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Pseudoalteromonas rubra S4059 produces the red pigment prodigiosin, which has pharmaceutical and industrial potential. Here, we targeted a putative prodigiosin-synthesizing transferase PigC, and a pigC in-frame deletion mutant did not produce prodigiosin. However, extractions of the pigC mutant cultures retained antibacterial activity, and bioassay-guided fractionation found antibacterial activity in two fractions of blue color. A precursor of prodigiosin, 4-methoxy-2,2′-bipyrrole-5-carbaldehyde (MBC), was the dominant compound in both the fractions and likely caused the antibacterial activity. Also, a stable blue pigment, di-pyrrolyl-dipyrromethene prodigiosin, was identified from the two fractions. We also discovered antibacterial activity in the sterile filtered (nonextracted) culture supernatant of both wild type and mutant, and both contained a heat-sensitive compound between 30 and 100 kDa. Deletion of prodigiosin production did not affect growth rate or biofilm formation of P. rubra and did not change its fitness, as the mutant and wild type coexisted in equal levels in mixed cultures. In conclusion, a prodigiosin biosynthetic gene cluster (BGC) was identified and verified genetically and chemically in P. rubra S4059 and a stable blue pigment was isolated from the pigC mutant of S4059, suggesting that this strain may produce several prodigiosin-derived compounds of pharmaceutical and/or industrial potential. IMPORTANCE Pigmented Pseudoalteromonas strains are renowned for their production of secondary metabolites, and genome mining has revealed a high number of biosynthetic gene clusters (BGCs) for which the chemistry is unknown. Identification of those BGCs is a prerequisite for linking products to gene clusters and for further exploitation through heterologous expression. In this study, we identified the BGCs for the red, bioactive pigment prodigiosin using genomic, genetic, and metabolomic approaches. We al

Plastic is omnipresent in the oceans and serves as surface for biofilm-forming microorganisms. Plastic debris comprises different polymers, which may influence microbial colonization and here, we evaluated if polymer type affects bacterial biofilm formation. Quantifying the biofilm on polyethylene (PE), polypropylene (PP) or polystyrene (PS) pellets by six marine bacterial strains (Vibrio, Pseudoalteromonas, Phaeobacter) demonstrated that each strain had a unique colonization behavior with either a preference for PS or PP over the other polymer types or no preference for a specific plastic type. PE, PP, and PS pellets were exposed to natural seawater microbiota using free-living or total communities as inoculum. Microbial assembly as determined by 16S rRNA (V4) amplicon sequencing was affected by the composition of the initial inoculum and also by the plastic type. Known polymer and hydrocarbon degraders such as Paraglaciecola, Oleibacter, and Hydrogenophaga were found in the plastic biofilms. Thus, on a community level, bacterial colonization on plastic is influenced by the microorganisms as well as the polymer type, and also individual strains can demonstrate polymer-specific colonization.

The Phaeobacter genus has been explored as probiotics in mariculture as a sustainable strategy for 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 P. inhibens 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 varying effects on the diversity and richness of the microbiomes. The structure of the live-feed microbiomes were significantly changed, while no effect was seen on the community structure associated with turbot larvae. The changes were highly targeted at particular taxa. The Rhodobacterales order were indigenous to all three microbiomes and decreased in relative abundance when P. inhibens was introduced in the copepod and turbot microbiomes, while they were unaffected in the microalgal microbiome. Altogether, the study demonstrates that 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 are particular towards 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 Phaeobac

Our understanding of microbial natural environments combines in situ experimentation with studies of specific interactions in laboratory-based setups. The purpose of this work was to develop, build and demonstrate the use of a microbial culture chamber enabling both in situ and laboratory-based studies. The design uses an enclosed chamber surrounded by two porous membranes that enables the comparison of growth of two separate microbial populations but allowing free exchange of small molecules. Initially, we tested if the presence of the macroalga Fucus vesicuiosus inside the chamber affected colonization of the outer membranes by marine bacteria. The alga did indeed enrich the total population of colonizing bacteria by more than a factor of four. These findings lead us to investigate the effect of the presence of the coccolithophoric alga Emiliania huxleyi on attachment and biofilm formation of the marine bacterium Phaeobacter mhibens DSM17395. These organisms co-exist in the marine environment and have a well-characterized interdependence on secondary metabolites. P. inhibens attached in significantly higher numbers when having access to E. huxleyi as compared to when exposed to sterile media. The experiment was carried out using a wild type (wt) strain as well as a TDA-deficient strain of P. inhibens. The ability of the bacterium to produce the antibacterial compound, tropodithietic acid (TDA) influenced its attachment since the P. mhibens DSM 17395 wt strain attached in higher numbers to a surface within the first 48 h of incubation with E. huxleyi as compared to a TDA-negative mutant. Whilst the attachment of the bacterium to a surface was facilitated by presence of the alga, however, we cannot conclude if this was directly affected by the algae or whether biofilm formation was dependent on the production of TDA by P. inhibens, which has been implied by previous studies. In the light of

It has previously been reported that some strains of the marine bacterium Pseudoalteromonas luteoviolacea produce the purple bioactive pigment violacein as well as the antibiotic compound indolmycin, hitherto only found in Streptomyces. The purpose of the present study was to determine the relative role of each of these two compounds as antibacterial compounds in P. luteoviolacea S4054. Using Tn10 transposon mutagenesis, a mutant strain that was significantly reduced in violacein production in mannose-containing substrates was created. Full genome analyses revealed that the vio-biosynthetic gene cluster was not interrupted by the transposon; instead the insertion was located to the maeA gene encoding the malic enzyme. Supernatant of the mutant strain inhibited Vibrio anguillarum and Staphylococcus aureus in well diffusion assays and in MIC assays at the same level as the wild type strain. The mutant strain killed V. anguillarum in co-culture experiments as efficiently as the wild type. Using UHPLC-UV/Vis analyses, we quantified violacein and indolmycin, and the mutant strain only produced 7-10% the amount of violacein compared to the wild type strain. In contrast, the amount of indolmycin produced by the mutant strain was about 300% that of the wild type. Since inhibition of V. anguillarum and S. aureus by the mutant strain was similar to that of the wild type, it is concluded that violacein is not the major antibacterial compound in P. luteoviolacea. We furthermore propose that production of violacein and indolmycin may be metabolically linked and that yet unidentified antibacterial compound(s) may be play a role in the antibacterial activity of P. luteoviolacea.

Microorganisms are a rich source of bioactives; however, chemical identification is a major bottleneck. Strategies that can prioritize the most prolific microbial strains and novel compounds are of great interest. Here, we present an integrated approach to evaluate the biosynthetic richness in bacteria and mine the associated chemical diversity. Thirteen strains closely related to Pseudoalteromonas luteoviolacea isolated from all over the Earth were analyzed using an untargeted metabolomics strategy, and metabolomic profiles were correlated with whole-genome sequences of the strains. We found considerable diversity: only 2% of the chemical features and 7% of the biosynthetic genes were common to all strains, while 30% of all features and 24% of the genes were unique to single strains. The list of chemical features was reduced to 50 discriminating features using a genetic algorithm and support vector machines. Features were dereplicated by tandem mass spectrometry (MS/MS) networking to identify molecular families of the same biosynthetic origin, and the associated pathways were probed using comparative genomics. Most of the discriminating features were related to antibacterial compounds, including the thiomarinols that were reported from P. luteoviolacea here for the first time. By comparative genomics, we identified the biosynthetic cluster responsible for the production of the antibiotic indolmycin, which could not be predicted with standard methods. In conclusion, we present an efficient, integrative strategy for elucidating the chemical richness of a given set of bacteria and link the chemistry to biosynthetic genes.

Background: Antibiotic resistance in bacteria spreads quickly, overtaking the pace at which new compounds are discovered and this emphasizes the immediate need to discover new compounds for control of infectious diseases. Terrestrial bacteria have for decades been investigated as a source of bioactive compounds leading to successful applications in pharmaceutical and biotech industries. Marine bacteria have so far not been exploited to the same extent; however, they are believed to harbor a multitude of novel bioactive chemistry. To explore this potential, genomes of 21 marine Alpha- and Gammaproteobacteria collected during the Galathea 3 expedition were sequenced and mined for natural product encoding gene clusters. Results: Independently of genome size, bacteria of all tested genera carried a large number of clusters encoding different potential bioactivities, especially within the Vibrionaceae and Pseudoalteromonadaceae families. A very high potential was identified in pigmented pseudoalteromonads with up to 20 clusters in a single strain, mostly NRPSs and NRPS-PKS hybrids. Furthermore, regulatory elements in bioactivity-related pathways including chitin metabolism, quorum sensing and iron scavenging systems were investigated both in silico and in vitro. Genes with siderophore function were identified in 50% of the strains, however, all but one harboured the ferric-uptake-regulator gene. Genes encoding the syntethase of acylated homoserine lactones were found in Roseobacter-clade bacteria, but not in the Vibrionaceae strains and only in one Pseudoalteromonas strains. The understanding and manipulation of these elements can help in the discovery and production of new compounds never identified under regular laboratory cultivation conditions. High chitinolytic potential was demonstrated and verified for Vibrio and Pseudoalteromonas species that commonly live in close association with eukaryotic organisms in the environment. Chitin regulation by the ChiS histidine-k

In many species of the marine Roseobacter clade, periods of attached life, in association with phytoplankton or particles, are interspersed with planktonic phases. The purpose of this study was to determine whether shifts between motile and sessile life in the globally abundant Roseobacter clade species Ruegeria mobilis are associated with intracellular concentrations of the signal compound cyclic dimeric guanosinmonophosphate (c-di-GMP), which in bacteria regulates transitions between motile and sessile life stages. Genes for diguanylate cyclases and phosphodiesterases, which are involved in c-di-GMP signalling, were found in the genome of R. mobilis strain F1926. Ion pair chromatography-tandem mass spectrometry revealed 20-fold higher c-di-GMP concentrations per cell in biofilm-containing cultures than in planktonic cells. An introduced diguanylate cyclase gene increased c-di-GMP and enhanced biofilm formation and production of the potent antibiotic tropodithietic acid (TDA). An introduced phosphodiesterase gene decreased c-di-GMP and reduced biofilm formation and TDA production. tdaC, a key gene for TDA biosynthesis, was expressed only in attached or biofilm-forming cells, and expression was induced immediately after initial attachment. In conclusion, c-di-GMP signalling controls biofilm formation and biofilm-associated traits in R. mobilis and, as suggested by presence of GGDEF and EAL domain protein genes, also in other Roseobacter clade species.

Bacterial quorum sensing (QS) and the corresponding signals, acyl homoserine lactones (AHLs), were first described for a luminescent Vibrio species. Since then, detailed knowledge has been gained on the functional level of QS; however, the abundance of AHLs in the family of Vibrionaceae in the environment has remained unclear. Three hundred and one Vibrionaceae strains were collected on a global research cruise and the prevalence and profile of AHL signals in this global collection were determined. AHLs were detected in 32 of the 301 strains using Agrobacterium tumefaciens and Chromobacterium violaceum reporter strains. Ethyl acetate extracts of the cultures were analysed by ultra-high performance liquid chromatography-high resolution mass spectrometry (MS) with automated tandem MS confirmation for AHLs. N-(3-hydroxy-hexanoyl) (OH-C6) and N-(3-hydroxy-decanoyl) (OH-C10) homoserine lactones were the most common AHLs found in 17 and 12 strains, respectively. Several strains produced a diversity of different AHLs, including N-heptanoyl (C7) HL. AHL-producing Vibrionaceae were found in polar, temperate and tropical waters. The AHL profiles correlated with strain phylogeny based on gene sequence homology, however not with geographical location. In conclusion, a wide range of AHL signals are produced by a number of clades in the Vibrionaceae family and these results will allow future investigations of inter- and intra-species interactions within this cosmopolitan family of marine bacteria.