Phagentaxonomie in der Next Generation Sequencing-Ära

Phages are the biggest known biological entity on earth (about 1031 particles). Due to next generation sequencing methods applied on environmental samples an unpreceeded amount of phage genome data is available. Due to their extreme diversity and the lack of monophyly a sequence based taxonomy is challenging. However, within the phages there are monophyletic subgroups that can be classified based on their genome sequence. A method that combines the shared gene content with taxon specific similarities enables a reliable identification of the phage family based exclusively on the corresponding genome sequence

Genomic variation among closely related Vibrio alginolyticus strains is located on mobile genetic elements

Background: Species of the genus Vibrio, one of the most diverse bacteria genera, have undergone niche adaptation followed by clonal expansion. Niche adaptation and ultimately the formation of ecotypes and speciation in this genus has been suggested to be mainly driven by horizontal gene transfer (HGT) through mobile genetic elements (MGEs). Our knowledge about the diversity and distribution of Vibrio MGEs is heavily biased towards human pathogens and our understanding of the distribution of core genomic signatures and accessory genes encoded on MGEs within specific Vibrio clades is still incomplete. We used nine different strains of the marine bacterium Vibrio alginolyticus isolated from pipefish in the Kiel-Fjord to perform a multiscale-comparative genomic approach that allowed us to investigate [1] those genomic signatures that characterize a habitat-specific ecotype and [2] the source of genomic variation within this ecotype. Results: We found that the nine isolates from the Kiel-Fjord have a closed-pangenome and did not differ based on core-genomic signatures. Unique genomic regions and a unique repertoire of MGEs within the Kiel-Fjord isolates suggest that the acquisition of gene-blocks by HGT played an important role in the evolution of this ecotype. Additionally, we found that ~ 90% of the genomic variation among the nine isolates is encoded on MGEs, which supports ongoing theory that accessory genes are predominately located on MGEs and shared by HGT. Lastly, we could show that these nine isolates share a unique virulence and resistance profile which clearly separates them from all other investigated V. alginolyticus strains and suggests that these are habitat-specific genes, required for a successful colonization of the pipefish, the niche of this ecotype. Conclusion We conclude that all nine V. alginolyticus strains from the Kiel-Fjord belong to a unique ecotype, which we named the Kiel-alginolyticus ecotype. The low sequence variation of the core-genome in combination with the presence of MGE encoded relevant traits, as well as the presence of a suitable niche (here the pipefish), suggest, that this ecotype might have evolved from a clonal expansion following HGT driven niche-adaptation

Tripartite species interaction : eukarotic hosts suffer more from phage susceptible than from phage resistant bacteria

Background: Evolutionary shifts in bacterial virulence are often associated with a third biological player, for instance temperate phages, that can act as hyperparasites. By integrating as prophages into the bacterial genome they can contribute accessory genes, which can enhance the fitness of their prokaryotic carrier (lysogenic conversion). Hyperparasitic influence in tripartite biotic interactions has so far been largely neglected in empirical host-parasite studies due to their inherent complexity. Here we experimentally address whether bacterial resistance to phages and bacterial harm to eukaryotic hosts is linked using a natural tri-partite system with bacteria of the genus Vibrio, temperate vibriophages and the pipefish Syngnathus typhle. We induced prophages from all bacterial isolates and constructed a three-fold replicated, fully reciprocal 75 × 75 phage-bacteria infection matrix. Results: According to their resistance to phages, bacteria could be grouped into three distinct categories: highly susceptible (HS-bacteria), intermediate susceptible (IS-bacteria), and resistant (R-bacteria). We experimentally challenged pipefish with three selected bacterial isolates from each of the three categories and determined the amount of viable Vibrio counts from infected pipefish and the expression of pipefish immune genes. While the amount of viable Vibrio counts did not differ between bacterial groups, we observed a significant difference in relative gene expression between pipefish infected with phage susceptible and phage resistant bacteria. Conclusion: These findings suggest that bacteria with a phage-susceptible phenotype are more harmful against a eukaryotic host, and support the importance of hyperparasitism and the need for an integrative view across more than two levels when studying host-parasite evolution

The Bacteroidetes Aequorivita sp. and Kaistella jeonii Produce Promiscuous Esterases With PET-Hydrolyzing Activity

Certain members of the Actinobacteria and Proteobacteria are known to degrade polyethylene terephthalate (PET). Here, we describe the first functional PET-active enzymes from the Bacteroidetes phylum. Using a PETase-specific Hidden-Markov-Model- (HMM-) based search algorithm, we identified several PETase candidates from Flavobacteriaceae and Porphyromonadaceae. Among them, two promiscuous and cold-active esterases derived from Aequorivita sp. (PET27) and Kaistella jeonii (PET30) showed depolymerizing activity on polycaprolactone (PCL), amorphous PET foil and on the polyester polyurethane Impranil® DLN. PET27 is a 37.8 kDa enzyme that released an average of 174.4 nmol terephthalic acid (TPA) after 120 h at 30°C from a 7 mg PET foil platelet in a 200 μl reaction volume, 38-times more than PET30 (37.4 kDa) released under the same conditions. The crystal structure of PET30 without its C-terminal Por-domain (PET30ΔPorC) was solved at 2.1 Å and displays high structural similarity to the IsPETase. PET30 shows a Phe-Met-Tyr substrate binding motif, which seems to be a unique feature, as IsPETase, LCC and PET2 all contain Tyr-Met-Trp binding residues, while PET27 possesses a Phe-Met-Trp motif that is identical to Cut190. Microscopic analyses showed that K. jeonii cells are indeed able to bind on and colonize PET surfaces after a few days of incubation. Homologs of PET27 and PET30 were detected in metagenomes, predominantly aquatic habitats, encompassing a wide range of different global climate zones and suggesting a hitherto unknown influence of this bacterial phylum on man-made polymer degradation

Classifying the Unclassified: A Phage Classification Method

This work reports the method ClassiPhage to classify phage genomes using sequence derived taxonomic features. ClassiPhage uses a set of phage specific Hidden Markov Models (HMMs) generated from clusters of related proteins. The method was validated on all publicly available genomes of phages that are known to infect Vibrionaceae. The phages belong to the well-described phage families of Myoviridae, Podoviridae, Siphoviridae, and Inoviridae. The achieved classification is consistent with the assignments of the International Committee on Taxonomy of Viruses (ICTV), all tested phages were assigned to the corresponding group of the ICTV-database. In addition, 44 out of 58 genomes of Vibrio phages not yet classified could be assigned to a phage family. The remaining 14 genomes may represent phages of new families or subfamilies. Comparative genomics indicates that the ability of the approach to identify and classify phages is correlated to the conserved genomic organization. ClassiPhage classifies phages exclusively based on genome sequence data and can be applied on distinct phage genomes as well as on prophage regions within host genomes. Possible applications include (a) classifying phages from assembled metagenomes; and (b) the identification and classification of integrated prophages and the splitting of phage families into subfamilies

Closely Related Vibrio alginolyticus Strains Encode an Identical Repertoire of Caudovirales-Like Regions and Filamentous Phages

Many filamentous vibriophages encode virulence genes that lead to the emergence of pathogenic bacteria. Most genomes of filamentous vibriophages characterized up until today were isolated from human pathogens. Despite genome-based predictions that environmental Vibrios also contain filamentous phages that contribute to bacterial virulence, empirical evidence is scarce. This study aimed to characterize the bacteriophages of a marine pathogen, Vibrio alginolyticus (Kiel-alginolyticus ecotype) and to determine their role in bacterial virulence. To do so, we sequenced the phage-containing supernatant of eight different V. alginolyticus strains, characterized the phages therein and performed infection experiments on juvenile pipefish to assess their contribution to bacterial virulence. We were able to identify two actively replicating filamentous phages. Unique to this study was that all eight bacteria of the Kiel-alginolyticus ecotype have identical bacteriophages, supporting our previously established theory of a clonal expansion of the Kiel-alginolyticus ecotype. We further found that in one of the two filamentous phages, two phage-morphogenesis proteins (Zot and Ace) share high sequence similarity with putative toxins encoded on the Vibrio cholerae phage CTXΦ. The coverage of this filamentous phage correlated positively with virulence (measured in controlled infection experiments on the eukaryotic host), suggesting that this phage contributes to bacterial virulence

Closely Related Vibrio alginolyticus Strains Encode an Identical Repertoire of Caudovirales-Like Regions and Filamentous Phages

Many filamentous vibriophages encode virulence genes that lead to the emergence of pathogenic bacteria. Most genomes of filamentous vibriophages characterized up until today were isolated from human pathogens. Despite genome-based predictions that environmental Vibrios also contain filamentous phages that contribute to bacterial virulence, empirical evidence is scarce. This study aimed to characterize the bacteriophages of a marine pathogen, Vibrio alginolyticus (Kiel-alginolyticus ecotype) and to determine their role in bacterial virulence. To do so, we sequenced the phage-containing supernatant of eight different V. alginolyticus strains, characterized the phages therein and performed infection experiments on juvenile pipefish to assess their contribution to bacterial virulence. We were able to identify two actively replicating filamentous phages. Unique to this study was that all eight bacteria of the Kiel-alginolyticus ecotype have identical bacteriophages, supporting our previously established theory of a clonal expansion of the Kiel-alginolyticus ecotype. We further found that in one of the two filamentous phages, two phage-morphogenesis proteins (Zot and Ace) share high sequence similarity with putative toxins encoded on the Vibrio cholerae phage CTXΦ. The coverage of this filamentous phage correlated positively with virulence (measured in controlled infection experiments on the eukaryotic host), suggesting that this phage contributes to bacterial virulence

The Life Cycle of Aurelia aurita Depends on the Presence of a Microbiome in Polyps Prior to Onset of Strobilation

ABSTRACT Aurelia aurita’s intricate life cycle alternates between benthic polyp and pelagic medusa stages. The strobilation process, a critical asexual reproduction mechanism in this jellyfish, is severely compromised in the absence of the natural polyp microbiome, with limited production and release of ephyrae. Yet, the recolonization of sterile polyps with a native polyp microbiome can correct this defect. Here, we investigated the precise timing necessary for recolonization as well as the host-associated molecular processes involved. We deciphered that a natural microbiota had to be present in polyps prior to the onset of strobilation to ensure normal asexual reproduction and a successful polyp-to-medusa transition. Providing the native microbiota to sterile polyps after the onset of strobilation failed to restore the normal strobilation process. The absence of a microbiome was associated with decreased transcription of developmental and strobilation genes as monitored by reverse transcription-quantitative PCR. Transcription of these genes was exclusively observed for native polyps and sterile polyps that were recolonized before the initiation of strobilation. We further propose that direct cell contact between the host and its associated bacteria is required for the normal production of offspring. Overall, our findings indicate that the presence of a native microbiome at the polyp stage prior to the onset of strobilation is essential to ensure a normal polyp-to-medusa transition. IMPORTANCE All multicellular organisms are associated with microorganisms that play fundamental roles in the health and fitness of the host. Notably, the native microbiome of the Cnidarian Aurelia aurita is crucial for the asexual reproduction by strobilation. Sterile polyps display malformed strobilae and a halt of ephyrae release, which is restored by recolonizing sterile polyps with a native microbiota. Despite that, little is known about the microbial impact on the strobilation process's timing and molecular consequences. The present study shows that A. aurita’s life cycle depends on the presence of the native microbiome at the polyp stage prior to the onset of strobilation to ensure the polyp-to-medusa transition. Moreover, sterile individuals correlate with reduced transcription levels of developmental and strobilation genes, evidencing the microbiome's impact on strobilation on the molecular level. Transcription of strobilation genes was exclusively detected in native polyps and those recolonized before initiating strobilation, suggesting microbiota-dependent gene regulation