probiotic and Pathogenic Bacteria on Eastern Oyster, Crassostrea Virginica, Larvae.

Several Vibrio spp. cause acute and severe mortality events in hatcheries where larvae of bivalve mollusks are reared, potentially leading to subsequent shortage of bivalve seed for the grow-out industry. In particular, strains of Vibrio coralliilyticus have been identified as a major cause of disease in Pacific, Crassostrea gigas, and eastern, C. virginica, oyster hatcheries in the United States of America. Probiotic bacteria are an inexpensive, practical, and natural method of disease control. Previous research shows that pretreatment of larval oysters with probiotic bacteria Bacillus pumilus RI06-95 (RI) and Phaeobacter inhibens S4 (S4) significantly decreases mortality caused by experimental challenge with the bacterial pathogen V. coralliilyticus RE22 (RE22). This study aims to characterize the immune response of 6-10-day-old eastern oyster larvae to experimental challenge with pathogen V. coralliilyticus RE22 and probionts RI and S4. Treatments included (a) pathogen and probiont exposure at a concentration of 5 × 104 CFU per mL (~2500 bacterial cells per larva) for a duration of 6 h, (b) probiont exposure at the same concentration for a duration of 24 h, and (c) probiont RI daily treatment of larvae in the hatchery for 4, 11, and 15 days. Differential gene expression analysis compared pathogen or probiotic-treated transcriptomes to unexposed controls. Probiotic and pathogen treatment led to upregulation of transcripts coding for several immune pattern recognition receptors (PRRs) involved in environmental sensing and detection of microbes in oyster larvae. Larval oyster responses to pathogen RE22 suggested suppression of expression of genes in immune signaling pathways (myd88, tak1, nkap), failure in upregulation of immune effector genes, high metabolic demand, and oxidative stress that potentially contributed to mortality. On the other hand, the transcriptomic response to probiotic bacteria RI and S4 suggested activation of immune signaling pathways and expression of immune effectors (e.g., Cv-spi2, mucins and perforin-2). These key features of the host immune response to probiotic bacteria were shared despite the length of probiotic exposure, probiotic species, and the type of environment in which exposures were conducted. This study suggests that pre-exposure of eastern oyster larvae to probiotics for 6-24 h prior to pathogenic challenge leads to a robust and effective immune response that may contribute to protecting larvae from subsequent challenge with V. coralliilyticus RE22. This research provides new insights into host-microbe interactions in larval oysters that could be applied in the management of vibriosis in bivalve hatcheries.

Global Response of Phaeobacter inhibens DSM 17395 to Deletion of Its 262-kb Chromid Encoding Antibiotic Synthesis.

The marine alphaproteobacterium Phaeobacter inhibens DSM 17395, a member of the Roseobacter group, was recently shown to markedly enhance growth upon deletion of its 262-kb chromid encoding biosynthesis of tropodithietic acid (TDA). To scrutinize the metabolic/regulatory adaptations that underlie enhanced growth of the Δ262 mutant, its transcriptome and proteome compared to the wild type were investigated in process-controlled bioreactors with Casamino Acids as growth substrate. Genome resequencing revealed only few additional genetic changes (a heterogenic insertion, prophage activation, and several point mutations) between wild type and Δ262 mutant, albeit with no conceivable effect on the studied growth physiology. The abundances of the vast majority of transcripts and proteins involved in the catabolic network for complete substrate oxidation to CO2 were found to be unchanged, suggesting that the enhanced amino acid utilization of the Δ262 mutant did not require elevated synthesis of most enzymes of the catabolic network. Similarly, constituents of genetic information processing and cellular processes remained mostly unchanged. In contrast, 426 genes displayed differential expression, of which 410 were localized on the 3.2-Mb chromosome, 5 on the 65-kb chromid, and 11 on the 78-kb chromid. Notably, the branched-chain amino transferase IlvE acting on rapidly utilized Val, Ile, and Leu was upregulated. Moreover, the transportome was reconfigured, as evidenced from increased abundances of transcripts and proteins of several uptake systems for amino acids and inorganic nutrients (e.g., phosphate). Some components of the respiratory chain were also upregulated, which correlates with the higher respiration rates of the Δ262 mutant. Furthermore, chromosomally encoded transcripts and proteins that are peripherally related to TDA biosynthesis (e.g., the serine acyl transferase CysE) were strongly downregulated in the Δ262 mutant. Taken together, these observations reflect adaptations to enhanced growth as well as the functional interconnectivity of the replicons of P. inhibens DSM 17395.

Changes in the Microbiome of Mariculture Feed Organisms after Treatment with a Potentially Probiotic Strain of Phaeobacter inhibens.

The Phaeobacter genus has been explored as probiotics in mariculture as a sustainable strategy for the prevention of bacterial infections. Its antagonistic effect against common fish pathogens is predominantly due to the production of the antibacterial compound tropodithietic acid (TDA), and TDA-producing strains have repeatedly been isolated from mariculture environments. Despite many in vitro trials targeting pathogens, little is known about its impact on host-associated microbiomes in mariculture. Hence, the purpose of this study was to investigate how the addition of a TDA-producing Phaeobacter inhibens strain affects the microbiomes of live feed organisms and fish larvae. We used 16S rRNA gene sequencing to characterize the bacterial diversity associated with live feed microalgae (Tetraselmis suecica), live feed copepod nauplii (Acartia tonsa), and turbot (Scophthalmus maximus) eggs/larvae. The microbial communities were unique to the three organisms investigated, and the addition of the probiotic bacterium had various effects on the diversity and richness of the microbiomes. The structure of the live feed microbiomes was significantly changed, while no effect was seen on the community structure associated with turbot larvae. The changes were seen primarily in particular taxa. The Rhodobacterales order was indigenous to all three microbiomes and decreased in relative abundance when P. inhibens was introduced in the copepod and turbot microbiomes, while it was unaffected in the microalgal microbiome. Altogether, the study demonstrates that the addition of P. inhibens in higher concentrations, as part of a probiotic regime, does not appear to cause major imbalances in the microbiome, but the effects were specific to closely related taxa.IMPORTANCE This work is an essential part of the risk assessment of the application of roseobacters as probiotics in mariculture. It provides insights into the impact of TDA-producing Phaeobacter inhibens on the commensal bacteria related to mariculture live feed and fish larvae. Also, the study provides a sequencing-based characterization of the microbiomes related to mariculture-relevant microalga, copepods, and turbot larvae.

Encapsulation of live marine bacteria for use in aquaculture facilities and process evaluation using response surface methodology.

New strategies are being proposed in marine aquaculture to use marine bacteria as alternative to antibiotics, as nutritional additive or as immune-stimulant. These approaches are particularly promising for larval and juvenile cultures. In many cases, the bacteria are released in the seawater, where they have to be at appropriate concentrations. In addition, only low-cost technologies are sustainable for this industry, without any complex requirements for use or storage. In this work, we explore the possibilities of preservation of a potential marine probiotic bacterium (Phaeobacter PP-154) as a product suitable for use in marine aquaculture by addition to the seawater. A method which guaranteed the preservation of the viable marine bacteria in a saline medium and their rapid release in the seawater was searched for. In a previous step, classical procedures (freeze-drying and freezing) had been explored, but undesirable results of the interaction of the products obtained with natural seawater led to investigate alternatives. We report the results of the immobilization of the marine bacteria in calcium alginate beads. The final product complies the salinity which allows the requirements of the bacteria without interference with alginate in the formation of beads, and a balanced hardness to retain the bacteria and to be easily released in the marine aquaculture environment. The process was evaluated using the central composite rotatable design (CCRD), a standard response surface methodology (RSM).

Amino Acid and Sugar Catabolism in the Marine Bacterium Phaeobacter inhibens DSM 17395 from an Energetic Viewpoint.

Growth energetics and metabolic efficiency contribute to the lifestyle and habitat imprint of microorganisms. Roseobacters constitute one of the most abundant and successful marine bacterioplankton groups. Here, we reflect on the energetics and metabolic efficiency of Phaeobacter inhibens DSM 17395, a versatile heterotrophic roseobacter. Fourteen different substrates (five sugars and nine amino acids) and their degradation pathways were assessed for energetic efficiencies based on catabolic ATP yields, calculated from net formed ATP and reducing equivalents. The latter were converted into ATP by employing the most divergent coupling ratios (i.e., ions per ATP) currently known for F1Fo ATP synthases in heterotrophic bacteria. The catabolic ATP yields of the pathways studied in P. inhibens differed ∼3-fold. The actual free energy costs for ATP synthesis were estimated at 81.6 kJ per mol ATP (3.3 ions per ATP) or 104.2 kJ per mol ATP (4.3 ions per ATP), yielding an average thermodynamic efficiency of ∼37.7% or ∼29.5%, respectively. Growth performance (rates, yields) and carbon assimilation efficiency were determined for P. inhibens growing in process-controlled bioreactors with 10 different single substrates (Glc, Man, N-acetylglucosamine [Nag], Phe, Trp, His, Lys, Thr, Val, or Leu) and with 2 defined substrate mixtures. The efficiencies of carbon assimilation into biomass ranged from ∼28% to 61%, with His/Trp and Thr/Leu yielding the lowest and highest levels. These efficiencies correlated with catabolic and ATP yields only to some extent. Substrate-specific metabolic demands and/or functions, as well as the compositions of the substrate mixtures, apparently affected the energetic costs of growth. These include energetic burdens associated with, e.g., slow growth, stress, and/or the production of tropodithietic acid.IMPORTANCE Heterotrophic members of the bacterioplankton serve the marine ecosystem by transforming organic matter, an activity that is governed by the bacterial growth efficiencies (BGEs) obtained under given environmental conditions. In marine ecology, the concept of BGE refers to the carbon assimilation efficiency within natural communities. The marine bacterium studied here, Phaeobacter inhibens DSM 17395, is a copiotrophic representative of the globally abundant Roseobacter group, and the 15 catabolic pathways investigated are widespread among these marine heterotrophs. Combining pathway-specific catabolic ATP yields with in-depth quantitative physiological data could (i) provide a new baseline for the study of growth energetics and efficiency in further Roseobacter group members and other copiotrophic marine bacteria in productive coastal ecosystems and (ii) contribute to a better understanding of the factors controlling BGE (including the additional energetic burden arising from widespread secondary-metabolite formation) based on laboratory studies with pure cultures.

The Probiotic Bacterium Phaeobacter inhibens Downregulates Virulence Factor Transcription in the Shellfish Pathogen Vibrio coralliilyticus by N-Acyl Homoserine Lactone Production.

Phaeobacter inhibens S4Sm acts as a probiotic bacterium against the oyster pathogen Vibrio coralliilyticus Here, we report that P. inhibens S4Sm secretes three molecules that downregulate the transcription of major virulence factors, metalloprotease genes, in V. coralliilyticus cultures. The effects of the S4Sm culture supernatant on the transcription of three genes involved in protease activity, namely, vcpA, vcpB, and vcpR (encoding metalloproteases A and B and their transcriptional regulator, respectively), were examined by reverse transcriptase quantitative PCR (qRT-PCR). The expression of vcpB and vcpR were reduced to 36% and 6.6%, respectively, compared to that in an untreated control. We constructed a V. coralliilyticus green fluorescent protein (GFP) reporter strain to detect the activity of inhibitory compounds. Using a bioassay-guided approach, the molecules responsible for V. coralliilyticus protease inhibition activity were isolated from S4Sm supernatant and identified as three N-acyl homoserine lactones (AHLs). The three AHLs are N-(3-hydroxydecanoyl)-l-homoserine lactone, N-(dodecanoyl-2,5-diene)-l-homoserine lactone, and N-(3-hydroxytetradecanoyl-7-ene)-l-homoserine lactone, and their half maximal inhibitory concentrations (IC50s) against V. coralliilyticus protease activity were 0.26 µM, 3.7 µM, and 2.9 µM, respectively. Our qRT-PCR data demonstrated that exposures to the individual AHLs reduced the transcription of vcpR and vcpB Combinations of the three AHLs (any two or all three AHLs) on V. coralliilyticus produced additive effects on protease inhibition activity. These AHL compounds may contribute to the host protective effects of S4Sm by disrupting the quorum sensing pathway that activates protease transcription of V. coralliilyticusIMPORTANCE Probiotics represent a promising alternative strategy to control infection and disease caused by marine pathogens of aquaculturally important species. Generally, the beneficial effects of probiotics include improved water quality, control of pathogenic bacteria and their virulence, stimulation of the immune system, and improved animal growth. Previously, we isolated a probiotic bacterium, Phaeobacter inhibens S4Sm, which protects oyster larvae from Vibrio coralliilyticus RE22Sm infection. We also demonstrated that both antibiotic secretion and biofilm formation play important roles in S4Sm probiotic activity. Here, we report that P. inhibens S4Sm, an alphaproteobacterium and member of the Roseobacter clade, also secretes secondary metabolites that hijack the quorum sensing ability of V. coralliilyticus RE22Sm, suppressing virulence gene expression. This finding demonstrates that probiotic bacteria can exert their host protection by using a multipronged array of behaviors that limit the ability of pathogens to become established and cause infection.

Causes and Consequences of a Variant Strain of Phaeobacter inhibens With Reduced Competition.

Phaeobacter inhibens 2.10 is an effective biofilm former and colonizer of marine surfaces and has the ability to outcompete other microbiota. During biofilm dispersal P. inhibens 2.10 produces heritable phenotypic variants, including those that have a reduced ability to inhibit the co-occurring bacterium Pseudoalteromonas tunicata. However, the genetic changes that underpin the phenotypic variation and what the ecological consequences are for variants within the population are unclear. To answer these questions we sequenced the genomes of strain NCV12a1, a biofilm variant of P. inhibens 2.10 with reduced inhibitory activity and the P. inhibens 2.10 WT parental strain. Genome wide analysis revealed point mutations in genes involved in synthesis of the antibacterial compound tropodithietic acid (TDA) and indirectly in extracellular polymeric substances (EPS) production. However, confocal laser scanning microscopy analyses found little differences in biofilm growth between P. inhibens 2.10 WT (parental) and NCV12a1. P. inhibens NCV12a1 was also not outcompeted in co-cultured biofilms with P. tunicata, despite its reduced inhibitory activity, rather these biofilms were thicker than those produced when the WT strain was co-cultured with P. tunicata. Notably, dispersal populations from biofilms of P. inhibens NCV12a1 had a higher proportion of WT-like morphotypes when co-cultured with P. tunicata. These observations may explain why the otherwise non-inhibiting variant persists in the presence of a natural competitor, adding to our understanding of the relative importance of genetic diversification in microbial biofilms.

A Novel Microbial Culture Chamber Co-cultivation System to Study Algal-Bacteria Interactions Using Emiliania huxleyi and Phaeobacter inhibens as Model Organisms.

Our understanding of microbial natural environments combines in situ experimentation with studies of specific interactions in laboratory-based setups. The purpose of this work was to develop, build and demonstrate the use of a microbial culture chamber enabling both in situ and laboratory-based studies. The design uses an enclosed chamber surrounded by two porous membranes that enables the comparison of growth of two separate microbial populations but allowing free exchange of small molecules. Initially, we tested if the presence of the macroalga Fucus vesiculosus inside the chamber affected colonization of the outer membranes by marine bacteria. The alga did indeed enrich the total population of colonizing bacteria by more than a factor of four. These findings lead us to investigate the effect of the presence of the coccolithophoric alga Emiliania huxleyi on attachment and biofilm formation of the marine bacterium Phaeobacter inhibens DSM17395. These organisms co-exist in the marine environment and have a well-characterized interdependence on secondary metabolites. P. inhibens attached in significantly higher numbers when having access to E. huxleyi as compared to when exposed to sterile media. The experiment was carried out using a wild type (wt) strain as well as a TDA-deficient strain of P. inhibens. The ability of the bacterium to produce the antibacterial compound, tropodithietic acid (TDA) influenced its attachment since the P. inhibens DSM17395 wt strain attached in higher numbers to a surface within the first 48 h of incubation with E. huxleyi as compared to a TDA-negative mutant. Whilst the attachment of the bacterium to a surface was facilitated by presence of the alga, however, we cannot conclude if this was directly affected by the algae or whether biofilm formation was dependent on the production of TDA by P. inhibens, which has been implied by previous studies. In the light of these results, other applications of immersed culture chambers are suggested.