Critical assessment of pan-genomics of metagenome-assembled genomes

Background: Large scale metagenome assembly and binning to generate metagenome-assembled genomes (MAGs) has become possible in the past five years. As a result, millions of MAGs have been produced and increasingly included in pan-genomics workflow. However, pan-genome analyses of MAGs may suffer from the known issues with MAGs: fragmentation, incompleteness, and contamination, due to mis-assembly and mis-binning. Here, we conducted a critical assessment of including MAGs in pan-genome analysis, by comparing pan-genome analysis results of complete bacterial genomes and simulated MAGs. Results: We found that incompleteness led to more significant core gene loss than fragmentation. Contamination had little effect on core genome size but had major influence on accessory genomes. The core gene loss remained when using different pan-genome analysis tools and when using a mixture of MAGs and complete genomes. Importantly, the core gene loss was partially alleviated by lowering the core gene threshold and using gene prediction algorithms that consider fragmented genes, but to a less degree when incompleteness was higher than 5%. The core gene loss also led to incorrect pan-genome functional predictions and inaccurate phylogenetic trees. Conclusions: We conclude that lowering core gene threshold and predicting genes in metagenome mode (as Anvio does with Prodigal) are necessary in pan-genome analysis of MAGs to alleviate the accuracy loss. Better quality control of MAGs and development of new pan-genome analysis tools specifically designed for MAGs are needed in future studies.

Enhanced loss of retinoic acid network genes in Xenopus laevis achieves a tighter signal regulation

Retinoic acid (RA) is a major regulatory signal during embryogenesis produced from vitamin A (retinol) by an extensive, autoregulating metabolic and signaling network to prevent fluctuations that result in developmental malformations. Xenopus laevis is an allotetraploid hybrid frog species whose genome includes L (long) and S (short) chromosomes from the originating species. Evolutionarily, the X. laevis subgenomes have been losing either L or S homoeologs in about 43% of genes to generate singletons. In the RA network, out of the 47 genes, about 46% have lost one of the homoeologs, like the genome average. In contrast, RA metabolism genes from storage (retinyl esters) to retinaldehyde production exhibit enhanced gene loss with 75% singletons out of 28 genes. The effect of this gene loss on RA signaling autoregulation was studied. Employing transient RA manipulations, homoeolog gene pairs were identified in which one homeolog exhibits enhanced responses or looser regulation than the other, while in other pairs both homoeologs exhibit similar RA responses. CRISPR/Cas9 targeting of individual homoeologs to reduce their activity supports the hypothesis where the RA metabolic network gene loss results in tighter network regulation and more efficient RA robustness responses to overcome complex regulation conditions.

Exploring the onset of B12-based mutualisms using a recently evolved Chlamydomonas auxotroph and B12-producing bacteria

Cobalamin (vitamin B12), is a cofactor for crucial metabolic reactions in multiple eukaryotic taxa, including major primary producers such as algae, and yet only prokaryotes can produce it. Many bacteria can colonise the algal phycosphere, forming stable communities that gain preferential access to exudates and in return provide compounds, such as B12. Extended coexistence can then drive gene loss, leading to greater algal-bacterial interdependence. In this study, we investigate how a recently evolved B12-dependent strain of Chlamydomonas reinhardtii, metE7, forms a mutualism with certain bacteria, including the rhizobium Mesorhizobium loti and even a strain of the gut bacterium E. coli engineered to produce cobalamin. Although metE7 was supported by B12 producers, its growth in co-culture was slower than the B12-independent wild-type, suggesting that high bacterial B12 provision may be necessary to favour B12 auxotrophs and their evolution. Moreover, we found that an E. coli strain that releases more B12 makes a better mutualistic partner, and although this trait may be more costly in isolation, greater B12 release provided an advantage in co-cultures. We hypothesise that, given the right conditions, bacteria that release more B12 may be selected for, particularly if they form close interactions with B12-dependent algae.

Evolution of plasmid mobility: origin and fate of non-conjugative plasmids

Conjugation drives horizontal gene transfer of many adaptive traits across prokaryotes. Yet, only a fourth of the plasmids encode the functions necessary to conjugate autonomously, others being non-mobile or mobilizable by other elements. How these different plasmids evolve is poorly understood. Here, we studied plasmid evolution in terms of their gene repertoires and relaxases. We observed that gene content in plasmid varies rapidly in relation to the rate of evolution of relaxases, such that plasmids with 95% identical relaxases have on average fewer than 50% of homologs. The identification of 249 recent transitions in terms of mobility types revealed that they are associated with even greater changes in gene repertoires, possibly mediated by transposable elements that are more abundant in such plasmids. These changes include pseudogenization of the conjugation locus, exchange of replication initiators, and extensive gene loss. In some instances, the transition between mobility types also leads to the genesis of novel plasmid taxonomic units. Most of these transitions are short-lived, suggesting a source-sink dynamic, where conjugative plasmids constantly generate mobilizable and putatively non-mobilizable plasmids by gene deletion. Yet, in few cases such transitions resulted in the emergence of large clades of relaxases present only in mobilizable plasmids, suggesting successful specialization of these families in the hijacking of diverse conjugative systems. Our results shed further light on the huge plasticity of plasmids, suggest that many non-conjugative plasmids emerged recently from conjugative elements and allowed to quantify how changes in plasmid mobility shape the variation of their gene repertoires.

Recurrent erosion of COA1/MITRAC15 exemplifies conditional gene dispensability in oxidative phosphorylation

Skeletal muscle fibers rely upon either oxidative phosphorylation or the glycolytic pathway with much less reliance on oxidative phosphorylation to achieve muscular contractions that power mechanical movements. Species with energy-intensive adaptive traits that require sudden bursts of energy have a greater dependency on glycolytic fibers. Glycolytic fibers have decreased reliance on OXPHOS and lower mitochondrial content compared to oxidative fibers. Hence, we hypothesized that gene loss might have occurred within the OXPHOS pathway in lineages that largely depend on glycolytic fibers. The protein encoded by the COA1/MITRAC15 gene with conserved orthologs found in budding yeast to humans promotes mitochondrial translation. We show that gene disrupting mutations have accumulated within the COA1 gene in the cheetah, several species of galliform birds, and rodents. The genomic region containing COA1 is a well-established evolutionary breakpoint region in mammals. Careful inspection of genome assemblies of closely related species of rodents and marsupials suggests two independent COA1 gene loss events co-occurring with chromosomal rearrangements. Besides recurrent gene loss events, we document changes in COA1 exon structure in primates and felids. The detailed evolutionary history presented in this study reveals the intricate link between skeletal muscle fiber composition and the occasional dispensability of the chaperone-like role of the COA1 gene.

Genome-wide characterization, evolution, structure, and expression analysis of the F-box genes in Caenorhabditis

Background F-box proteins represent a diverse class of adaptor proteins of the ubiquitin-proteasome system (UPS) that play critical roles in the cell cycle, signal transduction, and immune response by removing or modifying cellular regulators. Among closely related organisms of the Caenorhabditis genus, remarkable divergence in F-box gene copy numbers was caused by sizeable species-specific expansion and contraction. Although F-box gene number expansion plays a vital role in shaping genomic diversity, little is known about molecular evolutionary mechanisms responsible for substantial differences in gene number of F-box genes and their functional diversification in Caenorhabditis. Here, we performed a comprehensive evolution and underlying mechanism analysis of F-box genes in five species of Caenorhabditis genus, including C. brenneri, C. briggsae, C. elegans, C. japonica, and C. remanei. Results Herein, we identified and characterized 594, 192, 377, 39, 1426 F-box homologs encoding putative F-box proteins in the genome of C. brenneri, C. briggsae, C. elegans, C. japonica, and C. remanei, respectively. Our work suggested that extensive species-specific tandem duplication followed by a small amount of gene loss was the primary mechanism responsible for F-box gene number divergence in Caenorhabditis genus. After F-box gene duplication events occurred, multiple mechanisms have contributed to gene structure divergence, including exon/intron gain/loss, exonization/pseudoexonization, exon/intron boundaries alteration, exon splits, and intron elongation by tandem repeats. Based on high-throughput RNA sequencing data analysis, we proposed that F-box gene functions have diversified by sub-functionalization through highly divergent stage-specific expression patterns in Caenorhabditis species. Conclusions Massive species-specific tandem duplications and occasional gene loss drove the rapid evolution of the F-box gene family in Caenorhabditis, leading to complex gene structural variation and diversified functions affecting growth and development within and among Caenorhabditis species. In summary, our findings outline the evolution of F-box genes in the Caenorhabditis genome and lay the foundation for future functional studies.

Hidden diversity of double-stranded DNA phages in symbiotic Rhizobium species

In this study, we addressed the extent of diversification of phages associated with nitrogen-fixing symbiotic Rhizobium species. Despite the ecological and economic importance of the Rhizobium genus, little is known about the diversity of the associated phages. A thorough assessment of viral diversity requires investigating both lytic phages and prophages harboured in diverse Rhizobium genomes. Protein-sharing networks identified 56 viral clusters (VCs) among a set of 425 isolated phages and predicted prophages. The VCs formed by phages had more proteins in common and a higher degree of synteny, and they group together in clades in the associated phylogenetic tree. By contrast, the VCs of prophages showed significant genetic variation and gene loss, with selective pressure on the remaining genes. Some VCs were found in various Rhizobium species and geographical locations, suggesting that they have wide host ranges. Our results indicate that the VCs represent distinct taxonomic units, probably representing taxa equivalent to genera or even species. The finding of previously undescribed phage taxa indicates the need for further exploration of the diversity of phages associated with Rhizobium species. This article is part of the theme issue ‘The secret lives of microbial mobile genetic elements’.

New Genes in the Drosophila Y Chromosome: Lessons from D. willistoni

Y chromosomes play important roles in sex determination and male fertility. In several groups (e.g., mammals) there is strong evidence that they evolved through gene loss from a common X-Y ancestor, but in Drosophila the acquisition of new genes plays a major role. This conclusion came mostly from studies in two species. Here we report the identification of the 22 Y-linked genes in D. willistoni. They all fit the previously observed pattern of autosomal or X-linked testis-specific genes that duplicated to the Y. The ratio of gene gains to gene losses is ~25 in D. willistoni, confirming the prominent role of gene gains in the evolution of Drosophila Y chromosomes. We also found four large segmental duplications (ranging from 62 kb to 303 kb) from autosomal regions to the Y, containing ~58 genes. All but four of these duplicated genes became pseudogenes in the Y or disappeared. In the GK20609 gene the Y-linked copy remained functional, whereas its original autosomal copy degenerated, demonstrating how autosomal genes are transferred to the Y chromosome. Since the segmental duplication that carried GK20609 contained six other testis-specific genes, it seems that chance plays a significant role in the acquisition of new genes by the Drosophila Y chromosome.

Comparative plastomics of Amaryllidaceae: inverted repeat expansion and the degradation of the ndh genes in Strumaria truncata Jacq.

Amaryllidaceae is a widespread and distinctive plant family contributing both food and ornamental plants. Here we present an initial survey of plastomes across the family and report on both structural rearrangements and gene losses. Most plastomes in the family are of similar gene arrangement and content however some taxa have shown gains in plastome length while in several taxa there is evidence of gene loss. Strumaria truncata shows a substantial loss of ndh family genes while three other taxa show loss of cemA, which has been reported only rarely. Our sparse sampling of the family has detected sufficient variation to suggest further sampling across the family could be a rich source of new information on plastome variation and evolution.

Discovery of nondiazotrophic Trichodesmium species abundant and widespread in the open ocean

Filamentous and colony-forming cells within the cyanobacterial genus Trichodesmium might account for nearly half of nitrogen fixation in the sunlit ocean, a critical mechanism that sustains plankton’s primary productivity. Trichodesmium has long been portrayed as a diazotrophic genus. By means of genome-resolved metagenomics, here we reveal that nondiazotrophic Trichodesmium species not only exist but also are abundant and widespread in the open ocean, benefiting from a previously overlooked functional lifestyle to expand the biogeography of this prominent marine genus. Near-complete environmental genomes for those closely related candidate species reproducibly shared functional features including a lack of genes related to nitrogen fixation, hydrogen recycling, and hopanoid lipid production concomitant with the enrichment of nitrogen assimilation genes. Our results elucidate fieldwork observations of Trichodesmium cells fixing carbon but not nitrogen. The Black Queen hypothesis and burden of low-oxygen concentration requirements provide a rationale to explain gene loss linked to nitrogen fixation among Trichodesmium species. Disconnecting taxonomic signal for this genus from a microbial community’s ability to fix nitrogen will help refine our understanding of the marine nitrogen balance. Finally, we are reminded that established links between taxonomic lineages and functional traits do not always hold true.