Haiwei Luo

Single-cell genomics-based analysis of virus-host interactions in marine surface bacterioplankton

Viral infections dynamically alter the composition and metabolic potential of marine microbial communities and the evolutionary trajectories of host populations with resulting feedback on biogeochemical cycles. It is quite possible that all microbial populations in the ocean are impacted by viral infections. Our knowledge of virus–host relationships, however, has been limited to a minute fraction of cultivated host groups. Here, we utilized single-cell sequencing to obtain genomic blueprints of viruses inside or attached to individual bacterial and archaeal cells captured in their native environment, circumventing the need for host and virus cultivation. A combination of comparative genomics, metagenomic fragment recruitment, sequence anomalies and irregularities in sequence coverage depth and genome recovery were utilized to detect viruses and to decipher modes of virus–host interactions. Members of all three tailed phage families were identified in 20 out of 58 phylogenetically and geographically diverse single amplified genomes (SAGs) of marine bacteria and archaea. At least four phage–host interactions had the characteristics of late lytic infections, all of which were found in metabolically active cells. One virus had genetic potential for lysogeny. Our findings include first known viruses of Thaumarchaeota, Marinimicrobia, Verrucomicrobia and Gammaproteobacteria clusters SAR86 and SAR92. Viruses were also found in SAGs of Alphaproteobacteria and Bacteroidetes. A high fragment recruitment of viral metagenomic reads confirmed that most of the SAG-associated viruses are abundant in the ocean. Our study demonstrates that single-cell genomics, in conjunction with sequence-based computational tools, enable in situ, cultivation-independent insights into host–virus interactions in complex microbial communities.

Single-cell genomics shedding light on marine Thaumarchaeota diversification

Previous studies based on analysis of amoA, 16S ribosomal RNA or accA gene sequences have established that marine Thaumarchaeota fall into two phylogenetically distinct groups corresponding to shallow- and deep-water clades, but it is not clear how water depth interacts with other environmental factors, including light, temperature and location, to affect this pattern of diversification. Earlier studies focused on single-gene distributions were not able to link phylogenetic structure to other aspects of functional adaptation. Here, we analyzed the genome content of 46 uncultivated single Thaumarchaeota cells sampled from epi- and mesopelagic waters of subtropical, temperate and polar oceans. Phylogenomic analysis showed that populations diverged by depth, as expected, and that mesopelagic populations from different locations were well mixed. Functional analysis showed that some traits, including putative DNA photolyase and catalase genes that may be related to adaptive mechanisms to reduce light-induced damage, were found exclusively in members of the epipelagic clade. Our analysis of partial genomes has thus confirmed the depth differentiation of Thaumarchaeota populations observed previously, consistent with the distribution of putative mechanisms to reduce light-induced damage in shallow- and deep-water populations.

Evolutionary analysis of a streamlined lineage of surface ocean Roseobacters

The vast majority of surface ocean bacteria are uncultivated. Compared with their cultured relatives, they frequently exhibit a streamlined genome, reduced G+C content and distinct gene repertoire. These genomic traits are relevant to environmental adaptation, and have generally been thought to become fixed in marine bacterial populations through selection. Using single-cell genomics, we sequenced four uncultivated cells affiliated with the ecologically relevant Roseobacter clade and used a composition-heterogeneous Bayesian phylogenomic model to resolve these single-cell genomes into a new clade. This lineage has no representatives in culture, yet accounts for ∼35% of Roseobacters in some surface ocean waters. Analyses of multiple genomic traits, including genome size, G+C content and percentage of noncoding DNA, suggest that these single cells are representative of oceanic Roseobacters but divergent from isolates. Population genetic analyses showed that substitution of physicochemically dissimilar amino acids and replacement of G+C-rich to G+C-poor codons are accelerated in the uncultivated clade, processes that are explained equally well by genetic drift as by the more frequently invoked explanation of natural selection. The relative importance of drift vs selection in this clade, and perhaps in other marine bacterial clades with streamlined G+C-poor genomes, remains unresolved until more evidence is accumulated.

How do divergent ecological strategies emerge among marine bacterioplankton lineages

Heterotrophic bacteria in pelagic marine environments are frequently categorized into two canonical ecological groups: patch-associated and free-living. This framework provides a conceptual basis for understanding bacterial utilization of oceanic organic matter. Some patch-associated bacteria are ecologically linked with eukaryotic phytoplankton, and this observation fits with predicted coincidence of their genome expansion with marine phytoplankton diversification. By contrast, free-living bacteria in todays oceans typically live singly with streamlined metabolic and regulatory functions that allow them to grow in nutrient-poor seawater. Recent analyses of marine Alphaproteobacteria suggest that some free-living bacterioplankton lineages evolved from patch-associated ancestors up to several hundred million years ago. While evolutionary analyses agree with the hypothesis that natural selection has maintained these distinct ecological strategies and genomic traits in present-day populations, they do not rule out a major role for genetic drift in driving ancient ecological switches. These two evolutionary forces may have acted on ocean bacteria at different geological time scales and under different geochemical constraints, with possible implications for future adaptations to a changing ocean. New evolutionary models and genomic data are leading to a more comprehensive understanding of marine bacterioplankton evolutionary history.

Ecological Genomics of the Uncultivated Marine Roseobacter Lineage CHAB-I-5

ABSTRACT Members of the marine Roseobacter clade are major participants in global carbon and sulfur cycles. While roseobacters are well represented in cultures, several abundant pelagic lineages, including SAG-O19, DC5-80-3, and NAC11-7, remain largely uncultivated and show evidence of genome streamlining. Here, we analyzed the partial genomes of three single cells affiliated with CHAB-I-5, another abundant but exclusively uncultivated Roseobacter lineage. Members of this lineage encode several metabolic potentials that are absent in streamlined genomes. Examples are quorum sensing and type VI secretion systems, which enable them to effectively interact with host and other bacteria. Further analysis of the CHAB-I-5 single-cell amplified genomes (SAGs) predicted that this lineage comprises members with relatively large genomes (4.1 to 4.4 Mbp) and a high fraction of noncoding DNA (10 to 12%), which is similar to what is observed in many cultured, nonstreamlined Roseobacter lineages. The four uncultured lineages, while exhibiting highly variable geographic distributions, together represent >60% of the global pelagic roseobacters. They are consistently enriched in genes encoding the capabilities of light harvesting, oxidation of “energy-rich” reduced sulfur compounds and methylated amines, uptake and catabolism of various carbohydrates and osmolytes, and consumption of abundant exudates from phytoplankton. These traits may define the global prevalence of the four lineages among marine bacterioplankton.

Evolutionary origin of a streamlined marine bacterioplankton lineage

Planktonic bacterial lineages with streamlined genomes are prevalent in the ocean. The base composition of their DNA is often highly biased towards low G+C content, a possible source of systematic error in phylogenetic reconstruction. A total of 228 orthologous protein families were sampled that are shared among major lineages of Alphaproteobacteria, including the marine free-living SAR11 clade and the obligate endosymbiotic Rickettsiales. These two ecologically distinct lineages share genome sizes of <1.5 Mbp and genomic G+C content of <30%. Statistical analyses showed that only 28 protein families are composition-homogeneous, whereas the other 200 families significantly violate the composition-homogeneous assumption included in most phylogenetic methods. RAxML analysis based on the concatenation of 24 ribosomal proteins that fall into the heterogeneous protein category clustered the SAR11 and Rickettsiales lineages at the base of the Alphaproteobacteria tree, whereas that based on the concatenation of 28 homogeneous proteins (including 19 ribosomal proteins) disassociated the lineages and placed SAR11 at the base of the non-endosymbiotic lineages. When the two data sets were concatenated, only a model that accounted for compositional bias yielded a tree identical to the tree built with composition-homogeneous proteins. Ancestral genome analysis suggests that the first evolved SAR11 cell had a small genome streamlined from its ancestor by a factor of two and coinciding with an ecological transition, followed by further gradual streamlining towards the extant SAR11 populations.

Selection Maintains Low Genomic GC Content in Marine SAR11 Lineages

The genomic G+C content of ocean bacteria varies from below 30% to over 60%. This broad range of base composition is likely shaped by distinct mutational processes, recombination, effective population size, and selection driven by environmental factors. A number of studies have hypothesized that depletion of G/C in genomes of marine bacterioplankton cells is an adaptation to the nitrogen-poor pelagic oceans, but they failed to disentangle environmental factors from mutational biases and population history. Here, we reconstructed the evolutionary changes of bases at synonymous sites in genomes of two marine SAR11 populations and a freshwater counterpart with its evolutionary origin rooted in the marine lineage. Although they all have similar genome sizes, DNA repair gene repertoire, and base compositions, there is a stronger bias toward A/T changes, a reduced frequency of nitrogenous amino acids, and an exclusive occurrence of polyamine, opine, and taurine transport systems in the ocean populations, consistent with a greater nitrogen stress in surface oceans compared with freshwater lakes. Furthermore, the ratio of nonsynoymous to synonymous nucleotide diversity is not statistically distinguishable among these populations, suggesting that population history has a limited effect. Taken together, the ecological transition of SAR11 from ocean to freshwater habitats makes nitrogen more available to these organisms, and thus relaxation of purifying selection drove a genome-wide reduction in the frequency of G/C to A/T changes in the freshwater population.

Evolutionary determinants of genome-wide nucleotide composition

One of the long-standing mysteries of evolutionary genomics is the source of the wide phylogenetic diversity in genome nucleotide composition (G + C versus A + T), which must be a consequence of interspecific differences in mutation bias, the efficiency of selection for different nucleotides or a combination of the two. We demonstrate that although genomic G + C composition is strongly driven by mutation bias, it is also substantially modified by direct selection and/or as a by-product of biased gene conversion. Moreover, G + C composition at fourfold redundant sites is consistently elevated above the neutral expectation—more so than for any other class of sites.Genome-wide nucleotide composition varies greatly among species. Here, the authors show that genomic G + C composition is driven by mutation bias but is also modified by natural selection or biased gene conversion.

Excess of non-conservative amino acid changes in marine bacterioplankton lineages with reduced genomes

Surface ocean waters are dominated by planktonic bacterial lineages with highly reduced genomes. The best examples are the cyanobacterial genus Prochlorococcus, the alphaproteobacterial clade SAR11 and the gammaproteobacterial clade SAR86, which together represent over 50% of the cells in surface oceans. Several studies have identified signatures of selection on these lineages in todays ocean and have postulated selection as the primary force throughout their evolutionary history. However, massive loss of genomic DNA in these lineages often occurred in the distant past, and the selective pressures underlying these ancient events have not been assessed. Here, we probe ancient selective pressures by computing %GC-corrected rates of conservative and radical nonsynonymous nucleotide substitutions. Surprisingly, we found an excess of radical changes in several of these lineages in comparison to their relatives with larger genomes. Furthermore, analyses of allelic genome sequences of several populations within these lineages consistently supported that radical replacements are more likely to be deleterious than conservative changes. Our results suggest coincidence of massive genomic DNA losses and increased power of genetic drift, but we also suggest that additional evidence independent of the nucleotide substitution analyses is needed to support a primary role of genetic drift driving ancient genome reduction of marine bacterioplankton lineages.

Spontaneous mutations of a model heterotrophic marine bacterium

Heterotrophic marine bacterioplankton populations display substantive genomic diversity that is commonly explained to be the result of selective forces imposed by resource limitation or interactions with phage and predators. Here we use a mutation-accumulation experiment followed by whole-genome sequencing of mutation lines to determine an unbiased rate and molecular spectrum of spontaneous mutations for a model heterotrophic marine bacterium in the globally important Roseobacter clade, Ruegeria pomeroyi DSS-3. We find evidence for mutational bias towards deletions over insertions, and this process alone could account for a sizable portion of genome size diversity among roseobacters and also implies that lateral gene transfer and/or selection must also play a role in maintaining roseobacters with large genome sizes. We also find evidence for a mutational bias in favor of changes from A/T to G/C nucleobases, which explains widespread occurrences of G/C-enriched Roseobacter genomes. Using the calculated mutation rate of 1.39 × 10−10 per base per generation, we implement a ‘mutation-rate clock’ approach to date the evolution of roseobacters by assuming a constant mutation rate along their evolutionary history. This approach gives an estimated date of Roseobacter genome expansion in good agreement with an earlier fossil-based estimate of ~250 million years ago and is consistent with a hypothesis of a correlated evolutionary history between roseobacters and marine eukaryotic phytoplankton groups.