ISOLATION, GENOMICS AND ECOLOGY OF BACTERIOPHAGES INFECTING MARINE ROSEOBACTERS

Viruses are the most abundant biological entities in seawater. They influence the population dynamics, genetic heterogeneity, and biogeochemical cycles in marine ecosystems. Isolation and characterization of viruses which infect specific hosts have greatly advanced our knowledge on the biological and ecological interactions between viruses and their hosts. Roseobacter is an important lineage of marine bacteria which are genetically diverse, abundant and ubiquitous in the ocean. Roseobacters can make up to 25% of bacterial communities in coastal environments and play an active role in the marine sulfur cycle. However, only few bacteriophages which infect marine roseobacters had been isolated at the time when I began my studies. To understand the types of bacteriophages that infect roseobacters and how they interact with their hosts, I devoted my research to isolation and characterization of the bacteriophages infecting roseobacters (roseophages hereafter). In this dissertation, fourteen different phages infecting a marine strain, Ruegeria pomeroyi DSS-3, are described in terms of their morphology, growth, genomics and global distributions. These 14 roseophages were divided into four different groups: ssDNA, CbK-like, Chi-like, and N4-like roseophages. Two ssDNA phages belongs to an unclassified group of Microviridae. They contain only four ORFs with a genome size of 4.2 kb, representing the smallest and of all known ssDNA phage isolates. Interestingly, the ssDNA roseophages fall into a large group of uncultivated viral sequences identified by viral metagenomics. The isolation of CbK-like roseophages uncovers a new type of Siphoviridae infecting a member of Roseobacter lineage, Prior to this work, CbK-like phages had only been reported in a freshwater bacterium Caulobacter. The two CbK-like roseophage genomes are highly mosaic, containing features from siphoviruses, podoviruses, gene transfer agents, integrases and a large number of tRNAs. Chi-like siphophages are another newly discovered group of roseophages. Five different Chi-like phages (Siphoviridae) were isolated from DSS-3. A resistant strain of R. pomeroyi DSS-3 was found during superinfection with Chi-like roseophage DSS3Φ1. Genome sequencing confirmed that the resistant strain contains the intact genome of DSS3Φ1. The ability to integrate phage genome into host chromosome confirms that DSS3Φ1 is a temperate phage. Five N4-like roseophages of DSS-3 were isolated. They belong to the phage N4 lineage in Podoviridae. Genomes of N4-roseophages are highly syntenic, sharing a very similar genomic arrangement. The genomic conservation of N4-like phages allowed me to design N4-like phage specific primers based on their DNA polymerase genes. The primer set was used to PCR amplify the DNA pol gene of N4-like phages from 56 DNA samples to investigate the diversity and distribution of N4-like phages in the Chesapeake Bay. Surprisingly, N4-like phage sequences were only detected in the winter samples collected over two years. Metagenomic recruitments also confirmed that N4-like phages appear to prevail in the cold environment, such as Organic Lake, a hypersaline lake in Antarctica, where the temperature is usually below -10 C. According to metagenomic analyses, homologs of other DSS-3 phages (non-N4-like) are present in freshwater and marine habitats, Antarctica, human gut and feces, and coral-associated environments. This wide range distribution of roseophages seems to reflect the cosmopolitan nature of the Roseobacter clade. The discovery of different types of phages infecting a single strain and their wide distribution suggest that we are only seeing the tip of the iceberg of phages

A new family of globally distributed lytic roseophages with unusual deoxythymidine to deoxyuridine substitution

Marine bacterial viruses (bacteriophages) are abundant biological entities that are vital for shaping microbial diversity, impacting marine ecosystem function, and driving host evolution.1, 2, 3 The marine roseobacter clade (MRC) is a ubiquitous group of heterotrophic bacteria[4],[5] that are important in the elemental cycling of various nitrogen, sulfur, carbon, and phosphorus compounds.6, 7, 8, 9, 10 Bacteriophages infecting MRC (roseophages) have thus attracted much attention and more than 30 roseophages have been isolated,11, 12, 13 the majority of which belong to the N4-like group (Podoviridae family) or the Chi-like group (Siphoviridae family), although ssDNA-containing roseophages are also known.[14] In our attempts to isolate lytic roseophages, we obtained two new phages (DSS3_VP1 and DSS3_PM1) infecting the model MRC strain Ruegeria pomeroyi DSS-3. Here, we show that not only do these phages have unusual substitution of deoxythymidine with deoxyuridine (dU) in their DNA, but they are also phylogenetically distinct from any currently known double-stranded DNA bacteriophages, supporting the establishment of a novel family (“Naomiviridae”). These dU-containing phages possess DNA that is resistant to the commonly used library preparation method for metagenome sequencing, which may have caused significant underestimation of their presence in the environment. Nevertheless, our analysis of Tara Ocean metagenome datasets suggests that these unusual bacteriophages are of global importance and more diverse than other well-known bacteriophages, e.g., the Podoviridae in the oceans, pointing to an overlooked role for these novel phages in the environment

Optimizing de novo genome assembly from PCR-amplified metagenomes

Background Metagenomics has transformed our understanding of microbial diversity across ecosystems, with recent advances enabling de novo assembly of genomes from metagenomes. These metagenome-assembled genomes are critical to provide ecological, evolutionary, and metabolic context for all the microbes and viruses yet to be cultivated. Metagenomes can now be generated from nanogram to subnanogram amounts of DNA. However, these libraries require several rounds of PCR amplification before sequencing, and recent data suggest these typically yield smaller and more fragmented assemblies than regular metagenomes. Methods Here we evaluate de novo assembly methods of 169 PCR-amplified metagenomes, including 25 for which an unamplified counterpart is available, to optimize specific assembly approaches for PCR-amplified libraries. We first evaluated coverage bias by mapping reads from PCR-amplified metagenomes onto reference contigs obtained from unamplified metagenomes of the same samples. Then, we compared different assembly pipelines in terms of assembly size (number of bp in contigs ≥ 10 kb) and error rates to evaluate which are the best suited for PCR-amplified metagenomes. Results Read mapping analyses revealed that the depth of coverage within individual genomes is significantly more uneven in PCR-amplified datasets versus unamplified metagenomes, with regions of high depth of coverage enriched in short inserts. This enrichment scales with the number of PCR cycles performed, and is presumably due to preferential amplification of short inserts. Standard assembly pipelines are confounded by this type of coverage unevenness, so we evaluated other assembly options to mitigate these issues. We found that a pipeline combining read deduplication and an assembly algorithm originally designed to recover genomes from libraries generated after whole genome amplification (single-cell SPAdes) frequently improved assembly of contigs ≥10 kb by 10 to 100-fold for low input metagenomes. Conclusions PCR-amplified metagenomes have enabled scientists to explore communities traditionally challenging to describe, including some with extremely low biomass or from which DNA is particularly difficult to extract. Here we show that a modified assembly pipeline can lead to an improved de novo genome assembly from PCR-amplified datasets, and enables a better genome recovery from low input metagenomes

Succession of marine bacteria in response to Ulva prolifera-derived dissolved organic matter

Increasing macroalgal blooms as a consequence of climate warming and coastal eutrophication have profound effects on the marine environment. The outbreaks of Ulva prolifera in the Yellow Sea of China occurring every summer since 2007 to present have formed the world’s largest green tide. The green tide releases huge amounts of dissolved organic matter (DOM) to the seawater, causing an organic overload. However, how marine bacteria respond to this issue and the potential impact on the marine environment are still unclear. Here, we monitored the highly temporally resolved dynamics of marine bacterial community that occur in response to Ulva prolifera-derived DOM by performing a 168-h microcosm incubation experiment. DOM inputs significantly increased bacterial abundances within 6 h, decreased bacterial diversity and triggered clear community successions during the whole period of incubation. Vibrio of Gammaproteobacteria robustly and rapidly grew over short timescales (6–24 h), with its relative abundance accounting for up to 52.5% of active bacteria. From 24 to 48 h, some genera of Flavobacteriia grew rapidly, which was more conspicuous at a higher DOM concentration than at a lower concentration. The genus Donghicola of Alphaproteobacteria was predominant at later time points (>48 h). This bacterial community succession was accompanied by significant variations in the activity of 12 different extracellular enzymes, resulting in a rapid reduction of dissolved organic carbon by 74.5% within the first 36 h. In summary, our study demonstrates rapid successions of bacterial community and extracellular enzyme activity after DOM inputs, suggesting that the bacterial response to Ulva prolifera-derived organic matter may contribute to environmental restoration and may pose a health threat due to the bloom of potential pathogenic Vibrio

Optimizing de novo genome assembly from PCR-amplified metagenomes

Background. Metagenomics has transformed our understanding of microbial diversity across ecosystems, with recent advances enabling de novo assembly of genomes from metagenomes. These metagenome-assembled genomes are critical to provide ecological, evolutionary, and metabolic context for all the microbes and viruses yet to be cultivated. Metagenomes can now be generated from nanogram to subnanogram amounts of DNA. However, these libraries require several rounds of PCR amplification before sequencing, and recent data suggest these typically yield smaller and more fragmented assemblies than regular metagenomes. Methods. Here we evaluate de novo assembly methods of 169 PCR-amplified metagenomes, including 25 for which an unamplified counterpart is available, to optimize specific assembly approaches for PCR-amplified libraries. We first evaluated coverage bias by mapping reads from PCR-amplified metagenomes onto reference contigs obtained from unamplified metagenomes of the same samples. Then, we compared different assembly pipelines in terms of assembly size (number of bp in contigs ≥ 10kb) and error rates to evaluate which are the best suited for PCR-amplified metagenomes. Results. Read mapping analyses revealed that the depth of coverage within individual genomes is significantly more uneven in PCR-amplified datasets versus unamplified metagenomes, with regions of high depth of coverage enriched in short inserts. This enrichment scales with the number of PCR cycles performed, and is presumably due to preferential amplification of short inserts. Standard assembly pipelines are confounded by this type of coverage unevenness, so we evaluated other assembly options to mitigate these issues. We found that a pipeline combining read deduplication and an assembly algorithm originally designed to recover genomes from libraries generated after whole genome amplification (single-cell SPAdes) frequently improved assembly of contigs ≥ 10kb by 10 to 100-fold for low input metagenomes. Conclusions. PCR-amplified metagenomes have enabled scientists to explore communities traditionally challenging to describe, including some with extremely low biomass or from which DNA is particularly difficult to extract. Here we show that a modified assembly pipeline can lead to an improved de novo genome assembly from PCR-amplified datasets, and enables a better genome recovery from low input metagenomes

Optimizing de novo genome assembly from PCR-amplified metagenomes

Background: Metagenomics has transformed our understanding of microbial diversity across ecosystems, with recent advances enabling de novo assembly of genomes from metagenomes. These metagenome-assembled genomes are critical to provide ecological, evolutionary, and metabolic context for all the microbes and viruses yet to be cultivated. Metagenomes can now be generated from nanogram to subnanogram amounts of DNA. However, these libraries require several rounds of PCR amplification before sequencing, and recent data suggest these typically yield smaller and more fragmented assemblies than regular metagenomes. Methods: Here we evaluate de novo assembly methods of 169 PCR-amplified metagenomes, including 25 for which an unamplified counterpart is available, to optimize specific assembly approaches for PCR-amplified libraries. We first evaluated coverage bias by mapping reads from PCR-amplified metagenomes onto reference contigs obtained from unamplified metagenomes of the same samples. Then, we compared different assembly pipelines in terms of assembly size (number of bp in contigs ≥ 10kb) and error rates to evaluate which are the best suited for PCR-amplified metagenomes. Results: . Read mapping analyses revealed that the depth of coverage within individual genomes is significantly more uneven in PCR-amplified datasets versus unamplified metagenomes, with regions of high depth of coverage enriched in short inserts. This enrichment scales with the number of PCR cycles performed, and is presumably due to preferential amplification of short inserts. Standard assembly pipelines are confounded by this type of coverage unevenness, so we evaluated other assembly options to mitigate these issues. We found that a pipeline combining read deduplication and an assembly algorithm originally designed to recover genomes from libraries generated after whole genome amplification (single-cell SPAdes) frequently improved assembly of contigs ≥ 10kb by 10 to 100-fold for low input metagenomes. Conclusions: PCR-amplified metagenomes have enabled scientists to explore communities traditionally challenging to describe, including some with extremely low biomass or from which DNA is particularly difficult to extract. Here we show that a modified assembly pipeline can lead to an improved de novo genome assembly from PCR-amplified datasets, and enables a better genome recovery from low input metagenomes

Climate-driven flyway changes and memory-based long-distance migration

Millions of migratory birds occupy seasonally favourable breeding grounds in the Arctic1, but we know little about the formation, maintenance and future of the migration routes of Arctic birds and the genetic determinants of migratory distance. Here we established a continental-scale migration system that used satellite tracking to follow 56 peregrine falcons (Falco peregrinus) from 6 populations that breed in the Eurasian Arctic, and resequenced 35 genomes from 4 of these populations. The breeding populations used five migration routes across Eurasia, which were probably formed by longitudinal and latitudinal shifts in their breeding grounds during the transition from the Last Glacial Maximum to the Holocene epoch. Contemporary environmental divergence between the routes appears to maintain their distinctiveness. We found that the gene ADCY8 is associated with population-level differences in migratory distance. We investigated the regulatory mechanism of this gene, and found that long-term memory was the most likely selective agent for divergence in ADCY8 among the peregrine populations. Global warming is predicted to influence migration strategies and diminish the breeding ranges of peregrine populations of the Eurasian Arctic. Harnessing ecological interactions and evolutionary processes to study climate-driven changes in migration can facilitate the conservation of migratory birds