Publications

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    ABSTRACT: 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.
    The ISME Journal 04/2015; DOI:10.1038/ismej.2015.48 · 9.27 Impact Factor
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    Haiwei Luo
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    ABSTRACT: The vast majority of environmental bacteria and archaea remain uncultivated, yet their genome sequences are rapidly becoming available through single cell sequencing technologies. Reconstructing metabolism is one common way to make use of genome sequences of ecologically important bacteria, but molecular evolutionary analysis is another approach that, while currently underused, can reveal important insights into the function of these uncultivated microbes in nature. Because genome sequences from single cells are often incomplete, metabolic reconstruction based on genome content can be compromised. However, this problem does not necessarily impede the use of phylogenomic and population genomic approaches that are based on patterns of polymorphisms and substitutions at nucleotide and amino acid sites. These approaches explore how various evolutionary forces act to assemble genetic diversity within and between lineages. In this mini-review, I present examples illustrating the benefits of analyzing single cell genomes using evolutionary approaches.
    Frontiers in Microbiology 03/2015; 6:191. DOI:10.3389/fmicb.2015.00191 · 3.94 Impact Factor
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    Haiwei Luo, Mary Ann Moran
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    ABSTRACT: Members of the Roseobacter clade are equipped with a tremendous diversity of metabolic capabilities, which in part explains their success in so many different marine habitats. Ideas on how this diversity evolved and is maintained are reviewed, focusing on recent evolutionary studies exploring the timing and mechanisms of Roseobacter ecological diversification. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
    Microbiology and molecular biology reviews: MMBR 12/2014; 78(4):573-587. DOI:10.1128/MMBR.00020-14 · 15.26 Impact Factor
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    ABSTRACT: About half the carbon fixed by phytoplankton in the ocean is taken up and metabolized by marine bacteria, a transfer that is mediated through the seawater dissolved organic carbon (DOC) pool. The chemical complexity of marine DOC, along with a poor understanding of which compounds form the basis of trophic interactions between bacteria and phytoplankton, have impeded efforts to identify key currencies of this carbon cycle link. Here, we used transcriptional patterns in a bacterial-diatom model system based on vitamin B12 auxotrophy as a sensitive assay for metabolite exchange between marine plankton. The most highly up-regulated genes (up to 374-fold) by a marine Roseobacter clade bacterium when cocultured with the diatom Thalassiosira pseudonana were those encoding the transport and catabolism of 2,3-dihydroxypropane-1-sulfonate (DHPS). This compound has no currently recognized role in the marine microbial food web. As the genes for DHPS catabolism have limited distribution among bacterial taxa, T. pseudonana may use this sulfonate for targeted feeding of beneficial associates. Indeed, DHPS was both a major component of the T. pseudonana cytosol and an abundant microbial metabolite in a diatom bloom in the eastern North Pacific Ocean. Moreover, transcript analysis of the North Pacific samples provided evidence of DHPS catabolism by Roseobacter populations. Other such biogeochemically important metabolites may be common in the ocean but difficult to discriminate against the complex chemical background of seawater. Bacterial transformation of this diatom-derived sulfonate represents a previously unidentified and likely sizeable link in both the marine carbon and sulfur cycles.
    Proceedings of the National Academy of Sciences 12/2014; DOI:10.1073/pnas.1413137112 · 9.81 Impact Factor
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    Haiwei Luo
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    ABSTRACT: 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.The ISME Journal advance online publication, 28 November 2014; doi:10.1038/ismej.2014.227.
    The ISME Journal 11/2014; DOI:10.1038/ismej.2014.227 · 9.27 Impact Factor
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    ABSTRACT: Corallimorpharia is a small Order of skeleton-less animals that is closely related to the reef-building corals (Scleractinia) and of fundamental interest in the context of understanding the potential impacts of climate change in the future on coral reefs. The relationship between the nominal Orders Corallimorpharia and Scleractinia is controversial - the former is either the closest outgroup to the Scleractinia or, alternatively is derived from corals via skeleton loss. This latter scenario, the "naked coral" hypothesis, is strongly supported by analyses based on mitochondrial protein sequences, whereas the former is equally strongly supported by analyses of mitochondrial (mt) nucleotide sequences. The "naked coral" hypothesis seeks to link skeleton loss in the putative ancestor of corallimorpharians with a period of elevated oceanic CO2 during the Cretaceous, leading to the idea that these skeleton-less animals may be harbingers for the fate of coral reefs under global climate change. In an attempt to better understand their evolutionary relationships, we examined mitochondrial genome organization in a representative range (12 species, representing 3 of the 4 extant families) of corallimorpharians and compared these patterns to other Hexacorallia. The most surprising finding was that mt genome organization in Corallimorphus profundus, a deep-water species that is the most scleractinian-like of all corallimorpharians on the basis of morphology, was much more similar to the common scleractinian pattern than to those of other corallimorpharians. This finding is consistent with the idea that C. profundus represents a key position in the coral <-> corallimorpharian transition.
    Genome Biology and Evolution 04/2014; 6(5). DOI:10.1093/gbe/evu084 · 4.53 Impact Factor
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    ABSTRACT: Strain HIMB11 is a planktonic marine bacterium isolated from coastal seawater in Kaneohe Bay, Oahu, Hawaii belonging to the ubiquitous and versatile Roseobacter clade of the alphaproteobacterial family Rhodobacteraceae. Here we describe the preliminary characteristics of strain HIMB11, including annotation of the draft genome sequence and comparative genomic analysis with other members of the Roseobacter lineage. The 3,098,747 bp draft genome is arranged in 34 contigs and contains 3,183 protein-coding genes and 54 RNA genes. Phylogenomic and 16S rRNA gene analyses indicate that HIMB11 represents a unique sublineage within the Roseobacter clade. Comparison with other publicly available genome sequences from members of the Roseobacter lineage reveals that strain HIMB11 has the genomic potential to utilize a wide variety of energy sources (e.g. organic matter, reduced inorganic sulfur, light, carbon monoxide), while possessing a reduced number of substrate transporters.
    Standards in Genomic Sciences 02/2014; 9(3). DOI:10.4056/sigs.4998989 · 3.17 Impact Factor
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    ABSTRACT: 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.The ISME Journal advance online publication, 7 November 2013; doi:10.1038/ismej.2013.202.
    The ISME Journal 02/2014; 8:732-736. DOI:10.1038/ismej.2013.202 · 9.27 Impact Factor
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    ABSTRACT: 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.The ISME Journal advance online publication, 23 January 2014; doi:10.1038/ismej.2013.248.
    The ISME Journal 01/2014; DOI:10.1038/ismej.2013.248 · 9.27 Impact Factor
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    ABSTRACT: A fundamental question in marine microbial ecology is how microbes adapt to ocean environments. Although numerically dominant populations are typically considered more successful, higher census population sizes (Nc) do not equate directly to a greater capability for adaptation. Instead, effective population size (Ne) determines the fate of deleterious and favourable mutations, and thus is a key parameter for determining the adaptive potential of a population. In the case of the SAR11 and Roseobacter lineages, two abundant heterotrophic bacteria in ocean surface waters with contrasting life history strategies, culture-independent population surveys suggest that SAR11s have greater Nc than Roseobacters. To determine relative Ne, we compared the ratio of nonsynonymous to synonymous substitution rates (ω) of recently diverged lineages of these taxa. Values of ω associated with several of the Roseobacter subclades were lower than for SAR11 subclades, suggesting greater Ne in these cases. Most Roseobacter lineages also had smaller ω values compared with an atypical basal Roseobacter lineage with a large Nc. This finding provides insight into variability in Ne across two important marine bacterial lineages, and provides an evolutionary context for considering how heterotrophic marine bacteria may differ in their ability to adapt to changing ocean habitats.
    Environmental Microbiology Reports 11/2013; 6(2):167–172. DOI:10.1111/1758-2229.12129 · 3.26 Impact Factor
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    Haiwei Luo, Mary Ann Moran
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    ABSTRACT: Uncovering the metabolic capabilities of microbes is key to understanding global energy flux and nutrient transformations. Since the vast majority of environmental microorganisms are uncultured, metagenomics has become an important tool to genotype the microbial community. This study uses a recently developed computational method to confidently assign metagenomic reads to microbial clades without the requirement of metagenome assembly by comparing the evolutionary pattern of nucleotide sequences at non-synonymous sites between metagenomic and orthologous reference genes. We found evidence for new, ecologically relevant metabolic pathways in several lineages of surface ocean bacterioplankton using the Global Ocean Survey (GOS) metagenomic data, including assimilatory sulfate reduction and alkaline phosphatase capabilities in the alphaproteobacterial SAR11 clade, and proteorhodopsin-like genes in the cyanobacterial genus Prochlorococcus. These findings raise new hypotheses about microbial roles in energy flux and organic matter transformation in the ocean.
    Environmental Microbiology Reports 10/2013; 5(5):686-696. DOI:10.1111/1758-2229.12068 · 3.26 Impact Factor
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    ABSTRACT: Planktonic bacteria dominate surface ocean biomass and influence global biogeochemical processes, but remain poorly characterized owing to difficulties in cultivation. Using large-scale single cell genomics, we obtained insight into the genome content and biogeography of many bacterial lineages inhabiting the surface ocean. We found that, compared with existing cultures, natural bacterioplankton have smaller genomes, fewer gene duplications, and are depleted in guanine and cytosine, noncoding nucleotides, and genes encoding transcription, signal transduction, and noncytoplasmic proteins. These findings provide strong evidence that genome streamlining and oligotrophy are prevalent features among diverse, free-living bacterioplankton, whereas existing laboratory cultures consist primarily of copiotrophs. The apparent ubiquity of metabolic specialization and mixotrophy, as predicted from single cell genomes, also may contribute to the difficulty in bacterioplankton cultivation. Using metagenome fragment recruitment against single cell genomes, we show that the global distribution of surface ocean bacterioplankton correlates with temperature and latitude and is not limited by dispersal at the time scales required for nucleotide substitution to exceed the current operational definition of bacterial species. Single cell genomes with highly similar small subunit rRNA gene sequences exhibited significant genomic and biogeographic variability, highlighting challenges in the interpretation of individual gene surveys and metagenome assemblies in environmental microbiology. Our study demonstrates the utility of single cell genomics for gaining an improved understanding of the composition and dynamics of natural microbial assemblages.
    Proceedings of the National Academy of Sciences 06/2013; DOI:10.1073/pnas.1304246110 · 9.81 Impact Factor
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    ABSTRACT: Marine bacteria in the Roseobacter and SAR11 lineages successfully exploit the ocean habitat, together accounting for ~40% of bacteria in surface waters, yet have divergent life histories that exemplify patch-adapted versus free-living ecological roles. Here, we use a phylogenetic birth-and-death model to understand how genome content supporting different life history strategies evolved in these related alphaproteobacterial taxa, showing that the streamlined genomes of free-living SAR11 were gradually downsized from a common ancestral genome only slightly larger than the extant members (~2,000 genes), while the larger and variably sized genomes of roseobacters evolved along dynamic pathways from a sizeable common ancestor (~8,000 genes). Genome changes in the SAR11 lineage occurred gradually over ~800 million years, whereas Roseobacter genomes underwent more substantial modifications, including major periods of expansion, over ~260 million years. The timing of the first Roseobacter genome expansion was coincident with the predicted radiation of modern marine eukaryotic phytoplankton of sufficient size to create nutrient-enriched microzones and is consistent with present-day ecological associations between these microbial groups. We suggest that diversification of red-lineage phytoplankton is an important driver of divergent life history strategies among the heterotrophic bacterioplankton taxa that dominate the present-day ocean.
    mBio 06/2013; 4(4). DOI:10.1128/mBio.00373-13 · 6.88 Impact Factor
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    ABSTRACT: Proteorhodopsin (PR), a ubiquitous membrane photoprotein in marine environments, acts as a light-driven proton pump and can provide energy for bacterial cellular metabolism. However, knowledge of factors that regulate PR gene expression in different bacteria remains strongly limited. Here, experiments with Vibrio sp. AND4 showed that PR phototrophy promoted survival only in cells from stationary phase and not in actively growing cells. PR gene expression was tightly regulated, with very low values in exponential phase, a pronounced peak at the exponential/stationary phase intersection, and a marked decline in stationary phase. Thus, PR gene expression at the entry into stationary phase preceded, and could therefore largely explain, the stationary phase light-induced survival response in AND4. Further experiments revealed nutrient limitation, not light exposure, regulated this differential PR expression. Screening of available marine vibrios showed that the PR gene, and thus the potential for PR phototrophy, is found in at least three different clusters in the genus Vibrio. In an ecological context, our findings suggest that some PR-containing bacteria adapted to the exploitation of nutrient-rich micro-environments rely on a phase of relatively slowly declining resources to mount a cellular response preparing them for adverse conditions dispersed in the water column.
    Environmental Microbiology 01/2013; DOI:10.1111/1462-2920.12085 · 6.24 Impact Factor
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    Haiwei Luo, Austin L Hughes
    Molecular Systems Biology 11/2012; 8:625. DOI:10.1038/msb.2012.58 · 14.10 Impact Factor
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    ABSTRACT: A typical marine bacterial cell in coastal seawater contains only ∼200 molecules of mRNA, each of which lasts only a few minutes before being degraded. Such a surprisingly small and dynamic cellular mRNA reservoir has important implications for understanding the bacterium's responses to environmental signals, as well as for our ability to measure those responses. In this perspective, we review the available data on transcript dynamics in environmental bacteria, and then consider the consequences of a small and transient mRNA inventory for functional metagenomic studies of microbial communities.
    The ISME Journal 08/2012; 7(2). DOI:10.1038/ismej.2012.94 · 9.27 Impact Factor
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    ABSTRACT: Evolutionary relationships among placental mammalian orders have been controversial. Whole genome sequencing and new computational methods offer opportunities to resolve the relationships among 10 genomes belonging to the mammalian orders Primates, Rodentia, Carnivora, Perissodactyla and Artiodactyla. By application of the double cut and join distance metric, where gene order is the phylogenetic character, we computed genomic distances among the sampled mammalian genomes. With a marsupial outgroup, the gene order tree supported a topology in which Rodentia fell outside the cluster of Primates, Carnivora, Perissodactyla, and Artiodactyla. Results of breakpoint reuse rate and synteny block length analyses were consistent with the prediction of random breakage model, which provided a diagnostic test to support use of gene order as an appropriate phylogenetic character in this study. We discussed the influence of rate differences among lineages and other factors that may contribute to different resolutions of mammalian ordinal relationships by different methods of phylogenetic reconstruction.
    Molecular Phylogenetics and Evolution 08/2012; 65(3):871-82. DOI:10.1016/j.ympev.2012.08.008 · 4.02 Impact Factor
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    Haiwei Luo
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    ABSTRACT: Bacteria consume dissolved organic matter (DOM) through hydrolysis, transport and intracellular metabolism, and these activities occur in distinct subcellular localizations. Bacterial protein subcellular localizations for several major marine bacterial groups were predicted using genomic, metagenomic and metatranscriptomic data sets following modification of MetaP software for use with partial gene sequences. The most distinct pattern of subcellular localization was found for Bacteroidetes, whose genomes were substantially enriched with outer membrane and extracellular proteins but depleted of inner membrane proteins compared with five other taxa (SAR11, Roseobacter, Synechococcus, Prochlorococcus, oligotrophic marine Gammaproteobacteria). When subcellular localization patterns were compared between genes and transcripts, three taxa had expression biased toward proteins localized to cell locations outside of the cytosol (SAR11, Roseobacter, and Synechococcus), as expected based on the importance of carbon and nutrient acquisition in an oligotrophic ocean, but two taxa did not (oligotrophic marine Gammaproteobacteria and Bacteroidetes). Diel variations in the fraction and putative gene functions of transcripts encoding inner membrane and periplasmic proteins compared to cytoplasmic proteins suggest a close coupling of photosynthetic extracellular release and bacterial consumption, providing insights into interactions between phytoplankton, bacteria, and DOM.
    Applied and Environmental Microbiology 07/2012; 78(18):6550-7. DOI:10.1128/AEM.01406-12 · 3.95 Impact Factor
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    ABSTRACT: Understanding of the ecological roles and evolutionary histories of marine bacterial taxa can be complicated by mismatches in genome content between wild populations and their better-studied cultured relatives. We used computed patterns of non-synonymous (amino acid-altering) nucleotide diversity in marine metagenomic data to provide high-confidence identification of DNA fragments from uncultivated members of the Roseobacter clade, an abundant taxon of heterotrophic marine bacterioplankton in the world's oceans. Differences in gene stoichiometry in the Global Ocean Survey metagenomic data set compared with 39 sequenced isolates indicated that natural Roseobacter populations differ systematically in several genomic attributes from their cultured representatives, including fewer genes for signal transduction and cell surface modifications but more genes for Sec-like protein secretion systems, anaplerotic CO(2) incorporation, and phosphorus and sulfate uptake. Several of these trends match well with characteristics previously identified as distinguishing r- versus K-selected ecological strategies in bacteria, suggesting that the r-strategist model assigned to cultured roseobacters may be less applicable to their free-living oceanic counterparts. The metagenomic Roseobacter DNA fragments revealed several traits with evolutionary histories suggestive of horizontal gene transfer from other marine bacterioplankton taxa or viruses, including pyrophosphatases and glycosylation proteins.
    Environmental Microbiology 08/2011; 14(1):41-51. DOI:10.1111/j.1462-2920.2011.02528.x · 6.24 Impact Factor
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    ABSTRACT: Several isolates of the marine cyanobacterial genus Prochlorococcus have smaller genome sizes than those of the closely related genus Synechococcus. In order to test whether loss of protein-coding genes has contributed to genome size reduction in Prochlorococcus, we reconstructed events of gene family evolution over a strongly supported phylogeny of 12 Prochlorococcus genomes and 9 Synechococcus genomes. Significantly, more events both of loss of paralogs within gene families and of loss of entire gene families occurred in Prochlorococcus than in Synechococcus. The number of nonancestral gene families in genomes of both genera was positively correlated with the extent of genomic islands (GIs), consistent with the hypothesis that horizontal gene transfer (HGT) is associated with GIs. However, even when only isolates with comparable extents of GIs were compared, significantly more events of gene family loss and of paralog loss were seen in Prochlorococcus than in Synechococcus, implying that HGT is not the primary reason for the genome size difference between the two genera.
    Molecular Biology and Evolution 04/2011; 28(10):2751-60. DOI:10.1093/molbev/msr081 · 14.31 Impact Factor

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