Systematic Analysis of Diverse Polynucleotide Kinase Clp1 Family Proteins in Eukaryotes: Three Unique Clp1 Proteins of Trypanosoma brucei.

The Clp1 family proteins, consisting of the Clp1 and Nol9/Grc3 groups, have polynucleotide kinase (PNK) activity at the 5' end of RNA strands and are important enzymes in the processing of some precursor RNAs. However, it remains unclear how this enzyme family diversified in the eukaryotes. We performed a large-scale molecular evolutionary analysis of the full-length genomes of 358 eukaryotic species to classify the diverse Clp1 family proteins. The average number of Clp1 family proteins in eukaryotes was 2.3 ± 1.0, and most representative species had both Clp1 and Nol9/Grc3 proteins, suggesting that the Clp1 and Nol9/Grc3 groups were already formed in the eukaryotic ancestor by gene duplication. We also detected an average of 4.1 ± 0.4 Clp1 family proteins in members of the protist phylum Euglenozoa. For example, in Trypanosoma brucei, there are three genes of the Clp1 group and one gene of the Nol9/Grc3 group. In the Clp1 group proteins encoded by these three genes, the C-terminal domains have been replaced by unique characteristics domains, so we designated these proteins Tb-Clp1-t1, Tb-Clp1-t2, and Tb-Clp1-t3. Experimental validation showed that only Tb-Clp1-t2 has PNK activity against RNA strands. As in this example, N-terminal and C-terminal domain replacement also contributed to the diversification of the Clp1 family proteins in other eukaryotic species. Our analysis also revealed that the Clp1 family proteins in humans and plants diversified through isoforms created by alternative splicing.

Reconsideration of the previously classified incompatibility groups of plasmids, IncP-1 and IncP-11.

This study presents the reassessment of earlier published data with reference to the article published in Environmental Microbiology entitled 'IncP-type plasmids carrying genes for antibiotic resistance or aromatic compound degradation are prevalent in sequenced Aromatoleum and Thauera strains' by Lo et al. This correspondence clarifies misperceptions of plasmids classified under incompatibility (Inc) groups IncP-1 and IncP-11.

Prophages and plasmids can display opposite trends in the types of accessory genes they carry.

Mobile genetic elements (MGEs), such as phages and plasmids, often possess accessory genes encoding bacterial functions, facilitating bacterial evolution. Are there rules governing the arsenal of accessory genes MGEs carry? If such rules exist, they might be reflected in the types of accessory genes different MGEs carry. To test this hypothesis, we compare prophages and plasmids with respect to the frequencies at which they carry antibiotic resistance genes (ARGs) and virulence factor genes (VFGs) in the genomes of 21 pathogenic bacterial species using public databases. Our results indicate that prophages tend to carry VFGs more frequently than ARGs in three species, whereas plasmids tend to carry ARGs more frequently than VFGs in nine species, relative to genomic backgrounds. In Escherichia coli, where this prophage-plasmid disparity is detected, prophage-borne VFGs encode a much narrower range of functions than do plasmid-borne VFGs, typically involved in damaging host cells or modulating host immunity. In the species where the above disparity is not detected, ARGs and VFGs are barely found in prophages and plasmids. These results indicate that MGEs can differentiate in the types of accessory genes they carry depending on their infection strategies, suggesting a rule governing horizontal gene transfer mediated by MGEs.

Molecular dynamics study on the hydrogen bond formation between α-hydrogen atom of L-Phe and N5 atom of FAD in the enzyme-substrate complex of the L-Phe oxidase reaction.

It is difficult to observe the structure of the enzyme-substrate complex (ES complex) experimentally, since the complex changes to the enzyme and its product during observation. The molecular dynamics (MD) approach is ideal to observe the structural change of enzyme and of substrate in the ES complex. Analyses on the complex of L-Phe oxidase with L-Phe by MD showed 1) the distance between the α-hydrogen atom of L-Phe and the N5 atom of isoalloxazine ring of FAD to be 2.64 ± 0.19 Å, and 2) the angle CA-HA-N5 atoms to be 141.5 ± 10.7°. This result clearly showed that the α-hydrogen atom forms the hydrogen bond with the N5 atom of isoalloxazine ring of FAD in the enzyme-substrate complex. Thus, the complex is ready for the hydrogen transfer from substrate to FAD in the key step of the oxidation of substrate by the enzyme.

Annotating unknown species of urban microorganisms on a global scale unveils novel functional diversity and local environment association.

In urban ecosystems, microbes play a key role in maintaining major ecological functions that directly support human health and city life. However, the knowledge about the species composition and functions involved in urban environments is still limited, which is largely due to the lack of reference genomes in metagenomic studies comprises more than half of unclassified reads. Here we uncovered 732 novel bacterial species from 4728 samples collected from various common surface with the matching materials in the mass transit system across 60 cities by the MetaSUB Consortium. The number of novel species is significantly and positively correlated with the city population, and more novel species can be identified in the skin-associated samples. The in-depth analysis of the new gene catalog showed that the functional terms have a significant geographical distinguishability. Moreover, we revealed that more biosynthetic gene clusters (BGCs) can be found in novel species. The co-occurrence relationship between BGCs and genera and the geographical specificity of BGCs can also provide us more information for the synthesis pathways of natural products. Expanded the known urban microbiome diversity and suggested additional mechanisms for taxonomic and functional characterization of the urban microbiome. Considering the great impact of urban microbiomes on human life, our study can also facilitate the microbial interaction analysis between human and urban environment.

Comparative genomics of Bacteria commonly identified in the built environment.

BACKGROUND: The microbial community of the built environment (BE) can impact the lives of people and has been studied for a variety of indoor, outdoor, underground, and extreme locations. Thus far, these microorganisms have mainly been investigated by culture-based methods or amplicon sequencing. However, both methods have limitations, complicating multi-study comparisons and limiting the knowledge gained regarding in-situ microbial lifestyles. A greater understanding of BE microorganisms can be achieved through basic information derived from the complete genome. Here, we investigate the level of diversity and genomic features (genome size, GC content, replication strand skew, and codon usage bias) from complete genomes of bacteria commonly identified in the BE, providing a first step towards understanding these bacterial lifestyles. RESULTS: Here, we selected bacterial genera commonly identified in the BE (or "Common BE genomes") and compared them against other prokaryotic genera ("Other genomes"). The "Common BE genomes" were identified in various climates and in indoor, outdoor, underground, or extreme built environments. The diversity level of the 16S rRNA varied greatly between genera. The genome size, GC content and GC skew strength of the "Common BE genomes" were statistically larger than those of the "Other genomes" but were not practically significant. In contrast, the strength of selected codon usage bias (S value) was statistically higher with a large effect size in the "Common BE genomes" compared to the "Other genomes." CONCLUSION: Of the four genomic features tested, the S value could play a more important role in understanding the lifestyles of bacteria living in the BE. This parameter could be indicative of bacterial growth rates, gene expression, and other factors, potentially affected by BE growth conditions (e.g., temperature, humidity, and nutrients). However, further experimental evidence, species-level BE studies, and classification by BE location is needed to define the relationship between genomic features and the lifestyles of BE bacteria more robustly.

The recombination-cold region as an epidemiological marker of recombinogenic opportunistic pathogen Mycobacterium avium.

BACKGROUND: The rapid identification of lineage remains a challenge in the genotyping of clinical isolates of recombinogenic pathogens. The chromosome of Mycobacterium avium subsp. hominissuis (MAH), an agent of Mycobacterium avium complex (MAC) lung disease, is often mosaic and is composed of chromosomal segments originating from different lineages. This makes it difficult to infer the MAH lineage in a simple experimental set-up. To overcome this difficulty, we sought to identify chromosomal marker genes containing lineage-specific alleles by genome data mining. RESULTS: We conducted genetic population structure analysis, phylogenetic analysis, and a survey of historical recombination using data from 125 global MAH isolates. Six MAH lineages (EA1, EA2, SC1, SC2, SC3, and SC4) were identified in the current dataset. One P-450 gene (locus_tag MAH_0788/MAV_0940) in the recombination-cold region was found to have multiple alleles that could discriminate five lineages. By combining the information about allele type from one additional gene, the six MAH lineages as well as other M. avium subspecies were distinguishable. A recombination-cold region of 116 kb contains an insertion hotspot and is flanked by a mammalian cell-entry protein operon where allelic variants have previously been reported to occur. Hence, we speculate that the acquisition of lineage- or strain-specific insertions has introduced homology breaks in the chromosome, thereby reducing the chance of interlineage recombination. CONCLUSIONS: The allele types of the newly identified marker genes can be used to predict major lineages of M. avium. The single nucleotide polymorphism typing approach targeting multiallelic loci in recombination-cold regions will facilitate the epidemiological study of MAC, and may also be useful for equivalent studies of other nontuberculous mycobacteria potentially carrying mosaic genomes.

Collaborative environmental DNA sampling from petal surfaces of flowering cherry Cerasus × yedoensis 'Somei-yoshino' across the Japanese archipelago.

Recent studies have shown that environmental DNA is found almost everywhere. Flower petal surfaces are an attractive tissue to use for investigation of the dispersal of environmental DNA in nature as they are isolated from the external environment until the bud opens and only then can the petal surface accumulate environmental DNA. Here, we performed a crowdsourced experiment, the "Ohanami Project", to obtain environmental DNA samples from petal surfaces of Cerasus × yedoensis 'Somei-yoshino' across the Japanese archipelago during spring 2015. C. × yedoensis is the most popular garden cherry species in Japan and clones of this cultivar bloom simultaneously every spring. Data collection spanned almost every prefecture and totaled 577 DNA samples from 149 collaborators. Preliminary amplicon-sequencing analysis showed the rapid attachment of environmental DNA onto the petal surfaces. Notably, we found DNA of other common plant species in samples obtained from a wide distribution; this DNA likely originated from the pollen of the Japanese cedar. Our analysis supports our belief that petal surfaces after blossoming are a promising target to reveal the dynamics of environmental DNA in nature. The success of our experiment also shows that crowdsourced environmental DNA analyses have considerable value in ecological studies.

Comparative genomic and phenomic analysis of Clostridium difficile and Clostridium sordellii, two related pathogens with differing host tissue preference.

BACKGROUND: Clostridium difficile and C. sordellii are two anaerobic, spore forming, gram positive pathogens with a broad host range and the ability to cause lethal infections. Despite strong similarities between the two Clostridial strains, differences in their host tissue preference place C. difficile infections in the gastrointestinal tract and C. sordellii infections in soft tissues. RESULTS: In this study, to improve our understanding of C. sordellii and C. difficile virulence and pathogenesis, we have performed a comparative genomic and phenomic analysis of the two. The global phenomes of C. difficile and C. sordellii were compared using Biolog Phenotype microarrays. When compared to C. difficile, C. sordellii was found to better utilize more complex sources of carbon and nitrogen, including peptides. Phenotype microarray comparison also revealed that C. sordellii was better able to grow in acidic pH conditions. Using next generation sequencing technology, we determined the draft genome of C. sordellii strain 8483 and performed comparative genome analysis with C. difficile and other Clostridial genomes. Comparative genome analysis revealed the presence of several enzymes, including the urease gene cluster, specific to the C. sordellii genome that confer the ability of expanded peptide utilization and survival in acidic pH. CONCLUSIONS: The identified phenotypes of C. sordellii might be important in causing wound and vaginal infections respectively. Proteins involved in the metabolic differences between C. sordellii and C. difficile should be targets for further studies aimed at understanding C. difficile and C. sordellii infection site specificity and pathogenesis.

Comparative functional genomics of Lactobacillus spp. reveals possible mechanisms for specialization of vaginal lactobacilli to their environment.

Lactobacilli are found in a wide variety of habitats. Four species, Lactobacillus crispatus, L. gasseri, L. iners, and L. jensenii, are common and abundant in the human vagina and absent from other habitats. These may be adapted to the vagina and possess characteristics enabling them to thrive in that environment. Furthermore, stable codominance of multiple Lactobacillus species in a single community is infrequently observed. Thus, it is possible that individual vaginal Lactobacillus species possess unique characteristics that confer to them host-specific competitive advantages. We performed comparative functional genomic analyses of representatives of 25 species of Lactobacillus, searching for habitat-specific traits in the genomes of the vaginal lactobacilli. We found that the genomes of the vaginal species were significantly smaller and had significantly lower GC content than those of the nonvaginal species. No protein families were found to be specific to the vaginal species analyzed, but some were either over- or underrepresented relative to nonvaginal species. We also found that within the vaginal species, each genome coded for species-specific protein families. Our results suggest that even though the vaginal species show no general signatures of adaptation to the vaginal environment, each species has specific and perhaps unique ways of interacting with its environment, be it the host or other microbes in the community. These findings will serve as a foundation for further exploring the role of lactobacilli in the ecological dynamics of vaginal microbial communities and their ultimate impact on host health.

Comparison of gene expression of Paramecium bursaria with and without Chlorella variabilis symbionts.

BACKGROUND: The ciliate Paramecium bursaria harbors several hundred cells of the green-alga Chlorella sp. in their cytoplasm. Irrespective of the mutual relation between P. bursaria and the symbiotic algae, both cells retain the ability to grow without the partner. They can easily reestablish endosymbiosis when put in contact with each other. Consequently, P. bursaria is an excellent model for studying cell-cell interaction and the evolution of eukaryotic cells through secondary endosymbiosis between different protists. Despite the importance of this organism, no genomic resources have been identified for P. bursaria to date. This investigation compared gene expressions through RNA-Seq analysis and de novo transcriptome assembly of symbiont-free and symbiont-bearing host cells. RESULTS: To expedite the process of gene discovery related to the endosymbiosis, we have undertaken Illumina deep sequencing of mRNAs prepared from symbiont-bearing and symbiont-free P. bursaria cells. We assembled the reads de novo to build the transcriptome. Sequencing using Illumina HiSeq2000 platform yielded 232.3 million paired-end sequence reads. Clean reads filtered from the raw reads were assembled into 68,175 contig sequences. Of these, 10,557 representative sequences were retained after removing Chlorella sequences and lowly expressed sequences. Nearly 90% of these transcript sequences were annotated by similarity search against protein databases. We identified differentially expressed genes in the symbiont-bearing P. bursaria cells relative to the symbiont-free cells, including heat shock 70 kDa protein and glutathione S-transferase. CONCLUSIONS: This is the first reported comprehensive sequence resource of Paramecium - Chlorella endosymbiosis. Results provide some keys for the elucidation of secondary endosymbiosis in P. bursaria. We identified P. bursaria genes that are differentially expressed in symbiont-bearing and symbiont-free conditions.

Shotgun metagenomic sequencing of swab samples from Japanese university campuses.

We obtained shotgun metagenome sequences from swab samples obtained through 3-minute swabbing of different surfaces and the air within buildings at three university campuses in part of the Greater Tokyo Area in Japan. These data aid in understanding built environment microbial communities and elucidate various microbial profiles across different locations.

Landscape of global urban environmental resistome and its association with local socioeconomic and medical status.

Antimicrobial resistance (AMR) poses a critical threat to global health and development, with environmental factors-particularly in urban areas-contributing significantly to the spread of antibiotic resistance genes (ARGs). However, most research to date has been conducted at a local level, leaving significant gaps in our understanding of the global status of antibiotic resistance in urban environments. To address this issue, we thoroughly analyzed a total of 86,213 ARGs detected within 4,728 metagenome samples, which were collected by the MetaSUB International Consortium involving diverse urban environments in 60 cities of 27 countries, utilizing a deep-learning based methodology. Our findings demonstrated the strong geographical specificity of urban environmental resistome, and their correlation with various local socioeconomic and medical conditions. We also identified distinctive evolutionary patterns of ARG-related biosynthetic gene clusters (BGCs) across different countries, and discovered that the urban environment represents a rich source of novel antibiotics. Our study provides a comprehensive overview of the global urban environmental resistome, and fills a significant gap in our knowledge of large-scale urban antibiotic resistome analysis.

Characterization of the heart transcriptome of the white shark (Carcharodon carcharias).

BACKGROUND: The white shark (Carcharodon carcharias) is a globally distributed, apex predator possessing physical, physiological, and behavioral traits that have garnered it significant public attention. In addition to interest in the genetic basis of its form and function, as a representative of the oldest extant jawed vertebrate lineage, white sharks are also of conservation concern due to their small population size and threat from overfishing. Despite this, surprisingly little is known about the biology of white sharks, and genomic resources are unavailable. To address this deficit, we combined Roche-454 and Illumina sequencing technologies to characterize the first transciptome of any tissue for this species. RESULTS: From white shark heart cDNA we generated 665,399 Roche 454 reads (median length 387-bp) that were assembled into 141,626 contigs (mean length 503-bp). We also generated 78,566,588 Illumina reads, which we aligned to the 454 contigs producing 105,014 454/Illumina consensus sequences. To these, we added 3,432 non-singleton 454 contigs. By comparing these sequences to the UniProtKB/Swiss-Prot database we were able to annotate 21,019 translated open reading frames (ORFs) of ≥ 20 amino acids. Of these, 19,277 were additionally assigned Gene Ontology (GO) functional annotations. While acknowledging the limitations of our single tissue transcriptome, Fisher tests showed the white shark transcriptome to be significantly enriched for numerous metabolic GO terms compared to the zebra fish and human transcriptomes, with white shark showing more similarity to human than to zebra fish (i.e. fewer terms were significantly different). We also compared the transcriptome to other available elasmobranch sequences, for signatures of positive selection and identified several genes of putative adaptive significance on the white shark lineage. The white shark transcriptome also contained 8,404 microsatellites (dinucleotide, trinucleotide, or tetranucleotide motifs ≥ five perfect repeats). Detailed characterization of these microsatellites showed that ORFs with trinucleotide repeats, were significantly enriched for transcription regulatory roles and that trinucleotide frequency within ORFs was lower than for a wide range of taxonomic groups including other vertebrates. CONCLUSION: The white shark heart transcriptome represents a valuable resource for future elasmobranch functional and comparative genomic studies, as well as for population and other biological studies vital for effective conservation of this globally vulnerable species.

Transconjugant range of PromA plasmids in microbial communities is predicted by sequence similarity with the bacterial host chromosome.

Nucleotide sequence similarity, including k-mer plasmid composition, has been used for prediction of plasmid evolutionary host range, representing the hosts in which a plasmid has replicated at some point during its evolutionary history. However, the relationships between the bacterial taxa of experimentally identified transconjugants and the predicted evolutionary host ranges are poorly understood. Here, four different PromA group plasmids showing different k-mer compositions were used as model plasmids. Filter mating assays were performed with a donor harbouring plasmids and recipients of bacterial communities extracted from environmental samples. A broad range of transconjugants was obtained with different bacterial taxa. A calculation of the dissimilarities in k-mer compositions as Mahalanobis distance between the plasmid and its sequenced transconjugant chromosomes revealed that each plasmid and transconjugant were significantly more similar than the plasmid and other non-transconjugant chromosomes. These results indicate that plasmids with different k-mer compositions clearly have different host ranges to which the plasmid will be transferred and replicated. The similarity of the nucleotide compositions could be used for predicting not only the plasmid evolutionary host range but also future host ranges.

Urban Microbiomes in Narita, Chiba, Japan: Shotgun Metagenome Sequences of a Train Station.

Here, we performed shotgun metagenome sequencing of swab samples collected on floors at a train station in Narita City, Chiba, Japan. The taxonomic analysis revealed that Actinobacteria and Proteobacteria were the dominant phyla. The data will contribute to insight into the microbiome community on the surfaces of urban built environments.

Evaluation of rRNA depletion methods for capturing the RNA virome from environmental surfaces.

OBJECTIVE: Metatranscriptomic analysis of RNA viromes on built-environment surfaces is hampered by low RNA yields and high abundance of rRNA. Therefore, we evaluated the quality of libraries, efficiency of rRNA depletion, and viral detection sensitivity using a mock community and a melamine-coated table surface RNA with levels below those required (< 5 ng) with a library preparation kit (NEBNext Ultra II Directional RNA Library Prep Kit). RESULTS: Good-quality RNA libraries were obtained from 0.1 ng of mock community and table surface RNA by changing the adapter concentration and number of PCR cycles. Differences in the target species of the rRNA depletion method affected the community composition and sensitivity of virus detection. The percentage of viral occupancy in two replicates was 0.259 and 0.290% in both human and bacterial rRNA-depleted samples, a 3.4 and 3.8-fold increase compared with that for only bacterial rRNA-depleted samples. Comparison of SARS-CoV-2 spiked-in human rRNA and bacterial rRNA-depleted samples suggested that more SARS-CoV-2 reads were detected in bacterial rRNA-depleted samples. We demonstrated that metatranscriptome analysis of RNA viromes is possible from RNA isolated from an indoor surface (representing a built-environment surface) using a standard library preparation kit.

Comparative genomic analysis of the genus Staphylococcus including Staphylococcus aureus and its newly described sister species Staphylococcus simiae.

BACKGROUND: Staphylococcus belongs to the Gram-positive low G + C content group of the Firmicutes division of bacteria. Staphylococcus aureus is an important human and veterinary pathogen that causes a broad spectrum of diseases, and has developed important multidrug resistant forms such as methicillin-resistant S. aureus (MRSA). Staphylococcus simiae was isolated from South American squirrel monkeys in 2000, and is a coagulase-negative bacterium, closely related, and possibly the sister group, to S. aureus. Comparative genomic analyses of closely related bacteria with different phenotypes can provide information relevant to understanding adaptation to host environment and mechanisms of pathogenicity. RESULTS: We determined a Roche/454 draft genome sequence for S. simiae and included it in comparative genomic analyses with 11 other Staphylococcus species including S. aureus. A genome based phylogeny of the genus confirms that S. simiae is the sister group to S. aureus and indicates that the most basal Staphylococcus lineage is Staphylococcus pseudintermedius, followed by Staphylococcus carnosus. Given the primary niche of these two latter taxa, compared to the other species in the genus, this phylogeny suggests that human adaptation evolved after the split of S. carnosus. The two coagulase-positive species (S. aureus and S. pseudintermedius) are not phylogenetically closest but share many virulence factors exclusively, suggesting that these genes were acquired by horizontal transfer. Enrichment in genes related to mobile elements such as prophage in S. aureus relative to S. simiae suggests that pathogenesis in the S. aureus group has developed by gene gain through horizontal transfer, after the split of S. aureus and S. simiae from their common ancestor. CONCLUSIONS: Comparative genomic analyses across 12 Staphylococcus species provide hypotheses about lineages in which human adaptation has taken place and contributions of horizontal transfer in pathogenesis.

Sequence alignment of folk song melodies reveals cross-cultural regularities of musical evolution.

Culture evolves,1-5 but the existence of cross-culturally general regularities of cultural evolution is debated.6-8 As a diverse but universal cultural phenomenon, music provides a novel domain to test for the existence of such regularities.9-12 Folk song melodies can be thought of as culturally transmitted sequences of notes that change over time under the influence of cognitive and acoustic/physical constraints.9-15 Modeling melodies as evolving sequences constructed from an "alphabet" of 12 scale degrees16 allows us to quantitatively test for the presence of cross-cultural regularities using a sample of 10,062 melodies from musically divergent Japanese and English (British/American) folk song traditions.17,18 Our analysis identifies 328 pairs of highly related melodies, finding that note changes are more likely when they have smaller impacts on a song's melody. Specifically, (1) notes with stronger rhythmic functions are less likely to change, and (2) note substitutions are most likely between neighboring notes. We also find that note insertions/deletions ("indels") are more common than note substitutions, unlike genetic evolution where the reverse is true. Our results are consistent across English and Japanese samples despite major differences in their scales and tonal systems. These findings demonstrate that even a creative art form such as music is subject to evolutionary constraints analogous to those governing the evolution of genes, languages, and other domains of culture.