Tropodithietic Acid Research Articles

Draft genome of a Pseudovibrio sp. isolated from the skeleton of Pachyseris speciosa from a Singaporean reef.

A Pseudovibrio sp. was isolated from the skeleton of the heat resilient coral Pachyseris speciosa. Genome analysis revealed the presence of the complete denitrification pathway and potential dimethylsulfoniopropionate metabolism which enhance coral resilience and production of tropodithietic acid, an antibiotic implicated in host defense.

Co-existence of two antibiotic-producing marine bacteria: Pseudoalteromonas piscicida reduce gene expression and production of the antibacterial compound, tropodithietic acid, in Phaeobacter sp.

Many bacteria co-exist and produce antibiotics, yet we know little about how they cope and occupy the same niche. The purpose of the present study was to determine if and how two potent antibiotic-producing marine bacteria influence the secondary metabolome of each other. We established an agar- and broth-based system allowing co-existence of a Phaeobacter species and Pseudoalteromonas piscicida that, respectively, produce tropodithietic acid (TDA) and bromoalterochromides (BACs). Co-culturing of Phaeobacter sp. strain A36a-5a on Marine Agar with P. piscicida strain B39bio caused a reduction of TDA production in the Phaeobacter colony. We constructed a transcriptional gene reporter fusion in the tdaC gene in the TDA biosynthetic pathway in Phaeobacter and demonstrated that the reduction of TDA by P. piscicida was due to the suppression of the TDA biosynthesis. A stable liquid co-cultivation system was developed, and the expression of tdaC in Phaeobacter was reduced eightfold lower (per cell) in the co-culture compared to the monoculture. Mass spectrometry imaging of co-cultured colonies revealed a reduction of TDA and indicated that BACs diffused into the Phaeobacter colony. BACs were purified from Pseudoalteromonas; however, when added as pure compounds or a mixture they did not influence TDA production. In co-culture, the metabolome was dominated by Pseudoalteromonas features indicating that production of other Phaeobacter compounds besides TDA was reduced. In conclusion, co-existence of two antibiotic-producing bacteria may be allowed by one causing reduction in the antagonistic potential of the other. The reduction (here of TDA) was not caused by degradation but by a yet uncharacterized mechanism allowing Pseudoalteromonas to reduce expression of the TDA biosynthetic pathway.IMPORTANCEThe drug potential of antimicrobial secondary metabolites has been the main driver of research into these compounds. However, in recent years, their natural role in microbial systems and microbiomes has become important to determine the assembly and development of microbiomes. Herein, we demonstrate that two potent antibiotic-producing bacteria can co-exist, and one mechanism allowing the co-existence is the specific reduction of antibiotic production in one bacterium by the other. Understanding the molecular mechanisms in complex interactions provides insights for applied uses, such as when developing TDA-producing bacteria for use as biocontrol in aquaculture.

Unraveling the genomic secrets of Tritonibacter mobilis AK171: a plant growth-promoting bacterium isolated from Avicennia marina

The scarcity of freshwater resources resulting in a significant yield loss presents a pressing challenge in agriculture. To address this issue, utilizing abundantly available saline water could offer a smart solution. In this study, we demonstrate that the genome sequence rhizosphere bacterium Tritonibacter mobilis AK171, a halophilic marine bacterium recognized for its ability to thrive in saline and waterlogged environments, isolated from mangroves, has the remarkable ability to enable plant growth using saline irrigation. AK171 is characterized as rod-shaped cells, displays agile movement in free-living conditions, and adopts a rosette arrangement in static media. Moreover, The qualitative evaluation of PGP traits showed that AK171 could produce siderophores and IAA but could not solubilize phosphate nor produce hydrolytic enzymes it exhibits a remarkable tolerance to high temperatures and salinity. In this study, we conducted a comprehensive genome sequence analysis of T. mobilis AK171 to unravel the genetic mechanisms underlying its plant growth-promoting abilities in such challenging conditions. Our analysis revealed diverse genes and pathways involved in the bacterium’s adaptation to salinity and waterlogging stress. Notably, T. mobilis AK171 exhibited a high level of tolerance to salinity and waterlogging through the activation of stress-responsive genes and the production of specific enzymes and metabolites. Additionally, we identified genes associated with biofilm formation, indicating its potential role in establishing symbiotic relationships with host plants. Furthermore, our analysis unveiled the presence of genes responsible for synthesizing antimicrobial compounds, including tropodithietic acid (TDA), which can effectively control phytopathogens. This genomic insight into T. mobilis AK171 provides valuable information for understanding the molecular basis of plant-microbial interactions in saline and waterlogged environments. It offers potential applications for sustainable agriculture in challenging conditions.

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.

Biosynthesis of the bacterial antibiotic 3,7-dihydroxytropolone through enzymatic salvaging of catabolic shunt products.

The non-benzenoid aromatic tropone ring is a structural motif of numerous microbial and plant natural products with potent bioactivities. In bacteria, tropone biosynthesis involves early steps of the widespread CoA-dependent phenylacetic acid (paa) catabolon, from which a shunt product is sequestered and surprisingly further utilized as a universal precursor for structurally and functionally diverse tropone derivatives such as tropodithietic acid or (hydroxy)tropolones. Here, we elucidate the biosynthesis of the antibiotic 3,7-dihydroxytropolone in Actinobacteria by in vitro pathway reconstitution using paa catabolic enzymes as well as dedicated downstream tailoring enzymes, including a thioesterase (TrlF) and two flavoprotein monooxygenases (TrlCD and TrlE). We furthermore mechanistically and structurally characterize the multifunctional key enzyme TrlE, which mediates an unanticipated ipso-substitution involving a hydroxylation and subsequent decarboxylation of the CoA-freed side chain, followed by ring oxidation to afford tropolone. This study showcases a remarkably efficient strategy for 3,7-dihydroxytropolone biosynthesis and illuminates the functions of the involved biosynthetic enzymes.

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.

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.

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.

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.

Role is in the eye of the beholder-the multiple functions of the antibacterial compound tropodithietic acid produced by marine Rhodobacteraceae.

Many microbial secondary metabolites have been studied for decades primarily because of their antimicrobial properties. However, several of these metabolites also possess nonantimicrobial functions, both influencing the physiology of the producer and their ecological neighbors. An example of a versatile bacterial secondary metabolite with multiple functions is the tropone derivative tropodithietic acid (TDA). TDA is a broad-spectrum antimicrobial compound produced by several members of the Rhodobacteraceae family, a major marine bacterial lineage, within the genera Phaeobacter, Tritonibacter, and Pseudovibrio. The production of TDA is governed by the mode of growth and influenced by the availability of nutrient sources. The antibacterial effect of TDA is caused by disruption of the proton motive force of target microorganisms and, potentially, by its iron-chelating properties. TDA also acts as a signaling molecule, affecting gene expression in other bacteria, and altering phenotypic traits such as motility, biofilm formation, and antibiotic production in the producer. In microbial communities, TDA-producing bacteria cause a reduction of the relative abundance of closely related species and some fast-growing heterotrophic bacteria. Here, we summarize the current understanding of the chemical ecology of TDA, including the environmental niches of TDA-producing bacteria, and the molecular mechanisms governing the function and regulation of TDA.

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.

Three Rings to Rule Them All: How Versatile Flavoenzymes Orchestrate the Structural Diversification of Natural Products.

The structural diversification of natural products is instrumental to their versatile bioactivities. In this context, redox tailoring enzymes are commonly involved in the modification and functionalization of advanced pathway intermediates en route to the mature natural products. In recent years, flavoprotein monooxygenases have been shown to mediate numerous redox tailoring reactions that include not only (aromatic) hydroxylation, Baeyer–Villiger oxidation, or epoxidation reactions but also oxygenations that are coupled to extensive remodeling of the carbon backbone, which are often central to the installment of the respective pharmacophores. In this Perspective, we will highlight recent developments and discoveries in the field of flavoenzyme catalysis in bacterial natural product biosynthesis and illustrate how the flavin cofactor can be fine-tuned to enable chemo-, regio-, and stereospecific oxygenations via distinct flavin-C4a-peroxide and flavin-N5-(per)oxide species. Open questions remain, e.g., regarding the breadth of chemical reactions enabled particularly by the newly discovered flavin-N5-oxygen adducts and the role of the protein environment in steering such cascade-like reactions. Outstanding cases involving different flavin oxygenating species will be exemplified by the tailoring of bacterial aromatic polyketides, including enterocin, rubromycins, rishirilides, mithramycin, anthracyclins, chartreusin, jadomycin, and xantholipin. In addition, the biosynthesis of tropone natural products, including tropolone and tropodithietic acid, will be presented, which features a recently described prototypical flavoprotein dioxygenase that may combine flavin-N5-peroxide and flavin-N5-oxide chemistry. Finally, structural and mechanistic features of selected enzymes will be discussed as well as hurdles for their application in the formation of natural product derivatives via bioengineering.

Genome Analysis of the Janthinobacterium sp. Strain SLB01 from the Diseased Sponge of the Lubomirskia baicalensis.

The strain Janthinobacterium sp. SLB01 was isolated from the diseased freshwater sponge Lubomirskia baicalensis (Pallas, 1776) and the draft genome was published previously. The aim of this work is to analyze the genome of the Janthinobacterium sp. SLB01 to search for pathogenicity factors for Baikal sponges. We performed genomic analysis to determine virulence factors, comparing the genome of the strain SLB01 with genomes of other related J. lividum strains from the environment. The strain Janthinobacterium sp. SLB01 contained genes encoding violacein, alpha-amylases, phospholipases, chitinases, collagenases, hemolysin, and a type VI secretion system. In addition, the presence of conservative clusters of genes for the biosynthesis of secondary metabolites of tropodithietic acid and marinocine was found. We present genes for antibiotic resistance, including five genes encoding various lactamases and eight genes for penicillin-binding proteins, which are conserved in all analyzed strains. Major differences were found between the Janthinobacterium sp. SLB01 and J. lividum strains in the spectra of genes for glycosyltransferases and glycoside hydrolases, serine hydrolases, and trypsin-like peptidase, as well as some TonB-dependent siderophore receptors. Thus, the study of the analysis of the genome of the strain SLB01 allows us to conclude that the strain may be one of the pathogens of freshwater sponges.

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.

A Flavoprotein Dioxygenase Steers Bacterial Tropone Biosynthesis via Coenzyme A-Ester Oxygenolysis and Ring Epoxidation.

Bacterial tropone natural products such as tropolone, tropodithietic acid, or the roseobacticides play crucial roles in various terrestrial and marine symbiotic interactions as virulence factors, antibiotics, algaecides, or quorum sensing signals. We now show that their poorly understood biosynthesis depends on a shunt product from aerobic CoA-dependent phenylacetic acid catabolism that is salvaged by the dedicated acyl-CoA dehydrogenase-like flavoenzyme TdaE. Further characterization of TdaE revealed an unanticipated complex catalysis, comprising substrate dehydrogenation, noncanonical CoA-ester oxygenolysis, and final ring epoxidation. The enzyme thereby functions as an archetypal flavoprotein dioxygenase that incorporates both oxygen atoms from O2 into the substrate, most likely involving flavin-N5-peroxide and flavin-N5-oxide species for consecutive CoA-ester cleavage and epoxidation, respectively. The subsequent spontaneous decarboxylation of the reactive enzyme product yields tropolone, which serves as a key virulence factor in rice panicle blight caused by pathogenic edaphic Burkholderia plantarii. Alternatively, the TdaE product is most likely converted to more complex sulfur-containing secondary metabolites such as tropodithietic acid from predominant marine Rhodobacteraceae (e.g., Phaeobacter inhibens).

Pseudooceanicola algae sp. nov., isolated from the marine macroalga Fucus spiralis, shows genomic and physiological adaptations for an algae-associated lifestyle

The genus Pseudooceanicola from the alphaproteobacterial Roseobacter group currently includes ten validated species. We herein describe strain Lw-13eT, the first Pseudooceanicola species from marine macroalgae, isolated from the brown alga Fucus spiralis abundant at European and North American coasts. Physiological and pangenome analyses of Lw-13eT showed corresponding adaptive features. Adaptations to the tidal environment include a broad salinity tolerance, degradation of macroalgae-derived substrates (mannitol, mannose, proline), and resistance to several antibiotics and heavy metals. Notably, Lw-13eT can degrade oligomeric alginate via PL15 alginate lyase encoded in a polysaccharide utilization locus (PUL), rarely described for roseobacters to date. Plasmid localization of the PUL strengthens the importance of mobile genetic elements for evolutionary adaptations within the Roseobacter group. PL15 homologs were primarily detected in marine plant-associated metagenomes from coastal environments but not in the open ocean, corroborating its adaptive role in algae-rich habitats. Exceptional is the tolerance of Lw-13eT against the broad-spectrum antibiotic tropodithietic acid, produced by Phaeobacter spp. co-occurring in coastal habitats. Furthermore, Lw-13eT exhibits features resembling terrestrial plant-bacteria associations, i.e. biosynthesis of siderophores, terpenes and volatiles, which may contribute to mutual bacteria-algae interactions. Closest described relative of Lw-13eT is Pseudopuniceibacterium sediminis CY03T with 98.4% 16S rRNA gene sequence similarity. However, protein sequence-based core genome phylogeny and average nucleotide identity indicate affiliation of Lw-13eT with the genus Pseudooceanicola. Based on phylogenetic, physiological and (chemo)taxonomic distinctions, we propose strain Lw-13eT (=DSM 29013T=LMG 30557T) as a novel species with the name Pseudooceanicola algae.

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.

Bacterial Tropone Natural Products and Derivatives: Overview of their Biosynthesis, Bioactivities, Ecological Role and Biotechnological Potential.

Tropone natural products are non‐benzene aromatic compounds of significant ecological and pharmaceutical interest. Herein, we highlight current knowledge on bacterial tropones and their derivatives such as tropolones, tropodithietic acid, and roseobacticides. Their unusual biosynthesis depends on a universal CoA‐bound precursor featuring a seven‐membered carbon ring as backbone, which is generated by a side reaction of the phenylacetic acid catabolic pathway. Enzymes encoded by separate gene clusters then further modify this key intermediate by oxidation, CoA‐release, or incorporation of sulfur among other reactions. Tropones play important roles in the terrestrial and marine environment where they act as antibiotics, algaecides, or quorum sensing signals, while their bacterial producers are often involved in symbiotic interactions with plants and marine invertebrates (e. g., algae, corals, sponges, or mollusks). Because of their potent bioactivities and of slowly developing bacterial resistance, tropones and their derivatives hold great promise for biomedical or biotechnological applications, for instance as antibiotics in (shell)fish aquaculture.

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.