Phaeobacter Inhibens Research Articles

Genomic Features and Antimicrobial Activity of Phaeobacter inhibens Strains from Marine Biofilms.

Members of the genus Phaeobacter are widely distributed in the marine environment and are known for their ability to produce tropodithietic acid (TDA). Studies investigating the genomic and metabolic features of Phaeobacter strains from marine biofilms are sparse. Here, we analyze the complete genomes of 18 Phaeobacter strains isolated from biofilms on subtidal stones, with the aim of determining their potential to synthesize secondary metabolites. Based on whole-genome comparison and average nucleotide identity calculation, the isolated bacteria are classified as novel strains of Phaeobacter inhibens. Further analysis reveals a total of 153 biosynthetic gene clusters, which are assigned to 32 gene cluster families with low similarity to previously published ones. Complete TDA clusters are identified in 14 of the 18 strains, while in the other 4 strains the TDA clusters are rather incomplete and scattered across different chromosome and plasmid locations. Phylogenetic analysis suggests that their presence or absence may be potentially attributed to horizontal gene transfer. High-performance liquid chromatography-mass spectrometry analysis demonstrates the production of TDA in all the examined strains. Furthermore, the Phaeobacter strains have strong antibacterial activity against the pathogenic strain Vibrio owensii ems001, which is associated with acute hepatopancreatic necrosis in South American white shrimp. Altogether, this study ameliorates our knowledge of marine biofilm-associated Phaeobacter and offers new avenues for exploiting marine antimicrobial agents.

Disinfection Efficacy and Eventual Harmful Effect of Chemical Peracetic Acid (PAA) and Probiotic Phaeobacter inhibens Tested on Isochrisys galbana (var. T-ISO) Cultures

One of the main threats to aquaculture is represented by microbial pathogens, causing mass mortality episodes in hatcheries, which result in huge economic losses. Among the many disinfection methods applied to reduce this issue, the use of chemicals and beneficial microorganisms (probiotics) seems to be the most efficient. The aim of this study is to test the efficacy of two of them: a chemical, peracetic acid (PAA), and a probiotic, Phaeobacter inhibens. Tests were run on microalgae of the species Isochrysis galbana (var T-ISO). For both remedies, the microalgae survival rate and final cell concentration (cell/mL) were monitored. PAA analysis tested six different concentrations of the chemical: 7.5 µg, 10 µg/L, 20 µg/L, 30 µg/L, 40 µg/L, and 60 µg/L. Meanwhile, P. inhibens was tested with a concentration of 104 CFU/mL. Analysis for both the remedies was conducted on a laboratory scale using glass flasks, and on an industrial scale inside photobioreactors (PBRs). Among all the treatments, the one with PAA dosed with a concentration of 60 µg/L gave the best results, as the culture reached a final density of 8.61 × 106 cell/mL. However, none of the remedies involved in the experiment harmed microalgae or their growth. The results match perfectly with the condition requested for the tested remedies: to obtain an optimal breakdown of pathogens without interfering with culture growth. These features make PAA and P. inhibens good candidates for disinfection methods in aquaculture facilities.

Algal methylated compounds shorten the lag phase of Phaeobacter inhibens bacteria

The lag phase is key in resuming bacterial growth, but it remains underexplored particularly in environmental bacteria. Here we use transcriptomics and 13C-labelled metabolomics to show that the lag phase of the model marine bacterium Phaeobacter inhibens is shortened by methylated compounds produced by the microalgal partner, Emiliania huxleyi. Methylated compounds are abundantly produced and released by microalgae, and we show that their methyl groups can be collected by bacteria and assimilated through the methionine cycle. Our findings underscore the significance of methyl groups as a limiting factor during the lag phase and highlight the adjustability of this growth phase. In addition, we show that methylated compounds, typical of photosynthetic organisms, prompt diverse reductions in lag times in bacteria associated with algae and plants, potentially favouring early growth in some bacteria. These findings suggest ways to accelerate bacterial growth and underscore the significance of studying bacteria within an environmental context.

Antagonistic activity of Phaeobacter piscinae against the emerging fish pathogen Vibrio crassostreae in aquaculture feed algae.

Aquaculture provides a rich resource of high-quality protein; however, the production is challenged by emerging pathogens such as Vibrio crassostreae. While probiotic bacteria have been proposed as a sustainable solution to reduce pathogen load in aquaculture, their application requires a comprehensive assessment across the aquaculture food chain. The purpose of this study was to determine the antagonistic effect of the potential probiotic bacterium Phaeobacter piscinae against the emerging fish pathogen V. crassostreae in aquaculture feed algae that can be an entry point for pathogens in fish and shellfish aquaculture. P. piscinae strain S26 produces the antibacterial compound tropodithietic acid (TDA). In a plate-based assay, P. piscinae S26 was equally to more effective than the well-studied Phaeobacter inhibens DSM17395 in its inhibition of the fish pathogens Vibrio anguillarum 90-11-286 and V. crassostreae DMC-1. When co-cultured with the microalgae Tetraselmis suecica and Isochrysis galbana, P. piscinae S26 reduced the maximum cell density of V. crassostreae DMC-1 by 2 log and 3-4 log fold, respectively. A TDA-deficient mutant of P. piscinae S26 inhibited V. crassostreae DMC-1 to a lesser extent than the wild type, suggesting that the antagonistic effect involves TDA and other factors. TDA is the prime antagonistic agent of the inhibition of V. anguillarum 90-11-286. Comparative genomics of V. anguillarum 90-11-286 and V. crassostreae DMC-1 revealed that V. crassostreae DMC-1 carries a greater arsenal of antibiotic resistance genes potentially contributing to the reduced effect of TDA. In conclusion, P. piscinae S26 is a promising new candidate for inhibition of emerging pathogens such as V. crassostreae DMC-1 in algal feed systems and could contribute to a more sustainable aquaculture industry.IMPORTANCEThe globally important production of fish and shellfish in aquaculture is challenged by disease outbreaks caused by pathogens such as Vibrio crassostreae. These outbreaks not only lead to substantial economic loss and environmental damage, but treatment with antibiotics can also lead to antibiotic resistance affecting human health. Here, we evaluated the potential of probiotic bacteria, specifically the newly identified strain Phaeobacter piscinae S26, to counteract these threats in a sustainable manner. Through a systematic assessment of the antagonistic effect of P. piscinae S26 against V. crassostreae DMC-1, particularly within the context of algal feed systems, the study demonstrates the effectiveness of P. piscinae S26 as probiotic and thereby provides a strategic pathway for addressing disease outbreaks in aquaculture. This finding has the potential of significantly contributing to the long-term stability of the industry, highlighting the potential of probiotics as an efficient and environmentally conscious approach to safeguarding aquaculture productivity against the adverse impact of pathogens.

Novel metabolite madeirone and neomarinone extracted from Streptomyces aculeoletus as marine antibiofilm and antifouling agents.

Introduction: Biofouling poses a significant economic threat to various marine industries, leading to financial losses that can reach billions of euros annually. This study highlights the urgent need for effective alternatives to traditional antifouling agents, particularly following the global ban on organotin compounds. Material and methods: Streptomyces aculeolatus PTM-346 was isolated from sediment samples on the shores of the Madeira Archipelago, Portugal. The crude extract was fractionated using silica flash chromatography and preparative HPLC, resulting in two isolated marinone compounds: madeirone (1), a novel marinone derivative discovered in this study, and neomarinone (2). The antifouling activities of these compounds were tested against five marine bacterial species and the larvae of the mussel Mytilus galloprovincialis. Additionally, in silico and in vivo environmental toxicity evaluations of madeirone (1) and neomarinone (2) were conducted. Results: Madeirone (1) demonstrated significant antibiofilm efficacy, inhibiting Phaeobacter inhibens by up to 66%, Marinobacter hydrocarbonoclasticus by up to 60%, and Cobetia marina by up to 40%. Neomarinone (2) also exhibited substantial antibiofilm activity, with inhibition rates of up to 41% against P. inhibens, 40% against Pseudo-oceanicola batsensis, 56% against M. hydrocarbonoclasticus, 46% against C. marina, and 40% against Micrococcus luteus. The growth inhibition activity at the same concentrations of these compounds remained below 20% for the respective bacteria, highlighting their effectiveness as potent antibiofilm agents without significantly affecting bacterial viability. Additionally, both compounds showed potent effects against the settlement of Mytilus galloprovincialis larvae, with EC50 values of 1.76µg/mL and 0.12µg/mL for compounds (1) and (2), respectively, without impairing the viability of the targeted macrofouling species. In silico toxicity predictions and in vivo toxicity assays both support their potential for further development as antifouling agents. Conclusion: The newly discovered metabolite madeirone (1) and neomarinone (2) effectively inhibit both micro- and macrofouling. This distinct capability sets them apart from existing commercial antifouling agents and positions them as promising candidates for biofouling prevention. Consequently, these compounds represent a viable and environmentally friendly alternative for incorporation into paints, primers, varnishes, and sealants, offering significant advantages over traditional copper-based compounds.

The impact of interspecific competition on the genomic evolution of Phaeobacter inhibens and Pseudoalteromonas tunicata during biofilm growth.

Interspecific interactions in biofilms have been shown to cause the emergence of community-level properties. To understand the impact of interspecific competition on evolution, we deep-sequenced the dispersal population of mono- and co-culture biofilms of two antagonistic marine bacteria (Phaeobacter inhibens 2.10 and Pseudoalteromononas tunicata D2). Enhanced phenotypic and genomic diversification was observed in the P. tunicata D2 populations under both mono- and co-culture biofilms in comparison to P. inhibens 2.10. The genetic variation was exclusively due to single nucleotide variants and small deletions, and showed high variability between replicates, indicating their random emergence. Interspecific competition exerted an apparent strong positive selection on a subset of P. inhibens 2.10 genes (e.g., luxR, cobC, argH, and sinR) that could facilitate competition, while the P. tunicata D2 population was genetically constrained under competition conditions. In the absence of interspecific competition, the P. tunicata D2 replicate populations displayed high levels of mutations affecting the same genes involved in cell motility and biofilm formation. Our results show that interspecific biofilm competition has a complex impact on genomic diversification, which likely depends on the nature of the competing strains and their ability to generate genetic variants due to their genomic constraints.

Abundant Sulfitobacter marine bacteria protect Emiliania huxleyi algae from pathogenic bacteria

Emiliania huxleyi is a unicellular micro-alga that forms massive oceanic blooms and plays key roles in global biogeochemical cycles. Mounting studies demonstrate various stimulatory and inhibitory influences that bacteria have on the E. huxleyi physiology. To investigate these algal-bacterial interactions, laboratory co-cultures have been established by us and by others. Owing to these co-cultures, various mechanisms of algal-bacterial interactions have been revealed, many involving bacterial pathogenicity towards algae. However, co-cultures represent a significantly simplified system, lacking the complexity of bacterial communities. In order to investigate bacterial pathogenicity within an ecologically relevant context, it becomes imperative to enhance the microbial complexity of co-culture setups. Phaeobacter inhibens bacteria are known pathogens that cause the death of E. huxleyi algae in laboratory co-culture systems. The bacteria depend on algal exudates for growth, but when algae senesce, bacteria switch to a pathogenic state and induce algal death. Here we investigate whether P. inhibens bacteria can induce algal death in the presence of a complex bacterial community. We show that an E. huxleyi-associated bacterial community protects the alga from the pathogen, although the pathogen occurs within the community. To study how the bacterial community regulates pathogenicity, we reduced the complex bacterial community to a five-member synthetic community (syncom). The syncom is comprised of a single algal host and five isolated bacterial species, which represent major bacterial groups that are naturally associated with E. huxleyi. We discovered that a single bacterial species in the reduced community, Sulfitobacter pontiacus, protects the alga from the pathogen. We further found that algal protection from P. inhibens pathogenicity is a shared trait among several Sulfitobacter species. Algal protection by bacteria might be a common phenomenon with ecological significance, which is overlooked in reduced co-culture systems.

The Probiotic Phaeobacter inhibens Provokes Hypertrophic Growth via Activation of the IGF-1/Akt Pathway during the Process of Metamorphosis of Greater Amberjack (Seriola dumerili, Risso 1810)

Metamorphosis entails hormonally regulated morphological and physiological changes requiring high energy levels. Probiotics as feed supplements generate ameliorative effects on host nutrient digestion and absorption. Thereby, the aim of the present research was to investigate the impact of the probiotic Phaeobacter inhibens as a water additive on cellular signaling pathways in the metamorphosis of greater amberjack (Seriola dumerili). Activation of insulin-like growth factor type 1 receptor (IGF-1R), protein kinase B (Akt), mitogen-activated protein kinases (MAPKs) and AMP-activated protein kinase (AMPK), induction of heat shock proteins (Hsps), and programmed cell death were assessed through SDS-Page/immunoblot analysis, while energy metabolism was determined through enzymatic activities. According to the results, greater amberjack reared in P. inhibens-enriched water entered the metamorphic phase with greater body length, while protein synthesis was triggered to facilitate the hypertrophic growth as indicated by IGF-1/Akt activation and AMPK inhibition. Contrarily, MAPKs levels were reduced, whereas variations in Hsps response were evident in the probiotic treatment. Apoptosis and autophagy were mobilized potentially for the structural remodeling processes. Furthermore, the elevated enzymatic activities of intermediary metabolism highlighted the excess energy demands of metamorphosis. Collectively, the present findings demonstrate that P. inhibens may reinforce nutrient utilization, thus leading greater amberjack to an advanced growth and developmental state.

Aerobic bacteria produce nitric oxide via denitrification and promote algal population collapse

Microbial interactions govern marine biogeochemistry. These interactions are generally considered to rely on exchange of organic molecules. Here we report on a novel inorganic route of microbial communication, showing that algal-bacterial interactions between Phaeobacter inhibens bacteria and Gephyrocapsa huxleyi algae are mediated through inorganic nitrogen exchange. Under oxygen-rich conditions, aerobic bacteria reduce algal-secreted nitrite to nitric oxide (NO) through denitrification, a well-studied anaerobic respiratory mechanism. The bacterial NO is involved in triggering a cascade in algae akin to programmed cell death. During death, algae further generate NO, thereby propagating the signal in the algal population. Eventually, the algal population collapses, similar to the sudden demise of oceanic algal blooms. Our study suggests that the exchange of inorganic nitrogen species in oxygenated environments is a potentially significant route of microbial communication within and across kingdoms.

Development and evaluation of a formulation of probiont Phaeobacter inhibens S4 for the management of vibriosis in bivalve hatcheries

AbstractLarval eastern oysters (Crassostrea virginica) grown in shellfish hatcheries are susceptible to bacterial diseases, particularly vibriosis. Probiotics are microbes that confer health benefits to the host and have been identified as promising tools to manage diseases in aquaculture. The marine bacterium Phaeobacter inhibens S4 (S4) protects larval eastern oysters against challenge with the bacterial pathogen Vibrio coralliilyticus RE22 (RE22). A concentrated liquid formulation of probiont S4 that maintained high cell viability after long‐term storage was developed for commercial use in shellfish hatcheries. The safety and efficacy of the formulation were tested in six different trials in two hatcheries. The S4 formulation was added to C. virginica larvae culture tanks daily at 104 colony forming units (CFU)/mL from Day 1 post fertilisation until Day 6, 12 or 14, depending on the trial. Treatment of larvae in the hatchery with the S4 formulation did not significantly affect the survival and growth of the larvae. Formulated probiont S4 treatment in the hatchery led to a significant increase in relative percent survival (RPS) when larvae were subsequently challenged with the pathogen RE22 (105 CFU/mL) for 24 h in a laboratory challenge as compared to probiotic‐untreated RE22‐challenged larvae (RPS increase of 46%–74%, p < 0.05). These results suggest that this novel S4 formulation is a safe, easy‐to‐use and effective tool in preventing larval losses due to vibriosis in hatcheries.

The potential to produce tropodithietic acid by Phaeobacter inhibens affects the assembly of microbial biofilm communities in natural seawater

Microbial secondary metabolites play important roles in biotic interactions in microbial communities and yet, we do not understand how these compounds impact the assembly and development of microbial communities. To address the implications of microbial secondary metabolite production on biotic interactions in the assembly of natural seawater microbiomes, we constructed a model system where the assembly of a natural seawater biofilm community was influenced by the addition of the marine biofilm forming Phaeobacter inhibens that can produce the antibiotic secondary metabolite tropodithietic acid (TDA), or a mutant incapable of TDA production. Because of the broad antibiotic activity of TDA, we hypothesized that the potential of P. inhibens to produce TDA would strongly affect both biofilm and planktonic community assembly patterns. We show that 1.9 % of the microbial composition variance across both environments could be attributed to the presence of WT P. inhibens, and especially genera of the Bacteriodetes were increased by the presence of the TDA producer. Moreover, network analysis with inferred putative microbial interactions revealed that P. inhibens mainly displayed strong positive associations with genera of the Flavobacteriaceae and Alteromonadaceae, and that P. inhibens acts as a keystone OTU in the biofilm exclusively due to its potential to produce TDA. Our results demonstrate the potential impact of microbial secondary metabolites on microbial interactions and assembly dynamics of complex microbial communities.

Biosynthesis enhancement of tropodithietic acid (TDA) antibacterial compound through biofilm formation by marine bacteria Phaeobacter inhibens on micro-structured polymer surfaces

Biosynthesis of an antibacterial compound by marine bacteria is enhanced by growth of biofilms on micro-structured polymer surfaces.

The ability of Phaeobacter inhibens to produce tropodithietic acid influences the community dynamics of a microalgal microbiome

Microbial secondary metabolites facilitate microbial interactions and are crucial for understanding the complexity of microbial community dynamics. The purpose of the present study was to determine how a secondary metabolite producing marine bacteria or its metabolite deficient mutant affected the microbiome of the marine microalgae Tetraselmis suecica during a 70 day long co-evolution experiment. Using 16S rRNA gene amplicon sequencing, we found that neither the tropodithietic acid (TDA)-producing Phaeobacter inhibens wildtype nor the TDA-deficient mutant had major impacts on the community composition. However, a subset of strains, displayed temporally different relative abundance trajectories depending on the presence of P. inhibens. In particular, a Winogradskyella strain displayed temporal higher relative abundance when the TDA-producing wildtype was present. Numbers of the TDA-producing wildtype were reduced significantly more than those of the mutant over time indicating that TDA production was not an advantage. In communities without the P. inhibens wildtype strain, an indigenous population of Phaeobacter increased over time, indicating that indigenous Phaeobacter populations cannot co-exist with the TDA-producing wildtype. Despite that TDA was not detected chemically, we detected transcripts of the tdaC gene indicating that TDA could be produced in the microbial community associated with the algae. Our work highlights the importance of deciphering longitudinal strain dynamics when addressing the ecological effect of secondary metabolites in a relevant natural community.

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.Graphical abstract

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.

The expanded inhibitor of apoptosis gene family in oysters possesses novel domain architectures and may play diverse roles in apoptosis following immune challenge

BackgroundApoptosis plays important roles in a variety of functions, including immunity and response to environmental stress. The Inhibitor of Apoptosis (IAP) gene family of apoptosis regulators is expanded in molluscs, including eastern, Crassostrea virginica, and Pacific, Crassostrea gigas, oysters. The functional importance of IAP expansion in apoptosis and immunity in oysters remains unknown.ResultsPhylogenetic analysis of IAP genes in 10 molluscs identified lineage specific gene expansion in bivalve species. Greater IAP gene family expansion was observed in C. virginica than C. gigas (69 vs. 40), resulting mainly from tandem duplications. Functional domain analysis of oyster IAP proteins revealed 3 novel Baculoviral IAP Repeat (BIR) domain types and 14 domain architecture types across gene clusters, 4 of which are not present in model organisms. Phylogenetic analysis of bivalve IAPs suggests a complex history of domain loss and gain. Most IAP genes in oysters (76% of C. virginica and 82% of C. gigas), representing all domain architecture types, were expressed in response to immune challenge (Ostreid Herpesvirus OsHV-1, bacterial probionts Phaeobacter inhibens and Bacillus pumilus, several Vibrio spp., pathogenic Aliiroseovarius crassostreae, and protozoan parasite Perkinsus marinus). Patterns of IAP and apoptosis-related differential gene expression differed between the two oyster species, where C. virginica, in general, differentially expressed a unique set of IAP genes in each challenge, while C. gigas differentially expressed an overlapping set of IAP genes across challenges. Apoptosis gene expression patterns clustered mainly by resistance/susceptibility of the oyster host to immune challenge. Weighted Gene Correlation Network Analysis (WGCNA) revealed unique combinations of transcripts for 1 to 12 IAP domain architecture types, including novel types, were significantly co-expressed in response to immune challenge with transcripts in apoptosis-related pathways.ConclusionsUnprecedented diversity characterized by novel BIR domains and protein domain architectures was observed in oyster IAPs. Complex patterns of gene expression of novel and conserved IAPs in response to a variety of ecologically-relevant immune challenges, combined with evidence of direct co-expression of IAP genes with apoptosis-related transcripts, suggests IAP expansion facilitates complex and nuanced regulation of apoptosis and other immune responses in oysters.

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 ¼ OD_max were found to reside well within the range previously reported for E. coli and other bacteria, e.g., 3.28 µmol ATP (g cells_dry)⁻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.

Structural Elucidation of Cryptic Algaecides in Marine Algal‐Bacterial Symbioses by NMR Spectroscopy and MicroED

AbstractMicrobial secondary metabolite discovery is often conducted in pure monocultures. In a natural setting, however, where metabolites are constantly exchanged, biosynthetic precursors are likely provided by symbionts or hosts. In the current work, we report eight novel and architecturally unusual secondary metabolites synthesized by the bacterial symbiont Phaeobacter inhibens from precursors that, in a native context, would be provided by their algal hosts. Three of these were produced at low titres and their structures were determined de novo using the emerging microcrystal electron diffraction method. Some of the new metabolites exhibited potent algaecidal activity suggesting that the bacterial symbiont can convert algal precursors, tryptophan and sinapic acid, into complex cytotoxins. Our results have important implications for the parasitic phase of algal‐bacterial symbiotic interactions.

Structural Elucidation of Cryptic Algaecides in Marine Algal-Bacterial Symbioses by NMR Spectroscopy and MicroED.

Microbial secondary metabolite discovery is often conducted in pure monocultures. In a natural setting, however, where metabolites are constantly exchanged, biosynthetic precursors are likely provided by symbionts or hosts. In the current work, we report eight novel and architecturally unusual secondary metabolites synthesized by the bacterial symbiont Phaeobacter inhibens from precursors that, in a native context, would be provided by their algal hosts. Three of these were produced at low titres and their structures were determined de novo using the emerging microcrystal electron diffraction method. Some of the new metabolites exhibited potent algaecidal activity suggesting that the bacterial symbiont can convert algal precursors, tryptophan and sinapic acid, into complex cytotoxins. Our results have important implications for the parasitic phase of algal-bacterial symbiotic interactions.