Project description:We identify genetic determinants of Phaeobacter inhibens susceptibility to Phaeobacter phage MD18 Overall design: We screen Phaeobacter phage MD18 against a library of Phaeobacter inhibens barcoded transposon insertion mutants
Project description:The Roseobacter clade is a key group of bacteria in the ocean exhibiting diverse metabolic repertoires and a wide range of symbiotic life-styles. Many Roseobacters possess remarkable capabilities of attachment to both biotic and abiotic surfaces. When attached to each other, these bacteria form multi-cellular structures called rosettes. Phaeobacter inhibens, a well-studied Roseobacter, exhibits various cell sizes and morphologies that are either associated with rosettes or occur as single cells. Here we describe the distribution of P. inhibens morphologies and rosettes within a population. We detect an N-acetylglucosamine-containing polysaccharide on the poles of some cells and at the center of all rosettes. We demonstrate that rosettes are formed by the attachment of individual cells at the polysaccharide-containing pole rather than by cell division. Finally, we show that P. inhibens attachment to abiotic surfaces is hindered by the presence of DNA from itself, but not from other bacteria. Taken together, our findings demonstrate that cell adhesiveness is likely to play a significant role in the life cycle of P. inhibens as well as other Roseobacters.
Project description:Strain T5(T) is the type strain of the species Phaeobacter inhibens Martens et al. 2006, a secondary metabolite producing bacterium affiliated to the Roseobacter clade. Strain T5(T) was isolated from a water sample taken at the German Wadden Sea, southern North Sea. Here we describe the complete genome sequence and annotation of this bacterium with a special focus on the secondary metabolism and compare it with the genomes of the Phaeobacter inhibens strains DSM 17395 and DSM 24588 (2.10), selected because of the close phylogenetic relationship based on the 16S rRNA gene sequences of these three strains. The genome of strain T5(T) comprises 4,130,897 bp with 3.923 protein-coding genes and shows high similarities in genetic and genomic characteristics compared to P. inhibens DSM 17395 and DSM 24588 (2.10). Besides the chromosome, strain T5(T) possesses four plasmids, three of which show a high similarity to the plasmids of the strains DSM 17395 and DSM 24588 (2.10). Analysis of the fourth plasmid suggested horizontal gene transfer. Most of the genes on this plasmid are not present in the strains DSM 17395 and DSM 24588 (2.10) including a nitrous oxide reductase, which allows strain T5(T) a facultative anaerobic lifestyle. The G+C content was calculated from the genome sequence and differs significantly from the previously published value, thus warranting an emendation of the species description.
Project description:Phaeobacter inhibens DSM 17395, a model organism for marine Roseobacter group, was studied for its response to its own antimicrobial compound tropodithietic acid (TDA). TDA biosynthesis is encoded on the largest extrachromosomal element of P. inhibens, the 262 kb plasmid, whose curation leads to an increased growth and biomass yield. In this study, the plasmid-cured strain was compared to the wild-type strain and to transposon mutants lacking single genes of the TDA biosynthesis. The data show that the growth inhibition of the wild-type strain can be mainly attributed to the TDA produced by P. inhibens itself. Oxygen uptake rates remained constant in all strains but the growth rate dropped in the wild-type which supports the recently proposed mode of TDA action. Metabolome analysis showed no metabolic alterations that could be attributed directly to TDA. Taken together, the growth of P. inhibens is limited by its own antibacterial compound due to a partial destruction of the proton gradient which leads to a higher energetic demand. The universal presence of TDA biosynthesis in genome-sequenced isolates of the genus Phaeobacter shows that there must be a high benefit of TDA for P. inhibens in its ecological niche despite the drawback on its metabolism.
Project description:Phaeobacter inhibens 2.10 is an effective biofilm former and colonizer of marine surfaces and has the ability to outcompete other microbiota. During biofilm dispersal P. inhibens 2.10 produces heritable phenotypic variants, including those that have a reduced ability to inhibit the co-occurring bacterium Pseudoalteromonas tunicata. However, the genetic changes that underpin the phenotypic variation and what the ecological consequences are for variants within the population are unclear. To answer these questions we sequenced the genomes of strain NCV12a1, a biofilm variant of P. inhibens 2.10 with reduced inhibitory activity and the P. inhibens 2.10 WT parental strain. Genome wide analysis revealed point mutations in genes involved in synthesis of the antibacterial compound tropodithietic acid (TDA) and indirectly in extracellular polymeric substances (EPS) production. However, confocal laser scanning microscopy analyses found little differences in biofilm growth between P. inhibens 2.10 WT (parental) and NCV12a1. P. inhibens NCV12a1 was also not outcompeted in co-cultured biofilms with P. tunicata, despite its reduced inhibitory activity, rather these biofilms were thicker than those produced when the WT strain was co-cultured with P. tunicata. Notably, dispersal populations from biofilms of P. inhibens NCV12a1 had a higher proportion of WT-like morphotypes when co-cultured with P. tunicata. These observations may explain why the otherwise non-inhibiting variant persists in the presence of a natural competitor, adding to our understanding of the relative importance of genetic diversification in microbial biofilms.
Project description:The model coccolithophore, Emiliania huxleyi, forms expansive blooms dominated by the calcifying cell type, which produce calcite scales called coccoliths. Blooms last several weeks, after which the calcified algal cells rapidly die, descending into the deep ocean. E. huxleyi bloom collapse is attributed to E. huxleyi viruses (EhVs) that infect and kill calcifying cells, while other E. huxleyi pathogens, such as bacteria belonging to the roseobacter clade, are overlooked. EhVs kill calcifying E. huxleyi by inducing production of bioactive viral-glycosphingolipids (vGSLs), which trigger algal programmed cell death (PCD). The roseobacter Phaeobacter inhibens was recently shown to interact with and kill the calcifying cell type of E. huxleyi, but the mechanism of algal death remains unelucidated. Here we demonstrate that P. inhibens kills calcifying E. huxleyi by inducing a highly specific type of PCD called apoptosis-like-PCD (AL-PCD). Host death can successfully be abolished in the presence of a pan-caspase inhibitor, which prevents the activation of caspase-like molecules. This finding differentiates P. inhibens and EhV pathogenesis of E. huxleyi, by demonstrating that bacterial-induced AL-PCD requires active caspase-like molecules, while the viral pathogen does not. This is the first demonstration of a bacterium inducing AL-PCD in an algal host as a killing mechanism.
Project description: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.
Project description:The <i>Phaeobacter</i> 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 <i>in vitro</i> 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 <i>Phaeobacter inhibens</i> 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 (<i>Tetraselmis suecica</i>), live feed copepod nauplii (<i>Acartia tonsa</i>), and turbot (<i>Scophthalmus maximus</i>) 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 <i>Rhodobacterales</i> order was indigenous to all three microbiomes and decreased in relative abundance when <i>P. inhibens</i> was introduced in the copepod and turbot microbiomes, while it was unaffected in the microalgal microbiome. Altogether, the study demonstrates that the addition of <i>P. inhibens</i> 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.<b>IMPORTANCE</b> 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 <i>Phaeobacter inhibens</i> 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.
Project description:Tropodithietic acid (TDA) is an antibacterial compound produced by some Phaeobacter and Ruegeria spp. of the Roseobacter clade. TDA production is studied in marine broth or agar since antibacterial activity in other media is not observed. The purpose of this study was to determine how TDA production is influenced by substrate components. High concentrations of ferric citrate, as present in marine broth, or other iron sources were required for production of antibacterially active TDA. However, when supernatants of noninhibitory, low-iron cultures of Phaeobacter inhibens were acidified, antibacterial activity was detected in a bioassay. The absence of TDA in nonacidified cultures and the presence of TDA in acidified cultures were verified by liquid chromatography-high-resolution mass spectrometry. A noninhibitory TDA analog (pre-TDA) was produced by P. inhibens, Ruegeria mobilis F1926, and Phaeobacter sp. strain 27-4 under low-iron concentrations and was instantaneously converted to TDA when pH was lowered. Production of TDA in the presence of Fe(3+) coincides with formation of a dark brown substance, which could be precipitated by acid addition. From this brown pigment TDA could be liberated slowly with aqueous ammonia, and both direct-infusion mass spectrometry and elemental analysis indicated a [Fe(III)(TDA)2]x complex. The pigment could also be produced by precipitation of pure TDA with FeCl3. Our results raise questions about how biologically active TDA is produced in natural marine settings where iron is typically limited and whether the affinity of TDA to iron points to a physiological or ecological function of TDA other than as an antibacterial compound.