Metagenomic clustering links specific metabolic functions to globally relevant ecosystems.

Metagenomic sequencing has advanced our understanding of biogeochemical processes by providing an unprecedented view into the microbial composition of different ecosystems. While the amount of metagenomic data has grown rapidly, simple-to-use methods to analyze and compare across studies have lagged behind. Thus, tools expressing the metabolic traits of a community are needed to broaden the utility of existing data. Gene abundance profiles are a relatively low-dimensional embedding of a metagenome's functional potential and are, thus, tractable for comparison across many samples. Here, we compare the abundance of KEGG Ortholog Groups (KOs) from 6,539 metagenomes from the Joint Genome Institute's Integrated Microbial Genomes and Metagenomes (JGI IMG/M) database. We find that samples cluster into terrestrial, aquatic, and anaerobic ecosystems with marker KOs reflecting adaptations to these environments. For instance, functional clusters were differentiated by the metabolism of antibiotics, photosynthesis, methanogenesis, and surprisingly GC content. Using this functional gene approach, we reveal the broad-scale patterns shaping microbial communities and demonstrate the utility of ortholog abundance profiles for representing a rapidly expanding body of metagenomic data. IMPORTANCE: Metagenomics, or the sequencing of DNA from complex microbiomes, provides a view into the microbial composition of different environments. Metagenome databases were created to compile sequencing data across studies, but it remains challenging to compare and gain insight from these large data sets. Consequently, there is a need to develop accessible approaches to extract knowledge across metagenomes. The abundance of different orthologs (i.e., genes that perform a similar function across species) provides a simplified representation of a metagenome's metabolic potential that can easily be compared with others. In this study, we cluster the ortholog abundance profiles of thousands of metagenomes from diverse environments and uncover the traits that distinguish them. This work provides a simple to use framework for functional comparison and advances our understanding of how the environment shapes microbial communities.

Global and local genomic features together modulate the spontaneous single nucleotide mutation rate.

Spontaneous mutations are evolutionary engines as they generate variants for the evolutionary downstream processes that give rise to speciation and adaptation. Single nucleotide mutations (SNM) are the most abundant type of mutations among them. Here, we perform a meta-analysis to quantify the influence of selected global genomic parameters (genome size, genomic GC content, genomic repeat fraction, number of coding genes, gene count, and strand bias in prokaryotes) and local genomic features (local GC content, repeat content, CpG content and the number of SNM at CpG islands) on spontaneous SNM rates across the tree of life (prokaryotes, unicellular eukaryotes, multicellular eukaryotes) using wild-type sequence data in two different taxon classification systems. We find that the spontaneous SNM rates in our data are correlated with many genomic features in prokaryotes and unicellular eukaryotes irrespective of their sample sizes. On the other hand, only the number of coding genes was correlated with the spontaneous SNM rates in multicellular eukaryotes primarily contributed by vertebrates data. Considering local features, we notice that local GC content and CpG content significantly were correlated with the spontaneous SNM rates in the unicellular eukaryotes, while local repeat fraction is an important feature in prokaryotes and certain specific uni- and multi-cellular eukaryotes. Such predictive features of the spontaneous SNM rates often support non-linear models as the best fit compared to the linear model. We also observe that the strand asymmetry in prokaryotes plays an important role in determining the spontaneous SNM rates but the SNM spectrum does not.

Terminal regions of a protein are a hotspot for low complexity regions and selection.

Volatile low complexity regions (LCRs) are a novel source of adaptive variation, functional diversification and evolutionary novelty. An interplay of selection and mutation governs the composition and length of low complexity regions. High %GC and mutations provide length variability because of mechanisms like replication slippage. Owing to the complex dynamics between selection and mutation, we need a better understanding of their coexistence. Our findings underscore that positively selected sites (PSS) and low complexity regions prefer the terminal regions of genes, co-occurring in most Tetrapoda clades. We observed that positively selected sites within a gene have position-specific roles. Central-positively selected site genes primarily participate in defence responses, whereas terminal-positively selected site genes exhibit non-specific functions. Low complexity region-containing genes in the Tetrapoda clade exhibit a significantly higher %GC and lower ω (dN/dS: non-synonymous substitution rate/synonymous substitution rate) compared with genes without low complexity regions. This lower ω implies that despite providing rapid functional diversity, low complexity region-containing genes are subjected to intense purifying selection. Furthermore, we observe that low complexity regions consistently display ubiquitous prevalence at lower purity levels, but exhibit a preference for specific positions within a gene as the purity of the low complexity region stretch increases, implying a composition-dependent evolutionary role. Our findings collectively contribute to the understanding of how genetic diversity and adaptation are shaped by the interplay of selection and low complexity regions in the Tetrapoda clade.

New mitogenomes of Runcinidae and Facelinidae: two understudied heterobranch families (Mollusca: Gastropoda).

Here, we present the mitochondrial sequences of two sea slugs (Heterobranchia): Runcina aurata and Facelina auriculata, the latter being the type species of the family. The mitochondrial genomes are 14,282 and 14,171bp in length, respectively, with a complete set of 13 PCGs, 2 rRNAs, and 22 tRNAs. None of the mitogenomes show gene reorganization, keeping the standard mitogenomic structure of Heterobranchia. Nucleotide composition differs significantly between them, with R. aurata showing the most AT-rich mitogenome (25.7% GC content) reported to date in Heterobranchia, and F. auriculata showing a rich GC content (35%) compared with other heterobranch mitochondrial genomes.

The Genome of Plasmodium gonderi: Insights into the Evolution of Human Malaria Parasites.

Plasmodium species causing malaria in humans are not monophyletic, sharing common ancestors with nonhuman primate parasites. Plasmodium gonderi is one of the few known Plasmodium species infecting African old-world monkeys that are not found in apes. This study reports a de novo assembled P. gonderi genome with complete chromosomes. The P. gonderi genome shares codon usage, syntenic blocks, and other characteristics with the human parasites Plasmodium ovale s.l. and Plasmodium malariae, also of African origin, and the human parasite Plasmodium vivax and species found in nonhuman primates from Southeast Asia. Using phylogenetically aware methods, newly identified syntenic blocks were found enriched with conserved metabolic genes. Regions outside those blocks harbored genes encoding proteins involved in the vertebrate host-Plasmodium relationship undergoing faster evolution. Such genome architecture may have facilitated colonizing vertebrate hosts. Phylogenomic analyses estimated the common ancestor between P. vivax and an African ape parasite P. vivax-like, within the Asian nonhuman primates parasites clade. Time estimates incorporating P. gonderi placed the P. vivax and P. vivax-like common ancestor in the late Pleistocene, a time of active migration of hominids between Africa and Asia. Thus, phylogenomic and time-tree analyses are consistent with an Asian origin for P. vivax and an introduction of P. vivax-like into Africa. Unlike other studies, time estimates for the clade with Plasmodium falciparum, the most lethal human malaria parasite, coincide with their host species radiation, African hominids. Overall, the newly assembled genome presented here has the quality to support comparative genomic investigations in Plasmodium.

Significance of genetic code module structure in gene expression and GC content enhancement in RNA sequences.

The existent algebraic models of the genetic code contribute to the understanding of the physio-chemical characteristics of the amino acids. However, the process of translating a gene into a phenotype is highly complex. Moreover, the intricacy of gene expression gets further multiplied due to the biases in the codon usage. This paper explores an algebraic structure called module on the set of codons as well as on that of RNA sequences. We study the potential implications of these structures on gene expression and the GC content of an RNA sequence. The base order {C,U,G,A} appears to possess greater biological significance than many of the orders previously studied. We have developed a novel algorithm to generate RNA sequences with high GC content, aiming to enhance the thermostability of biomolecules. The insights gained from this investigation may have applications in biomolecular modeling and docking, protein engineering, drug development, and related fields.

Optimized transgene expression in the red alga Porphyridium purpureum and efficient recombinant protein secretion into the culture medium.

Microalgae represent a promising but yet underexplored production platform for biotechnology. The vast majority of studies on recombinant protein expression in algae have been conducted in a single species, the green alga Chlamydomonas reinhardtii. However, due to epigenetic silencing, transgene expression in Chlamydomonas is often inefficient. Here we have investigated parameters that govern efficient transgene expression in the red microalga Porphyridium purpureum. Porphyridium is unique in that the introduced transformation vectors are episomally maintained as autonomously replicating plasmids in the nucleus. We show that full codon optimization to the preferred codon usage in the Porphyridium genome confers superior transgene expression, not only at the level of protein accumulation, but also at the level of mRNA accumulation, indicating that high translation rates increase mRNA stability. Our optimized expression constructs resulted in YFP accumulation to unprecedented levels of up to 5% of the total soluble protein. We also designed expression cassettes that target foreign proteins to the secretory pathway and lead to efficient protein secretion into the culture medium, thus simplifying recombinant protein harvest and purification. Our study paves the way to the exploration of red microalgae as expression hosts in molecular farming for recombinant proteins and metabolites.

Dinucleotide biases in the genomes of prokaryotic and eukaryotic dsDNA viruses and their hosts.

The genomes of cellular organisms display CpG and TpA dinucleotide composition biases. Such biases have been poorly investigated in dsDNA viruses. Here, we show that in dsDNA virus, bacterial, and eukaryotic genomes, the representation of TpA and CpG dinucleotides is strongly dependent on genomic G + C content. Thus, the classical observed/expected ratios do not fully capture dinucleotide biases across genomes. Because a larger portion of the variance in TpA frequency was explained by G + C content, we explored which additional factors drive the distribution of CpG dinucleotides. Using the residuals of the linear regressions as a measure of dinucleotide abundance and ancestral state reconstruction across eukaryotic and prokaryotic virus trees, we identified an important role for phylogeny in driving CpG representation. Nonetheless, phylogenetic ANOVA analyses showed that few host associations also account for significant variations. Among eukaryotic viruses, most significant differences were observed between arthropod-infecting viruses and viruses that infect vertebrates or unicellular organisms. However, an effect of viral DNA methylation status (either driven by the host or by viral-encoded methyltransferases) is also likely. Among prokaryotic viruses, cyanobacteria-infecting phages resulted to be significantly CpG-depleted, whereas phages that infect bacteria in the genera Burkolderia and Staphylococcus were CpG-rich. Comparison with bacterial genomes indicated that this effect is largely driven by the general tendency for phages to resemble the host's genomic CpG content. Notably, such tendency is stronger for temperate than for lytic phages. Our data shed light into the processes that shape virus genome composition and inform manipulation strategies for biotechnological applications.

mRNA nuclear export: how mRNA identity features distinguish functional RNAs from junk transcripts.

The division of the cellular space into nucleoplasm and cytoplasm promotes quality control mechanisms that prevent misprocessed mRNAs and junk RNAs from gaining access to the translational machinery. Here, we explore how properly processed mRNAs are distinguished from both misprocessed mRNAs and junk RNAs by the presence or absence of various 'identity features'.

Effects of error, chimera, bias, and GC content on the accuracy of amplicon sequencing.

IMPORTANCE: Amplicon sequencing of targeted genes is the predominant approach to estimate the membership and structure of microbial communities. However, accurate reconstruction of community composition is difficult due to sequencing errors, and other methodological biases and effective approaches to overcome these challenges are essential. Using a mock community of 33 phylogenetically diverse strains, this study evaluated the effect of GC content on sequencing results and tested different approaches to improve overall sequencing accuracy while characterizing the pros and cons of popular amplicon sequence data processing approaches. The sequencing results from this study can serve as a benchmarking data set for future algorithmic improvements. Furthermore, the new insights on sequencing error, chimera formation, and GC bias from this study will help enhance the quality of amplicon sequencing studies and support the development of new data analysis approaches.

Systematic analysis of plasmids of the Serratia marcescens complex using 142 closed genomes.

Plasmids play important roles in bacterial genome diversification. In the Serratia marcescens complex (SMC), a notable contribution of plasmids to genome diversification was also suggested by our recent analysis of >600 draft genomes. As accurate analyses of plasmids in draft genomes are difficult, in this study we analysed 142 closed genomes covering the entire complex, 67 of which were obtained in this study, and identified 132 plasmids (1.9-244.4 kb in length) in 77 strains. While the average numbers of plasmids in clinical and non-clinical strains showed no significant difference, strains belonging to clade 2 (one of the two hospital-adapted lineages) contained more plasmids than the others. Pangenome analysis revealed that of the 28 954 genes identified, 12.8 % were plasmid-specific, and 1.4 % were present in plasmids or chromosomes depending on the strain. In the latter group, while transposon-related genes were most prevalent (31.4 % of the function-predicted genes), genes related to antimicrobial resistance and heavy metal resistance accounted for a notable proportion (22.7 %). Mash distance-based clustering separated the 132 plasmids into 23 clusters and 50 singletons. Most clusters/singletons showed notably different GC contents compared to those of host chromosomes, suggesting their recent or relatively recent appearance in the SMC. Among the 23 clusters, 17 were found in only clinical or only non-clinical strains, suggesting the possible preference of their distribution on the environmental niches of host strains. Regarding the host strain phylogeny, 16 clusters were distributed in two or more clades, suggesting their interclade transmission. Moreover, for many plasmids, highly homologous plasmids were found in other species, indicating the broadness of their potential host ranges, beyond the genus, family, order, class or even phylum level. Importantly, highly homologous plasmids were most frequently found in Klebsiella pneumoniae and other species in the family Enterobacteriaceae, suggesting that this family, particularly K. pneumoniae, is the main source for plasmid exchanges with the SMC. These results highlight the power of closed genome-based analysis in the investigation of plasmids and provide important insights into the nature of plasmids distributed in the SMC.

Human histone H1 variants impact splicing outcome by controlling RNA polymerase II elongation.

Histones shape chromatin structure and the epigenetic landscape. H1, the most diverse histone in the human genome, has 11 variants. Due to the high structural similarity between the H1s, their unique functions in transferring information from the chromatin to mRNA-processing machineries have remained elusive. Here, we generated human cell lines lacking up to five H1 subtypes, allowing us to characterize the genomic binding profiles of six H1 variants. Most H1s bind to specific sites, and binding depends on multiple factors, including GC content. The highly expressed H1.2 has a high affinity for exons, whereas H1.3 binds intronic sequences. H1s are major splicing regulators, especially of exon skipping and intron retention events, through their effects on the elongation of RNA polymerase II (RNAPII). Thus, H1 variants determine splicing fate by modulating RNAPII elongation.

Overlaps Between CDS Regions of Protein-Coding Genes in the Human Genome: A Case Study on the NR1D1-THRA Gene Pair.

For several decades, it has been known that a substantial number of genes within human DNA exhibit overlap; however, the biological and evolutionary significance of these overlaps remain poorly understood. This study focused on investigating specific instances of overlap where the overlapping DNA region encompasses the coding DNA sequences (CDSs) of protein-coding genes. The results revealed that proteins encoded by overlapping CDSs exhibit greater disorder than those from nonoverlapping CDSs. Additionally, these DNA regions were identified as GC-rich. This could be partially attributed to the absence of stop codons from two distinct reading frames rather than one. Furthermore, these regions were found to harbour fewer single-nucleotide polymorphism (SNP) sites, possibly due to constraints arising from the overlapping state where mutations could affect two genes simultaneously.While elucidating these properties, the NR1D1-THRA gene pair emerged as an exceptional case with highly structured proteins and a distinctly conserved sequence across eutherian mammals. Both NR1D1 and THRA are nuclear receptors lacking a ligand-binding domain at their C-terminus, which is the region where these gene pairs overlap. The NR1D1 gene is involved in the regulation of circadian rhythm, while the THRA gene encodes a thyroid hormone receptor, and both play crucial roles in various physiological processes. This study suggests that, in addition to their well-established functions, the specifically overlapping CDS regions of these genes may encode protein segments with additional, yet undiscovered, biological roles.

Evolution and diversity of nucleotide and dinucleotide composition in poxviruses.

Poxviruses (family Poxviridae) have long dsDNA genomes and infect a wide range of hosts, including insects, birds, reptiles and mammals. These viruses have substantial incidence, prevalence and disease burden in humans and in other animals. Nucleotide and dinucleotide composition, mostly CpG and TpA, have been largely studied in viral genomes because of their evolutionary and functional implications. We analysed here the nucleotide and dinucleotide composition, as well as codon usage bias, of a set of representative poxvirus genomes, with a very diverse host spectrum. After correcting for overall nucleotide composition, entomopoxviruses displayed low overall GC content, no enrichment in TpA and large variation in CpG enrichment, while chordopoxviruses showed large variation in nucleotide composition, no obvious depletion in CpG and a weak trend for TpA depletion in GC-rich genomes. Overall, intergenome variation in dinucleotide composition in poxviruses is largely accounted for by variation in overall genomic GC levels. Nonetheless, using vaccinia virus as a model, we found that genes expressed at the earliest times in infection are more CpG-depleted than genes expressed at later stages. This observation has parallels in betahepesviruses (also large dsDNA viruses) and suggests an antiviral role for the innate immune system (e.g. via the zinc-finger antiviral protein ZAP) in the early phases of poxvirus infection. We also analysed codon usage bias in poxviruses and we observed that it is mostly determined by genomic GC content, and that stratification after host taxonomy does not contribute to explaining codon usage bias diversity. By analysis of within-species diversity, we show that genomic GC content is the result of mutational biases. Poxvirus genomes that encode a DNA ligase are significantly AT-richer than those that do not, suggesting that DNA repair systems shape mutation biases. Our data shed light on the evolution of poxviruses and inform strategies for their genetic manipulation for therapeutic purposes.

Microsatellite diversity in four cultivated species of Actinidiaceae and Rutaceae.

Microsatellites or Simple Sequence Repeats (SSRs) are short iterations of 1-6 bp in the genomes of almost all living organisms. Our study aimed to explore the microsatellite diversity in four cultivated species, namely Actinidia chinensis, Actinidia eriantha, Citrus maxima, and Citrus sinensis of the Actinidiaceae and Rutaceae families. We present a comprehensive analysis of microsatellite abundance, distribution, and motif composition in the genomes of these species. The association of microsatellite abundance with genomic features such as genome size, GC content, number of microsatellites, relative abundance, and relative density was also examined. The results revealed significant variations in the frequency and distribution of microsatellites across the genomes of these four species. Notably, a positive correlation was observed between genome size and microsatellite number as well as with GC content, indicating that larger genomes provide more opportunities for the accumulation of microsatellites. Furthermore, a negative correlation of genome size with relative microsatellite abundance and relative density was observed. These findings provide new insights into the microsatellite landscape of Actinidiaceae and Rutaceae, which could be explored for the development of microsatellite markers for diverse applications in the characterization of genetic diversity, molecular plant breeding, and phylogenetic analysis.

CRISPR/Cas9 Editing Sites Identification and Multi-Elements Association Analysis in Camellia sinensis.

CRISPR/Cas9 is an efficient genome-editing tool, and the identification of editing sites and potential influences in the Camellia sinensis genome have not been investigated. In this study, bioinformatics methods were used to characterise the Camellia sinensis genome including editing sites, simple sequence repeats (SSRs), G-quadruplexes (GQ), gene density, and their relationships. A total of 248,134,838 potential editing sites were identified in the genome, and five PAM types, AGG, TGG, CGG, GGG, and NGG, were observed, of which 66,665,912 were found to be specific, and they were present in all structural elements of the genes. The characteristic region of high GC content, GQ density, and PAM density in contrast to low gene density and SSR density was identified in the chromosomes in the joint analysis, and it was associated with secondary metabolites and amino acid biosynthesis pathways. CRISPR/Cas9, as a technology to drive crop improvement, with the identified editing sites and effector elements, provides valuable tools for functional studies and molecular breeding in Camellia sinensis.

Abandoning the Isochore Theory Can Help Explain Genome Compositional Organization in Fish.

The organization of the genome nucleotide (AT/GC) composition in vertebrates remains poorly understood despite the numerous genome assemblies available. Particularly, the origin of the AT/GC heterogeneity in amniotes, in comparison to the homogeneity in anamniotes, is controversial. Recently, several exceptions to this dichotomy were confirmed in an ancient fish lineage with mammalian AT/GC heterogeneity. Hence, our current knowledge necessitates a reevaluation considering this fact and utilizing newly available data and tools. We analyzed fish genomes in silico with as low user input as possible to compare previous approaches to assessing genome composition. Our results revealed a disparity between previously used plots of GC% and histograms representing the authentic distribution of GC% values in genomes. Previous plots heavily reduced the range of GC% values in fish to comply with the alleged AT/GC homogeneity and AT-richness of their genomes. We illustrate how the selected sequence size influences the clustering of GC% values. Previous approaches that disregarded chromosome and genome sizes, which are about three times smaller in fish than in mammals, distorted their results and contributed to the persisting confusion about fish genome composition. Chromosome size and their transposons may drive the AT/GC heterogeneity apparent on mammalian chromosomes, whereas far less in fishes.

High subtelomeric GC content in the genome of a zoonotic Cryptosporidium species.

Cryptosporidium canis is a zoonotic species causing cryptosporidiosis in humans in addition to its natural hosts dogs and other fur animals. To understand the genetic basis for host adaptation, we sequenced the genomes of C. canis from dogs, minks, and foxes and conducted a comparative genomics analysis. While the genomes of C. canis have similar gene contents and organisations, they (~41.0 %) and C. felis (39.6 %) have GC content much higher than other Cryptosporidium spp. (24.3-32.9 %) sequenced to date. The high GC content is mostly restricted to subtelomeric regions of the eight chromosomes. Most of these GC-balanced genes encode Cryptosporidium-specific proteins that have intrinsically disordered regions and are involved in host-parasite interactions. Natural selection appears to play a more important role in the evolution of codon usage in GC-balanced C. canis, and most of the GC-balanced genes have undergone positive selection. While the identity in whole genome sequences between the mink- and dog-derived isolates is 99.9 % (9365 SNVs), it is only 96.0 % (362 894 SNVs) between them and the fox-derived isolate. In agreement with this, the fox-derived isolate possesses more subtelomeric genes encoding invasion-related protein families. Therefore, the change in subtelomeric GC content appears to be responsible for the more GC-balanced C. canis genomes, and the fox-derived isolate could represent a new Cryptosporidium species.

Genome size and GC content of myxomycetes.

More than 1272 myxomycetes species have been described, accounting for more than half of all Amoebozoa species. However, the genome size of only three myxomycetes species has been reported. Therefore, we used flow cytometry to present an extensive survey and a phylogeny-based analysis of genome size and GC content evolution in 144 myxomycetes species. The genome size of myxomycetes ranged from 18.7 Mb to 470.3 Mb, and the GC content ranged from 38.7% to 70.1%. Bright-spored clade showed larger genome sizes and more intra-order genome size variations than the dark-spored clade. GC content and genome size were positively correlated in both bright-spored and dark-spored clades, and spore size was positively correlated with genome size and GC content in the bright-spored clade. We provided the first genome size data set in Myxomycetes, and our results will provide helpful information for future Myxomycetes studies, such as genome sequencing.

Characterization and Diversity of Klebsiella pneumoniae Prophages.

Klebsiella pneumoniae is a common human commensal and opportunistic pathogen. In recent years, the clinical isolation and resistance rates of K. pneumoniae have shown a yearly increase, leading to a special interest in mobile genetic elements. Prophages are a representative class of mobile genetic elements that can carry host-friendly genes, transfer horizontally between strains, and coevolve with the host's genome. In this study, we identified 15,946 prophages from the genomes of 1437 fully assembled K. pneumoniae deposited in the NCBI database, with 9755 prophages on chromosomes and 6191 prophages on plasmids. We found prophages to be notably diverse and widely disseminated in the K. pneumoniae genomes. The K. pneumoniae prophages encoded multiple putative virulence factors and antibiotic resistance genes. The comparison of strain types with prophage types suggests that the two may be related. The differences in GC content between the same type of prophages and the genomic region in which they were located indicates the alien properties of the prophages. The overall distribution of GC content suggests that prophages integrated on chromosomes and plasmids may have different evolutionary characteristics. These results suggest a high prevalence of prophages in the K. pneumoniae genome and highlight the effect of prophages on strain characterization.