Production and regulation of fouling inhibitory compounds by the marine bacterium Pseudoalteromonas tunicata

The marine surface-associated bacterium Pseudoaltermonas tunicata, produces a range of compounds that inhibit fouling organisms, including invertebrate larvae, bacteria, algal spores and fungi. In addition to these antifouling compounds P. tunicata cells produce both a yellow and a purple pigment. The aim of this study was to further characterise the antifouling activities, their regulation and relationship with pigmentation, and the ecological significance of P. tunicata and related organisms. It was discovered that the anti-algal compound was extracellular, heat sensitive, polar and between 3 and 10 kDa in size. The anti-fungal compound was found to be the yellow pigment and active against a wide range of fungal and yeast isolates. Chemical analysis suggests that this compound consists of a carbon ring bound to a fatty-acid side chain. Genetic analysis supports the chemical data for the active compound as a mutant in a gene encoding for a long-chain fatty-acid CoA ligase was deficient for anti-fungal activity. To address the regulation of antifouling compounds and their relationship to pigmentation transposon mutagenesis of P. tunicata was performed. Mutants lacking the yellow pigment displayed a reduced ability to inhibit fouling organisms. Further analysis of these mutants identified genes involved with the synthesis and regulation of synthesis of pigment and antifouling compounds. One of these mutants was disrupted in a gene (wmpR) with similarity to the transcriptional regulators ToxR from Vibrio cholerae and CadC from Escherichia coli. Analysis of global protein expression using two-dimensional gel electrophoresis showed that WmpR is essential for the expression of at least fifteen proteins important for the synthesis of fouling inhibitors. The ecological significance of antifouling bacteria was addressed by assessing the antifouling capabilities of a collection of bacteria isolated from different marine surfaces. Overall, isolates from living surfaces displayed more antifouling traits then strains isolated from non-living surfaces. Five dark-pigmented strains originating from the alga Ulva lactuca were further studied. Phylogenetic and phenotypic analysis revealed that they were all members of the genus Pseudoalteromonas and were closely related to P. tunicata. Two strains represented a novel species within the genus and were taxonomically defined as P. ulvae sp. nov

Development of Novel Drugs from Marine Surface Associated Microorganisms

While the oceans cover more than 70% of the Earth’s surface, marine derived microbial natural products have been largely unexplored. The marine environment is a habitat for many unique microorganisms, which produce biologically active compounds (“bioactives”) to adapt to particular environmental conditions. For example, marine surface associated microorganisms have proven to be a rich source for novel bioactives because of the necessity to evolve allelochemicals capable of protecting the producer from the fierce competition that exists between microorganisms on the surfaces of marine eukaryotes. Chemically driven interactions are also important for the establishment of cross-relationships between microbes and their eukaryotic hosts, in which organisms producing antimicrobial compounds (“antimicrobials”), may protect the host surface against over colonisation in return for a nutrient rich environment. As is the case for bioactive discovery in general, progress in the detection and characterization of marine microbial bioactives has been limited by a number of obstacles, such as unsuitable culture conditions, laborious purification processes, and a lack of de-replication. However many of these limitations are now being overcome due to improved microbial cultivation techniques, microbial (meta-) genomic analysis and novel sensitive analytical tools for structural elucidation. Here we discuss how these technical advances, together with a better understanding of microbial and chemical ecology, will inevitably translate into an increase in the discovery and development of novel drugs from marine microbial sources in the future

Phylogenetic screening of a bacterial, metagenomic library using homing endonuclease restriction and marker insertion

Metagenomics provides access to the uncultured majority of the microbial world. The approaches employed in this field have, however, had limited success in linking functional genes to the taxonomic or phylogenetic origin of the organism they belong to. Here we present an efficient strategy to recover environmental DNA fragments that contain phylogenetic marker genes from metagenomic libraries. Our method involves the cleavage of 23S ribsosmal RNA (rRNA) genes within pooled library clones by the homing endonuclease I-CeuI followed by the insertion and selection of an antibiotic resistance cassette. This approach was applied to screen a library of 6500 fosmid clones derived from the microbial community associated with the sponge Cymbastela concentrica. Several fosmid clones were recovered after the screen and detailed phylogenetic and taxonomic assignment based on the rRNA gene showed that they belong to previously unknown organisms. In addition, compositional features of these fosmid clones were used to classify and taxonomically assign a dataset of environmental shotgun sequences. Our approach represents a valuable tool for the analysis of rapidly increasing, environmental DNA sequencing information

Identification of the Antibacterial Compound Produced by the Marine Epiphytic Bacterium Pseudovibrio sp. D323 and Related Sponge-Associated Bacteria

Surface-associated marine bacteria often produce secondary metabolites with antagonistic activities. In this study, tropodithietic acid (TDA) was identified to be responsible for the antibacterial activity of the marine epiphytic bacterium Pseudovibrio sp. D323 and related strains. Phenol was also produced by these bacteria but was not directly related to the antibacterial activity. TDA was shown to effectively inhibit a range of marine bacteria from various phylogenetic groups. However TDA-producers themselves were resistant and are likely to possess resistance mechanism preventing autoinhibition. We propose that TDA in isolate D323 and related eukaryote-associated bacteria plays a role in defending the host organism against unwanted microbial colonisation and, possibly, bacterial pathogens

Developing a protocol for the effective aplication of Phaeobacter in Ulva cultures for pathogen control in IMTA-RAS

Poster.-- COST ACTION CA 20106: SeaWheat Ulva: Tomorrows "Wheat of the Sea", a Model for an Innovative Mariculture, 1st Conference: From fundamental biology to aquaculture: state-of-the-art, bottlenecks and gaps - Cádiz, Spain, 12-15 September 2022N

Shedding light on the Ulva holobiont: Alga-bacteria interactions with implications for integrated multitrofic aquaculture

Poster.-- Close Encounters IIM (3rd Kind), Vigo, 23 June 2022Macroalgae, like Ulva genus, provides an important niche for epiphytic biofilm-forming bacteria, including those of the genus Phaeobacter with the ability to antagonize fish pathogens such as Vibrio anguillarum, through the production of tropodithietic acid (TDA) [1–3]. P. gallaeciensis has previously demonstrated its effectiveness as a probiotic in aquaculture by reducing mortality in fish larvae experimentally infected with this pathogen as well as its colonization of U. ohnoi surface [1]. This colonization can be used as apathogen control strategy in multitrophic fish-algae cultures in recirculatingwater systems (IMTA-RAS), improving the health of the fish (Fig. 1).However, the optimal conditions for the culture of U. ohnoi could have a determining influence both on the maintenance of these biofilms and on the production of TDA, especially the intensity of lightN

A horizon scan of priorities for coastal marine microbiome research

Research into the microbiomes of natural environments is changing the way ecologists and evolutionary biologists view the importance of microbes in ecosystem function. This is particularly relevant in ocean environments, where microbes constitute the majority of biomass and control most of the major biogeochemical cycles, including those that regulate the Earth's climate. Coastal marine environments provide goods and services that are imperative to human survival and well-being (e.g. fisheries, water purification), and emerging evidence indicates that these ecosystem services often depend on complex relationships between communities of microorganisms (the ‘microbiome’) and their hosts or environment – termed the ‘holobiont’. Understanding of coastal ecosystem function must therefore be framed under the holobiont concept, whereby macroorganisms and their associated microbiomes are considered as a synergistic ecological unit. Here we evaluated the current state of knowledge on coastal marine microbiome research and identified key questions within this growing research area. Although the list of questions is broad and ambitious, progress in the field is increasing exponentially, and the emergence of large, international collaborative networks and well-executed manipulative experiments are rapidly advancing the field of coastal marine microbiome research

A horizon scan of priorities for coastal marine microbiome research

Research into the microbiomes of natural environments is changing the way ecologists and evolutionary biologists view the importance of microbes in ecosystem function. This is particularly relevant in ocean environments, where microbes constitute the majority of biomass and control most of the major biogeochemical cycles, including those that regulate the Earth's climate. Coastal marine environments provide goods and services that are imperative to human survival and well-being (e.g. fisheries, water purification), and emerging evidence indicates that these ecosystem services often depend on complex relationships between communities of microorganisms (the ‘microbiome’) and their hosts or environment – termed the ‘holobiont’. Understanding of coastal ecosystem function must therefore be framed under the holobiont concept, whereby macroorganisms and their associated microbiomes are considered as a synergistic ecological unit. Here we evaluated the current state of knowledge on coastal marine microbiome research and identified key questions within this growing research area. Although the list of questions is broad and ambitious, progress in the field is increasing exponentially, and the emergence of large, international collaborative networks and well-executed manipulative experiments are rapidly advancing the field of coastal marine microbiome research