RESUMO
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.
Assuntos
Doenças dos Peixes , Probióticos , Roseobacter , Tenacibaculum , Animais , Aquicultura , Doenças dos Peixes/microbiologia , Doenças dos Peixes/prevenção & controle , Peixes/microbiologia , Tenacibaculum/genéticaRESUMO
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.
Assuntos
Clorófitas/microbiologia , Copépodes/microbiologia , Linguados/microbiologia , Microbiota , Probióticos/farmacologia , Rhodobacteraceae/química , Ração Animal , Animais , Bactérias/isolamento & purificação , Copépodes/crescimento & desenvolvimento , Linguados/crescimento & desenvolvimento , Larva/microbiologia , Microalgas/microbiologia , Óvulo/microbiologia , RNA Bacteriano/análise , RNA Ribossômico 16S/análiseRESUMO
Covering: up to 2019Humanity is in dire need for novel medicinal compounds with biological activities ranging from antibiotic to anticancer and anti-dementia effects. Recent developments in genome sequencing and mining have revealed an unappreciated potential for bioactive molecule production in marine Proteobacteria. Also, novel bioactive compounds have been discovered through molecular manipulations of either the original marine host bacteria or in heterologous hosts. Nevertheless, in contrast to the large repertoire of such molecules as predicted by in silico analysis, few marine bioactive compounds have been reported. This review summarizes the recent advances in the study of natural products from marine Proteobacteria. Here we present successful examples on genetic engineering of biosynthetic gene clusters of natural products from marine Proteobacteria. We also discuss the future prospects of discovering novel bioactive molecules via both heterologous production methodology and the development of marine Proteobacteria as new cell factories.
Assuntos
Organismos Aquáticos/metabolismo , Produtos Biológicos/metabolismo , Engenharia Metabólica , Proteobactérias/metabolismo , Organismos Aquáticos/genética , Engenharia Metabólica/métodos , Proteobactérias/genéticaRESUMO
An S-methylated analogue of tropodithietic acid (TDA, 1), methyl troposulfenin (2), was isolated from the marine alphaproteobacterium Phaeobacter inhibens. The structure was elucidated by NMR and HRMS. Its inhibitory effect against the fish pathogen Vibrio anguillarum was 4-fold to 100-fold lower than that of the known antibacterial compound TDA. Methyl troposulfenin lacks the acidic proton of TDA, indicating that the methylation turns the potent antibacterial TDA into an inactive compound, and thereby, this analysis supports the proposed mode of action of TDA.
Assuntos
Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Rhodobacteraceae/química , Compostos de Sulfidrila/isolamento & purificação , Compostos de Sulfidrila/farmacologia , Tropolona/análogos & derivados , Animais , Doenças dos Peixes/microbiologia , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Metilação , Testes de Sensibilidade Microbiana , Estrutura Molecular , Tropolona/isolamento & purificação , Tropolona/farmacologia , Vibrio/efeitos dos fármacosRESUMO
Only 1% of marine bacteria are currently culturable using standard laboratory procedures, and this is a major obstacle for our understanding of the biology of marine microorganisms and for the discovery of novel microbial natural products. Therefore, the purpose of this study was to investigate if improved cultivation conditions, including the use of an alternative gelling agent and supplementation with signaling molecules, improve the culturability of bacteria from seawater. Replacing agar with gellan gum improved viable counts 3- to 40-fold, depending on medium composition and incubation conditions, with a maximum of 6.6% culturability relative to direct cell counts. Through V4 amplicon sequencing we found that culturable diversity was also affected by a change in gelling agent, facilitating the growth of orders not culturable on agar-based substrates. Community analyses showed that communities grown on gellan gum substrates were significantly different from communities grown on agar and that they covered a larger fraction of the seawater community. Other factors, such as incubation temperature and time, had less obvious effects on viable counts and culturable diversity. Supplementation with acylated homoserine lactones (AHLs) did not have a positive effect on total viable counts or a strong effect on culturable diversity. However, low concentrations of AHLs increased the relative abundance of sphingobacteria. Hence, with alternative growth substrates, it is possible to significantly increase the number and diversity of cultured marine bacteria.IMPORTANCE Serious challenges to human health, such as the occurrence and spread of antibiotic resistance and an aging human population in need of bioactive pharmaceuticals, have revitalized the search for natural microbial products. The marine environment, representing the largest ecosystem in the biosphere, harbors an immense and virtually untapped microbial diversity producing unique bioactive compounds. However, we are currently able to cultivate only a minute fraction of this diversity. The lack of cultivated microbes is hampering not only bioprospecting efforts but also our general understanding of marine microbes. In this study, we present a means to increase the number and diversity of cultured bacteria from seawater, showing that relatively simple changes to medium components may facilitate the isolation and growth of hitherto unknown bacterial orders.
Assuntos
Organismos Aquáticos/efeitos dos fármacos , Organismos Aquáticos/crescimento & desenvolvimento , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Técnicas Bacteriológicas/métodos , Meios de Cultura/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Biodiversidade , Viabilidade Microbiana , Polissacarídeos Bacterianos/metabolismoRESUMO
UNLABELLED: Minimizing the use of antibiotics in the food production chain is essential for limiting the development and spread of antibiotic-resistant bacteria. One alternative intervention strategy is the use of probiotic bacteria, and bacteria of the marine Roseobacter clade are capable of antagonizing fish-pathogenic vibrios in fish larvae and live feed cultures for fish larvae. The antibacterial compound tropodithietic acid (TDA), an antiporter that disrupts the proton motive force, is key in the antibacterial activity of several roseobacters. Introducing probiotics on a larger scale requires understanding of any potential side effects of long-term exposure of the pathogen to the probionts or any compounds they produce. Here we exposed the fish pathogen Vibrio anguillarum to TDA for several hundred generations in an adaptive evolution experiment. No tolerance or resistance arose during the 90 days of exposure, and whole-genome sequencing of TDA-exposed lineages and clones revealed few mutational changes, compared to lineages grown without TDA. Amino acid-changing mutations were found in two to six different genes per clone; however, no mutations appeared unique to the TDA-exposed lineages or clones. None of the virulence genes of V. anguillarum was affected, and infectivity assays using fish cell lines indicated that the TDA-exposed lineages and clones were less invasive than the wild-type strain. Thus, long-term TDA exposure does not appear to result in TDA resistance and the physiology of V. anguillarum appears unaffected, supporting the application of TDA-producing roseobacters as probiotics in aquaculture. IMPORTANCE: It is important to limit the use of antibiotics in our food production, to reduce the risk of bacteria developing antibiotic resistance. We showed previously that marine bacteria of the Roseobacter clade can prevent or reduce bacterial diseases in fish larvae, acting as probiotics. Roseobacters produce the antimicrobial compound tropodithietic acid (TDA), and we were concerned regarding whether long-term exposure to this compound could induce resistance or affect the disease-causing ability of the fish pathogen. Therefore, we exposed the fish pathogen Vibrio anguillarum to increasing TDA concentrations over 3 months. We did not see the development of any resistance to TDA, and subsequent infection assays revealed that none of the TDA-exposed clones had increased virulence toward fish cells. Hence, this study supports the use of roseobacters as a non-risk-based disease control measure in aquaculture.
Assuntos
Antibacterianos/farmacologia , Doenças dos Peixes/microbiologia , Tropolona/análogos & derivados , Vibrioses/veterinária , Vibrio/efeitos dos fármacos , Animais , Farmacorresistência Bacteriana , Peixes , Genótipo , Fenótipo , Tropolona/farmacologia , Vibrio/genética , Vibrio/patogenicidade , Vibrio/fisiologia , Vibrioses/microbiologia , Virulência/efeitos dos fármacosRESUMO
The community composition of N2 -fixing microorganisms (diazotrophs) was investigated in copepods (primarily Acartia spp.) in parallel to that of seawater in coastal waters off Denmark (Øresund) and New England, USA. The unicellular cyanobacterial diazotroph UCYN-A was detected from seawater and full-gut copepods, suggesting that the new N contributed by UCYN-A is directly transferred to higher trophic levels in these waters. Deltaproteobacterial and Cluster 3 nifH sequences were detected in > 1 µm seawater particles and full-gut copepods, suggesting that they associate with copepods primarily via feeding. The dominant communities in starved copepods were Vibrio spp. and related Gammaproteobacteria, suggesting they represent the most permanent diazotroph associations in the copepods. N2 fixation rates were up to 3.02 pmol N copepod(-1) day(-1). Although at a typical copepod density in estuarine waters, these volumetric rates are low; considering the small size of a copepod, these mesozooplanktonic crustaceans may serve as hotspots of N2 fixation, at 12.9-71.9 µmol N dm(-3) copepod biomass day(-1). Taken together, diazotroph associations range from more permanent attachments to copepod feeding on some groups. Similar diazotroph groups detected on the eastern and western Atlantic Ocean suggest that these associations are a general phenomenon and play a role in the coastal N cycles.
Assuntos
Copépodes/microbiologia , Cianobactérias/metabolismo , Gammaproteobacteria/genética , Fixação de Nitrogênio/fisiologia , Nitrogênio/metabolismo , Alphaproteobacteria/genética , Alphaproteobacteria/metabolismo , Animais , Oceano Atlântico , Biomassa , Cianobactérias/genética , Deltaproteobacteria/genética , Deltaproteobacteria/metabolismo , Dinamarca , Gammaproteobacteria/isolamento & purificação , Gammaproteobacteria/metabolismo , Dados de Sequência Molecular , New England , Água do Mar/microbiologia , Vibrio/genética , Vibrio/isolamento & purificação , Vibrio/metabolismoRESUMO
The wide distribution of diverse nitrogenase (nifH) genes affiliated with those of heterotrophic bacteria in marine and estuarine waters indicates ubiquity and an ecologically relevant role for heterotrophic N2 -fixers (diazotrophs) in aquatic nitrogen (N) cycling. However, the lack of cultivated representatives currently precludes an evaluation of their N2 -fixing capacity. In this study, microoxic or anoxic N-free media were inoculated with estuarine Baltic Sea surface water to select for N2 -fixers. After visible growth and isolation of single colonies on oxic plates or in anoxic agar tubes, nifH gene amplicons were obtained from 64 strains and nitrogenase activity, applying the acetylene reduction assay, was confirmed for 40 strains. Two strains, one Gammaproteobacterium affiliated with Pseudomonas and one Alphaproteobacterium affiliated with Rhodopseudomonas were shown to represent established members of the indigenous diazotrophic community in the Baltic Sea, with abundances of up to 7.9 × 10(4) and 4.7 × 10(4) nifH copies l(-1) respectively. This study reports media for successful isolation of heterotrophic diazotrophs. The applied methodology and the obtained strains will facilitate future identification of factors controlling heterotrophic diazotrophic activity in aquatic environments, which is a prerequisite for understanding and evaluating their ecology and contribution to N cycling at local and regional scales.
Assuntos
Alphaproteobacteria/isolamento & purificação , Estuários , Gammaproteobacteria/isolamento & purificação , Fixação de Nitrogênio , Oxirredutases/genética , Água do Mar/microbiologia , Alphaproteobacteria/classificação , Alphaproteobacteria/genética , Bactérias/classificação , Bactérias/enzimologia , Bactérias/genética , Bactérias/isolamento & purificação , Gammaproteobacteria/classificação , Gammaproteobacteria/genética , Processos Heterotróficos , Fixação de Nitrogênio/genética , Filogenia , Microbiologia da ÁguaRESUMO
Our understanding of the role of secondary metabolites in microbial communities is challenged by intrinsic limitations of culturing bacteria under laboratory conditions and hence cultivation independent approaches are needed. Here, we present a protocol termed Secondary Metabolite FISH (SecMet-FISH), combining advantages of gene-targeted fluorescence in situ hybridization (geneFISH) with in-solution methods (in-solution FISH) to detect and quantify cells based on their genetic capacity to produce secondary metabolites. The approach capitalizes on the conserved nature of biosynthetic gene clusters (BGCs) encoding adenylation (AD) and ketosynthase (KS) domains, and thus selectively targets the genetic basis of non-ribosomal peptide and polyketide biosynthesis. The concept relies on the generation of amplicon pools using degenerate primers broadly targeting AD and KS domains followed by fluorescent labeling, detection, and quantification. Initially, we obtained AD and KS amplicons from Pseuodoalteromonas rubra, which allowed us to successfully label and visualize BGCs within P. rubra cells, demonstrating the feasibility of SecMet-FISH. Next, we adapted the protocol and optimized it for hybridization in both Gram-negative and Gram-positive bacterial cell suspensions, enabling high-throughput single cell analysis by flow cytometry. Ultimately, we used SecMet-FISH to successfully distinguish secondary metabolite producers from non-producers in a five-member synthetic community.
Assuntos
Família Multigênica , Hibridização in Situ Fluorescente/métodos , Citometria de FluxoRESUMO
Microalgal microbiomes play vital roles in the growth and health of their host, however, their composition and functions remain only partially characterized, especially across microalgal phyla. In this study, a natural seawater microbiome was introduced to three distinct, axenic species of microalgae, the haptophyte Isochrysis galbana, the chlorophyte Tetraselmis suecica, and the diatom Conticribra weissflogii (previously Thalassiosira), and its divergence and assembly under constant illumination was monitored over 49 days using 16S rRNA amplicon and metagenomic analyses. The microbiomes had a high degree of host specificity in terms of taxonomic composition and potential functions, including CAZymes profiles. Rhodobacteraceae and Flavobacteriaceae families were abundant across all microalgal hosts, but I. galbana microbiomes diverged further from T. suecica and C. weissflogii microbiomes. I. galbana microbiomes had a much higher relative abundance of Flavobacteriaceae, whereas the two other algal microbiomes had higher relative abundances of Rhodobacteraceae. This could be due to the bacterivorous mixotrophic nature of I. galbana affecting the carbohydrate composition available to the microbiomes, which was supported by the CAZymes profile of I. galbana microbiomes diverging further from those of T. suecica and C. weissflogii microbiomes. Finally, the presence of denitrification and other anaerobic pathways was found exclusively in the microbiomes of C. weissflogii, which we speculate could be a result of anoxic microenvironments forming in aggregates formed by this diatom during the experiment. These results underline the significant role of the microalgal host species on microbiome composition and functional profiles along with other factors, such as the trophic mode of the microalgal host. IMPORTANCE: As the main primary producers of the oceans, microalgae serve as cornerstones of the ecosystems they are part of. Additionally, they are increasingly used for biotechnological purposes such as the production of nutraceuticals, pigments, and antioxidants. Since the bacterial microbiomes of microalgae can affect their hosts in beneficial and detrimental ways, understanding these microbiomes is crucial to both the ecological and applied roles of microalgae. The present study advances the understanding of microalgal microbiome assembly, composition, and functionality across microalgal phyla, which may inform the modeling and engineering of microalgal microbiomes for biotechnological purposes.
Assuntos
Diatomáceas , Haptófitas , Microalgas , Microbiota , RNA Ribossômico 16S , Microbiota/fisiologia , RNA Ribossômico 16S/genética , Clorófitas/microbiologia , Água do Mar/microbiologiaRESUMO
In the search for novel drug candidates, diverse environmental microbiomes have been surveyed for their secondary metabolite biosynthesis potential, yet little is known about the biosynthetic diversity encoded by divergent microbiomes from different ecosystems, and the environmental parameters driving this diversity. Here, we used targeted amplicon sequencing of adenylation (AD) and ketosynthase (KS) domains along with 16S sequencing to delineate the unique biosynthetic potential of microbiomes from three separate habitats (soil, water, and sediments) exhibiting unique small spatial scale physicochemical gradients. The estimated richness of AD domains was highest in marine sediments with 656 ± 58 operational biosynthetic units (OBUs), while the KS domain richness was highest in soil microbiomes with 388 ± 67 OBUs. Microbiomes with rich and diverse bacterial communities displayed the highest PK potential across all ecosystems, and on a small spatial scale, pH and salinity were significantly, positively correlated to KS domain richness in soil and aquatic systems, respectively. Integrating our findings, we were able to predict the KS domain richness with a RMSE of 31 OBUs and a R2 of 0.91, and by the use of publicly available information on bacterial richness and diversity, we identified grassland biomes as being particularly promising sites for the discovery of novel polyketides. Furthermore, a focus on acidobacterial taxa is likely to be fruitful, as these were responsible for most of the variation in biosynthetic diversity. Overall, our results highlight the importance of sampling diverse environments with high taxonomic diversity in the pursuit for novel secondary metabolites. IMPORTANCE To counteract the antibiotic resistance crisis, novel anti-infective agents need to be discovered and brought to market. Microbial secondary metabolites have been important sources of inspiration for small-molecule therapeutics. However, the isolation of novel antibiotics is difficult, and the risk of rediscovery is high. With the overarching purpose of identifying promising microbiomes for discovery of novel bioactivity, we mapped out the most significant drivers of biosynthetic diversity across divergent microbiomes. We found the biosynthetic potential to be unique to individual ecosystems, and to depend on bacterial taxonomic diversity. Within systems, and on small spatial scales, pH and salinity correlated positively to the biosynthetic richness of the microbiomes, Acidobacteria representing the taxa most highly associated with biosynthetic diversity. Ultimately, understanding the key drivers of the biosynthesis potential of environmental microbiomes will allow us to focus bioprospecting efforts and facilitate the discovery of novel therapeutics.
Assuntos
Microbiota , Microbiota/genética , Bactérias/genética , Acidobacteria , Solo/química , Metabolismo SecundárioRESUMO
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.
Assuntos
Biofilmes , Microbiota , Antibacterianos , Água do MarRESUMO
As we stand on the brink of the post-antibiotic era, we are in dire need of novel antimicrobial compounds. Microorganisms produce a wealth of so-called secondary metabolites and have been our most prolific source of antibiotics so far. However, rediscovery of known antibiotics from well-studied cultured microorganisms, and the fact that the majority of microorganisms in the environment are out of reach by means of conventional cultivation techniques, have led to the exploration of the biosynthetic potential in natural microbial communities by novel approaches. In this mini review we discuss how sequence-based analyses have exposed an unprecedented wealth of potential for secondary metabolite production in soil, marine, and host-associated microbiomes, with a focus on the biosynthesis of non-ribosomal peptides and polyketides. Furthermore, we discuss how the complexity of natural microbiomes and the lack of standardized methodology has complicated comparisons across biomes. Yet, as even the most commonly sampled microbiomes hold promise of providing novel classes of natural products, we lastly discuss the development of approaches applied in the translation of the immense biosynthetic diversity of natural microbiomes to the procurement of novel antibiotics.
RESUMO
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.
Assuntos
Microbiota , Rhodobacteraceae , Antibacterianos , Fenótipo , Rhodobacteraceae/genética , Rhodobacteraceae/metabolismo , Tropolona/análogos & derivadosRESUMO
Novel natural products have traditionally been sourced from culturable soil microorganisms, whereas marine sources have been less explored. The purpose of this study was to profile the microbial biosynthetic potential in coastal surface seawater and sandy sediment samples and to evaluate the feasibility of capturing this potential using traditional culturing methods. Amplicon sequencing of conserved ketosynthase (KS) and adenylation (AD) domains within polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes showed that seawater and, in particular, sandy sediment had a high biosynthetic potential with 6,065 and 11,072 KS operational biosynthetic units (OBUs) and 3,292 and 5,691 AD OBUs, respectively, compared to that of four soil samples collected by Charlop-Powers et al. (Z. Charlop-Powers, C. C. Pregitzer, C. Lemetre, M. A. Ternei, et al., Proc Natl Acad Sci U S A 113:14811-14816, 2016, https://doi.org/10.1073/pnas.1615581113) with 7,067 KS and 1,629 AD OBUs. All three niches harbored unique OBUs (P = 0.001 for KS and P = 0.002 for AD by permutational multivariate analysis of variance [PERMANOVA]). The total colonial growth captured 1.9% of KS and 13.6% of AD OBUs from seawater and 2.2% KS and 12.5% AD OBUs from sediment. In a subset of bioactive isolates, only four KS OBUs and one AD OBU were recovered from whole-genome sequencing (WGS) of seven seawater-derived strains and one AD OBU from a sediment-derived strain, adding up to 0.028% of the original OBU diversity. Using a pairwise regression model of classified amplicon sequence variants (ASVs) to the species level, and OBUs, we suggest a method to estimate possible links between taxonomy and biosynthetic potential, which indicated that low abundance organisms may hold a disproportional share of the biosynthetic potential. Thus, marine microorganisms are a rich source of novel bioactive potential, which is difficult to access with traditional culturing methods.IMPORTANCE Since bacterial resistance to antibiotics is developing worldwide, new antibiotics are needed. Most antibiotics discovered so far have been found in soil-dwelling bacteria, so we instead targeted marine environments as a novel source of bioactive potential. We used amplicon sequencing of bioactive gene clusters in the microbiome of coastal seawater and sandy sediments and found the bioactive potential to be comparable to, but distinct from, the bioactive potential of selected soil microbiomes. Moreover, most of this potential is not captured by culturing. Comparing the biosynthetic potential to the corresponding microbiome composition suggested that minor constituents of the microbiome likely hold a disproportionally large fraction of the biosynthesis potential.
RESUMO
Bacteria-host interactions are universal in nature and have significant effects on host functionality. Bacterial secondary metabolites are believed to play key roles in such interactions as well as in interactions within the host-associated microbial community. Hence, prominent secondary metabolite-producing bacteria may be strong drivers of microbial community composition in natural host-associated microbiomes. This has, however, not been rigorously tested, and the purpose of this study was to investigate how the secondary metabolite producer Phaeobacter inhibens affects the diversity and composition of microbiomes associated with the microalga Emiliania huxleyi and the European flat oyster, Ostrea edulis. Roseobacters were indigenous to both communities exhibiting relative abundances between 2.8% and 7.0%. Addition of P. inhibens caused substantial changes in the overall structure of the low-complexity microbiome of E. huxleyi, but did not shape microbial community structure to the same degree in the more complex oyster microbiomes. Species-specific interactions occurred in both microbiomes and specifically the abundances of other putative secondary metabolite-producers such as vibrios and pseudoalteromonads were reduced. Thus, the impact of a bioactive strain like P. inhibens on host-associated microbiomes depends on the complexity and composition of the existing microbiome.
Assuntos
Microbiota/fisiologia , Rhodobacteraceae/fisiologia , Animais , Biodiversidade , Haptófitas/microbiologia , Especificidade de Hospedeiro , Microalgas/microbiologia , Interações Microbianas , Microbiota/genética , Ostreidae/microbiologia , RNA Ribossômico 16S/genéticaRESUMO
The Roseobacter group is a widespread marine bacterial group, of which some species produce the broad-spectrum antibiotic tropodithietic acid (TDA). A mode of action for TDA has previously been proposed in Escherichia coli, but little is known about its effect on non-producing marine bacteria at in situ concentrations. The purpose of this study was to investigate how a sub-lethal level of TDA affects Vibrio vulnificus at different time points (30 and 60 min) using a transcriptomic approach. Exposure to TDA for as little as 30 min resulted in the differential expression of genes associated with cell regeneration, including the up-regulation of those involved in biogenesis of the cell envelope. Defence mechanisms including oxidative stress defence proteins and iron uptake systems were also up-regulated in response to TDA, while motility-related genes were down-regulated. Gene expression data and scanning electron microscopy imaging revealed a switch to a biofilm phenotype in the presence of TDA. Our study shows that a low concentration of this antibiotic triggers a defence response to reactive oxygen species and iron depletion in V. vulnificus, which indicates that the mode of action of TDA is likely more complex in this bacterium than what is known for E. coli.
Assuntos
Antibacterianos/farmacologia , Expressão Gênica/efeitos dos fármacos , Tropolona/análogos & derivados , Vibrio vulnificus/efeitos dos fármacos , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Transporte Biológico/genética , Membrana Celular/metabolismo , Parede Celular/metabolismo , Perfilação da Expressão Gênica , Ferro/metabolismo , Estresse Oxidativo/genética , Tropolona/farmacologia , Vibrio vulnificus/genética , Vibrio vulnificus/metabolismo , Vibrio vulnificus/ultraestruturaRESUMO
The expanding aquaculture industry plays an important role in feeding the growing human population and with the expansion, sustainable bacterial disease control, such as probiotics, becomes increasingly important. Tropodithietic acid (TDA)-producing Phaeobacter spp. can protect live feed, for example rotifers and Artemia as well as larvae of turbot and cod against pathogenic vibrios. Here, we show that the emerging live feed, copepods, is unaffected by colonization of the fish pathogen Vibrio anguillarum, making them potential infection vectors. However, TDA-producing Phaeobacter inhibens was able to significantly inhibit V. anguillarum in non-axenic cultures of copepod Acartia tonsa and the copepod feed Rhodomonas salina. Vibrio grew to 106 CFU ml-1 and 107 CFU ml-1 in copepod and R. salina cultures, respectively. However, vibrio counts remained at the inoculum level (104 CFU ml-1 ) when P. inhibens was also added. We further developed a semi-strain-specific qPCR for V. anguillarum to detect and quantify the pathogen in non-axenic systems. In conclusion, P. inhibens efficiently inhibits the fish larval pathogen V. anguillarum in the emerging live feed, copepods, supporting its use as a probiotic in aquaculture. Furthermore, qPCR provides an effective method for detecting vibrio pathogens in complex non-axenic live feed systems.
Assuntos
Copépodes/crescimento & desenvolvimento , Criptófitas/crescimento & desenvolvimento , Doenças dos Peixes/microbiologia , Rhodobacteraceae/metabolismo , Tropolona/análogos & derivados , Vibrioses/veterinária , Vibrio/fisiologia , Ração Animal/análise , Animais , Aquicultura , Copépodes/fisiologia , Criptófitas/fisiologia , Doenças dos Peixes/metabolismo , Doenças dos Peixes/prevenção & controle , Linguados/metabolismo , Linguados/microbiologia , Probióticos/administração & dosagem , Tropolona/metabolismo , Vibrio/crescimento & desenvolvimento , Vibrioses/metabolismo , Vibrioses/microbiologia , Vibrioses/prevenção & controleRESUMO
The Roseobacter-group species Phaeobacter inhibens produces the antibacterial tropodithietic acid (TDA) and the algaecidal roseobacticides with both compound classes sharing part of the same biosynthetic pathway. The purpose of this study was to investigate the production of roseobacticides more broadly in TDA-producing roseobacters and to compare the effect of producers and non-producers on microalgae. Of 33 roseobacters analyzed, roseobacticide production was a unique feature of TDA-producing P. inhibens, P. gallaeciensis and P. piscinae strains. One TDA-producing Phaeobacter, 27-4, did not produce roseobacticides, possibly due to a transposable element. TDA-producing Ruegeria and Pseudovibrio did not produce roseobacticides. Addition of roseobacticide-containing bacterial extracts affected the growth of the microalgae Rhodomonas salina, Thalassiosira pseudonana and Emiliania huxleyi, while growth of Tetraselmis suecica was unaffected. During co-cultivation, growth of E. huxleyi was initially stimulated by the roseobacticide producer DSM 17395, while the subsequent decline in algal cell numbers during senescence was enhanced. Strain 27-4 that does not produce roseobacticides had no effect on algal growth. Both bacterial strains, DSM 17395 and 27-4, grew during co-cultivation presumably utilizing algal exudates. Furthermore, TDA-producing roseobacters have potential as probiotics in marine larviculture and it is promising that the live feed Tetraselmis was unaffected by roseobacticides-containing extracts.
Assuntos
Microalgas/crescimento & desenvolvimento , Microalgas/microbiologia , Roseobacter/metabolismo , Tropolona/análogos & derivados , Vias Biossintéticas , Filogenia , Roseobacter/classificação , Tropolona/metabolismoRESUMO
Microorganisms are of great importance to aquaculture where they occur naturally, and can be added artificially, fulfilling different roles. They recycle nutrients, degrade organic matter and, occasionally, they infect and kill the fish, their larvae or the live feed. Also, some microorganisms may protect fish and larvae against disease. Hence, monitoring and manipulating the microbial communities in aquaculture environments hold great potential; both in terms of assessing and improving water quality, but also in terms of controlling the development of microbial infections. Using microbial communities to monitor water quality and to efficiently carry out ecosystem services within the aquaculture systems may only be a few years away. Initially, however, we need to thoroughly understand the microbiomes of both healthy and diseased aquaculture systems, and we need to determine how to successfully manipulate and engineer these microbiomes. Similarly, we can reduce the need to apply antibiotics in aquaculture through manipulation of the microbiome, i.e. by the use of probiotic bacteria. Recent studies have demonstrated that fish pathogenic bacteria in live feed can be controlled by probiotics and that mortality of infected fish larvae can be reduced significantly by probiotic bacteria. However, the successful management of the aquaculture microbiota is currently hampered by our lack of knowledge of relevant microbial interactions and the overall ecology of these systems.