Putative Origin Of Replication Research Articles

Specificities and commonalities of the Planctomycetes plasmidome.

Plasmids, despite their critical role in antibiotic resistance and modern biotechnology, are understood in only a few bacterial groups in terms of their natural ecological dynamics. The bacterial phylum Planctomycetes, known for its unique molecular and cellular biology, has a largely unexplored plasmidome. This study offers a thorough exploration of the diversity of natural plasmids within Planctomycetes, which could serve as a foundation for developing various genetic research tools for this phylum. Planctomycetes plasmids encode a broad range of biological functions and appear to have coevolved significantly with their host chromosomes, sharing many homologues. Recent transfer events of insertion sequences between cohabiting chromosomes and plasmids were also observed. Interestingly, 64% of plasmid genes are distantly related to either chromosomally encoded genes or have homologues in plasmids from other bacterial groups. The planctomycetal plasmidome is composed of 36% exclusive proteins. Most planctomycetal plasmids encode a replication initiation protein from the Replication Protein A family near a putative iteron-containing replication origin, as well as active type I partition systems. The identification of one conjugative and three mobilizable plasmids suggests the occurrence of horizontal gene transfer via conjugation within this phylum. This comprehensive description enhances our understanding of the plasmidome of Planctomycetes and its potential implications in antibiotic resistance and biotechnology.

Phylogenetic relationships and introduction history inferred from complete mitochondrial genomes of four smelts (Osmeridae) of the modern San Francisco Estuary

We announce complete mitogenomes of four smelts (Osmeridae) of the San Francisco Estuary (SFE): endemic Hypomesus transpacificus, native H. pretiosus and Spirinchus thaleichthys, and non-native H. nipponensis. The genomes each contained 13 protein coding, 2 ribosomal RNA and 22 transfer RNA genes, a control region (D-loop), and a putative origin of replication of the light strand (OL); gene order followed other osmerids. Total genomes were 16,767 base-pairs (bp) for H. transpacificus; 16,672bp for H. pretiosus; 16,811bp for S. thaleichthys; and 16,779bp for H. nipponensis. GC content was from 47.8% (S. thaleichthys) to 49.4% (H. pretiosus). Phylogenetic analyses of mitogenomes placed H. transpacificus sequences within the H. pretiosus [Oregon + SFE] clade and most closely related to H. pretiosus [SFE], whereas affinities of derivative cytochrome b sequences supported a Lake Suwa origin for SFE H. nipponensis.

Genome-wide binding sites of Plasmodium falciparum mini chromosome maintenance protein MCM6 show new insights into parasite DNA replication.

Multiple rounds of DNA replication take place in various stages of the life cycle in the human malaria parasite Plasmodium falciparum. Previous bioinformatics analysis has shown the presence of putative Autonomously Replicating Sequence (ARS) like sequences in the Plasmodium genome. However, the actual sites and frequency of replication origins in the P. falciparum genome based on experimental data still remain elusive. Minichromosome maintenance (MCM) proteins are recruited by the Origin recognition complex (ORC) to the origins of replication in eukaryotes including P. falciparum. We used PfMCM6 for chromatin immunoprecipitation followed by sequencing (ChIP-seq) in the quest for identification of putative replication origins in the parasite. PfMCM6 DNA binding sites annotation revealed high enrichment at exon regions. This is contrary to higher eukaryotes that show an inclination of origin sites towards transcriptional start sites. ChIP-seq results were further validated by ChIP-qPCR results as well as nascent strand abundance assay at the selected PfMCM6 enriched sites that also showed preferential binding of PfORC1 suggesting potential of these sites as origin sites. Further, PfMCM6 ChIP-seq data showed a positive correlation with previously published histone H4K8Ac genome-wide binding sites but not with H3K9Ac sites suggesting epigenetic control of replication initiation sites in the parasites. Overall, our data show the genome-wide distribution of PfMCM6 binding sites with their potential as replication origins in this deadly human pathogen that not only broadens our knowledge of parasite DNA replication and its unique biology, it may help to find new avenues for intervention processes.

Comparative analyses of the complete mitochondrial genomes of two southern African endemic guitarfish, Acroteriobatus annulatus and A. blochii

Shark-like rays (order Rhinopristiformes) are among the most threatened cartilaginous fish globally. Despite this, unresolved taxonomic issues still exist within the group. To date, no studies have used complete mitochondrial genomes to assess the phylogenetic placement of Acroteriobatus within the non-monophyletic family Rhinobatidae. The current study reports the first complete mitochondrial genomes for Acroteriobatus annulatus and A. blochii. Similar to other rhinopristiforms, the complete sequences of A. annulatus (16,773 bp) and A. blochii (16,771 bp) were circular molecules with gene organisations identical to that of the typical vertebrate mitogenome. The A + T content was higher than the G + C content, with a bias towards A and C nucleotides observed in all complete mitogenomes. The stem-and-loop secondary structures of the putative origin of light-strand replication were found to have highly conserved synthesis and stem regions, with all substitutions and indels restricted to the loop structure. The ratios of non-synonymous to synonymous substitution rates indicated that purifying selection has been the dominant driver of evolution in rhinopristiform mitogenomes. Phylogenetic reconstructions placed Acroteriobatus as a sister-group to Rhinobatos, confirming its affiliation with the family Rhinobatidae. However, based on its apparent polyphyly with the aforementioned genera, the familial assignment of Pseudobatos is not fully resolved and requires further investigation.

Widespread phages of endosymbionts: Phage WO genomics and the proposed taxonomic classification of Symbioviridae.

Wolbachia are the most common obligate, intracellular bacteria in animals. They exist worldwide in arthropod and nematode hosts in which they commonly act as reproductive parasites or mutualists, respectively. Bacteriophage WO, the largest of Wolbachia’s mobile elements, includes reproductive parasitism genes, serves as a hotspot for genetic divergence and genomic rearrangement of the bacterial chromosome, and uniquely encodes a Eukaryotic Association Module with eukaryotic-like genes and an ensemble of putative host interaction genes. Despite WO’s relevance to genome evolution, selfish genetics, and symbiotic applications, relatively little is known about its origin, host range, diversification, and taxonomic classification. Here we analyze the most comprehensive set of 150 Wolbachia and phage WO assemblies to provide a framework for discretely organizing and naming integrated phage WO genomes. We demonstrate that WO is principally in arthropod Wolbachia with relatives in diverse endosymbionts and metagenomes, organized into four variants related by gene synteny, often oriented opposite the putative origin of replication in the Wolbachia chromosome, and the large serine recombinase is an ideal typing tool to distinguish the four variants. We identify a novel, putative lytic cassette and WO’s association with a conserved eleven gene island, termed Undecim Cluster, that is enriched with virulence-like genes. Finally, we evaluate WO-like Islands in the Wolbachia genome and discuss a new model in which Octomom, a notable WO-like Island, arose from a split with WO. Together, these findings establish the first comprehensive Linnaean taxonomic classification of endosymbiont phages, including non-Wolbachia phages from aquatic environments, that includes a new family and two new genera to capture the collective relatedness of these viruses.

Novel Cyclovirus Species in Dogs with Hemorrhagic Gastroenteritis.

Nested PCRs with circovirus/cyclovirus pan-rep (replicase gene) primers detected eukaryotic circular Rep-encoding single-stranded DNA (CRESS DNA) viruses in three (samples CN9E, CN16E and CN34) of 18 canine parvovirus-2-positive fecal samples from household dogs with hemorrhagic gastroenteritis on the Caribbean island of Nevis. The complete genomes of CRESS DNA virus CN9E, CN16E and CN34 were determined by inverse nested PCRs. Based on (i) genome organization, (ii) location of the putative origin of replication, (iii) pairwise genome-wide sequence identities, (iv) the presence of conserved motifs in the putative replication-associated protein (Rep) and the arginine-rich region in the amino terminus of the putative capsid protein (Cp) and (v) a phylogenetic analysis, CN9E, CN16E and CN34 were classified as cycloviruses. Canine-associated cycloviruses CN16E and CN34 were closely related to each other and shared low genome-wide nucleotide (59.642–59.704%), deduced Rep (35.018–35.379%) and Cp (26.601%) amino acid sequence identities with CN9E. All the three canine-associated cycloviruses shared < 80% genome-wide pairwise nucleotide sequence identities with cycloviruses from other animals/environmental samples, constituting two novel species (CN9E and CN16E/34) within the genus Cyclovirus. Considering the feeding habits of dogs, we could not determine whether the cycloviruses were of dietary origin or infected the host. Interestingly, the CN9E putative Rep-encoding open reading frame was found to use the invertebrate mitochondrial genetic code with an alternative initiation codon (ATA) for translation, corroborating the hypothesis that cycloviruses are actually arthropod-infecting viruses. To our knowledge, this is the first report on the detection and complete genome analysis of cycloviruses from domestic dogs.

Detection and Complete Genome Analysis of Circoviruses and Cycloviruses in the Small Indian Mongoose (Urva auropunctata): Identification of Novel Species

Fecal samples from 76 of 83 apparently healthy small Indian mongooses (Urva auropunctata) were PCR positive with circovirus/cyclovirus pan-rep (replicase gene) primers. In this case, 30 samples yielded high quality partial rep sequences (~400 bp), of which 26 sequences shared maximum homology with cycloviruses from an arthropod, bats, humans or a sheep. Three sequences exhibited maximum identities with a bat circovirus, whilst a single sequence could not be assigned to either genus. Using inverse nested PCRs, the complete genomes of mongoose associated circoviruses (Mon-1, -29 and -66) and cycloviruses (Mon-20, -24, -32, -58, -60 and -62) were determined. Mon-1, -20, -24, -29, -32 and -66 shared <80% maximum genome-wide pairwise nucleotide sequence identities with circoviruses/cycloviruses from other animals/sources, and were assigned to novel circovirus, or cyclovirus species. Mon-58, -60 and -62 shared maximum pairwise identities of 79.90–80.20% with human and bat cycloviruses, which were borderline to the cut-off identity value for assigning novel cycloviral species. Despite high genetic diversity, the mongoose associated circoviruses/cycloviruses retained the various features that are conserved among members of the family Circoviridae, such as presence of the putative origin of replication (ori) in the 5′-intergenic region, conserved motifs in the putative replication-associated protein and an arginine rich region in the amino terminus of the putative capsid protein. Since only fecal samples were tested, and mongooses are polyphagous predators, we could not determine whether the mongoose associated circoviruses/cycloviruses were of dietary origin, or actually infected the host. To our knowledge, this is the first report on detection and complete genome analysis of circoviruses/cycloviruses in the small Indian mongoose, warranting further studies in other species of mongooses.

Identification of the Unwinding Region in the Clostridioides difficile Chromosomal Origin of Replication.

Faithful DNA replication is crucial for viability of cells across all kingdoms. Targeting DNA replication is a viable strategy for inhibition of bacterial pathogens. Clostridioides difficile is an important enteropathogen that causes potentially fatal intestinal inflammation. Knowledge about DNA replication in this organism is limited and no data is available on the very first steps of DNA replication. Here, we use a combination of in silico predictions and in vitro experiments to demonstrate that C. difficile employs a bipartite origin of replication that shows DnaA-dependent melting at oriC2, located in the dnaA-dnaN intergenic region. Analysis of putative origins of replication in different clostridia suggests that the main features of the origin architecture are conserved. This study is the first to characterize aspects of the origin region of C. difficile and contributes to our understanding of the initiation of DNA replication in clostridia.

Replication of theSalmonella Genomic Island 1 (SGI1) triggered by helper IncC conjugative plasmids promotes incompatibility and plasmid loss.

The mobilizable resistance island Salmonella genomic island 1 (SGI1) is specifically mobilized by IncA and IncC conjugative plasmids. SGI1, its variants and IncC plasmids propagate multidrug resistance in pathogenic enterobacteria such as Salmonella enterica serovars and Proteus mirabilis. SGI1 modifies and uses the conjugation apparatus encoded by the helper IncC plasmid, thus enhancing its own propagation. Remarkably, although SGI1 needs a coresident IncC plasmid to excise from the chromosome and transfer to a new host, these elements have been reported to be incompatible. Here, the stability of SGI1 and its helper IncC plasmid, each expressing a different fluorescent reporter protein, was monitored using fluorescence-activated cell sorting (FACS). Without selective pressure, 95% of the cells segregated into two subpopulations containing either SGI1 or the helper plasmid. Furthermore, FACS analysis revealed a high level of SGI1-specific fluorescence in IncC+ cells, suggesting that SGI1 undergoes active replication in the presence of the helper plasmid. SGI1 replication was confirmed by quantitative PCR assays, and extraction and restriction of its plasmid form. Deletion of genes involved in SGI1 excision from the chromosome allowed a stable coexistence of SGI1 with its helper plasmid without selective pressure. In addition, deletion of S003 (rep) or of a downstream putative iteron-based origin of replication, while allowing SGI1 excision, abolished its replication, alleviated the incompatibility with the helper plasmid and enabled its cotransfer to a new host. Like SGI1 excision functions, rep expression was found to be controlled by AcaCD, the master activator of IncC plasmid transfer. Transient SGI1 replication seems to be a key feature of the life cycle of this family of genomic islands. Sequence database analysis revealed that SGI1 variants encode either a replication initiator protein with a RepA_C domain, or an alternative replication protein with N-terminal replicase and primase C terminal 1 domains.

Characterization of the Whole Mitogenome of Largehead Hairtail Trichiurus lepturus (Trichiuridae): Insights into Special Characteristics.

Mitochondrial genomes are valuable for making inferences regarding population genetics, phylogenetics and recently in understanding adaptive variation in OXPHOS genes. Ribbon fish, Trichiurus lepturus is distributed in the Indian Ocean and contributes to a major share of pelagic fish landings. Considering the importance, we characterized the complete mitogenome of large head hairtail T. lepturus from Indian waters by PCR amplification and sequencing. The entire mitogenome is 16,840bp in length, with 13 protein coding genes, 2 subunit ribosomal RNA genes, and 1 control region similar to other vertebrates. Genome contains only 21 tRNAs (tRNAPro is absent) as against 22 tRNAs reported in other vertebrates. The putative origin of L-strand replication (OL) was located in a cluster of five tRNA genes (WANCY region) and may not be functional due to the variation of conserved motif (5'-GCCGG-3') and loop. These two unusual features are common for Trichiurus mitogenomes. Phylogenetic analysis supported the monophyly of Trichiuridae. This study will provide information for further studies on taxonomy, phylogeny and patterns of adaptive evolution in the mitogenome of Trichiurus species.

The complete mitochondrial genome of Sphenomorphus indicus (Reptilia: Scincidae)

The complete mitochondrial genome (mitogenome) sequence of Sphenomorphus indicus was sequenced and characterized by next-generation sequencing technology. The total length of mitogenome is 17,027 bp and contains 13 protein-coding genes, 22 tRNA genes, 2 ribosome RNA genes, and 2 non-coding regions (the control region and the putative L-strand replication origin). Most of the genes of S. indicus are encoded on the H-strand, except for the ND6 subunit gene and 8 tRNA genes which is distributed on the L-strand. Phylogenetic reconstruction suggested that S. indicus is the sister group of the S. incognitus within the genus Sphenomorphus. The complete mitochondrial genome sequence presented here will be useful to study the evolutionary relationships and genetic diversity of S. indicus.

Complete mitochondrial genome of the Western Capercaillie Tetrao urogallus (Phasianidae, Tetraoninae).

The Western Capercaillie (Tetrao urogallus) is a galliform bird of boreal climax forests from Scandinavia to eastern Siberia, with a fragmented population in southwestern Europe. We extracted the DNA of T. urogallus aquitanicus and obtained the complete mitochondrial genome (mitogenome) sequence by combining Illumina and Sanger sequencing sequence data. The mitochondrial genome of T. urogallus is 16,683 bp long and is very similar to that of Lyrurus tetrix (16,677 bp). The T. urogallus mitogenome contains the normal 13 protein-coding genes (PCGs), 22 transfer RNAs, 2 ribosomal RNAs, and the control region. The number, order, and orientation of the mitochondrial genes are the same as in L. tetrix and in other species of the same and other bird families. The three domains of the control region contained conserved sequences (ETAS; CSBs), boxes (F, E, D, C, B, BS box), the putative origin of replication of the H-strand (OH) and bidirectional promoters of translation (LSP/HSP).

The complete mitochondrial genome of Microdous chalmersi (Gobiiformes: Odontobutidae)

Microdous chalmersi is an Odontobutidae fish that mainly inhabits in East and Southeast Asia. In this study, the complete mitochondrial genome of M. chalmersi was first determined and described. It was 16,485 bp in length and composed by 13 protein-coding genes, 22 tRNA genes, two rRNA genes, a putative light strand origin of replication (OL) and a putative control region (D-loop). The overall nucleotide composition was 28.9% of A, 16.0% of G, 25.0% of T, and 30.1% of C, with a total of 53.9% A + T content. Phylogenetic analysis suggested that M. chalmersi had the closest evolutionary relationship with Micropercops swinhonis. The availability of the M. chalmersi mitogenome would contribute to further phylogenetic research of Odontobutidae and genetics studies of this species.

The complete mitochondrial genome of the critically endangered Cuora mccordi (Reptilia: Geoemydidae)

The complete mitochondrial genome (mitogenome) sequence of the MeCord's box turtle (Cuora mccordi) was firstly determined by shotgun sequencing. The total length of mitogenome is 16,688 bp, and contains 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 2 main noncoding regions (the control region and the putative L-strand replication origin). The majority genes (12 protein-coding genes, 2 rRNA and 14 tRNA) were distributed on the H-strand, except for the ND6 subunit gene and eight tRNA genes which were encoded on the L-strand. The phylogenetic tree of C. mccordi and 10 other closely related species was built. The DNA data presented here will be useful to study the evolutionary relationships and genetic diversity of C. mccordi.

The complete mitochondrial genome of Sphenomorphus incognitus (Reptilia: Scincidae)

The complete mitochondrial genome (mitogenome) sequence of Sphenomorphus incognitus was determined by shotgun sequencing. The total length of mitogenome is 17 417 bp, and contains 13 protein-coding genes, 22 tRNA genes, two ribosome RNA genes and two non-coding regions (the control region and the putative L-strand replication origin). Most of the genes of S. incognitus were distributed on the H-strand, except for the ND6 subunit gene and eight tRNA genes which were encoded on the L-strand. The phylogenetic tree of S. incognitus and 8 other closely species was built. The DNA data presented here will be useful to study the evolutionary relationships and genetic diversity of S. incognitus.

The complete mitochondrial genome of the horned lizard Phrynosoma blainvillii (Squamata: Phrynosomatidae) from California, USA

Analysis of Phrynosoma blainvillii Gray from Marina, Monterey County, California, using 150 bp paired-end Illumina sequences (Illumina, San Diego, CA) resulted in the assembly of its complete mitogenome. The mitogenome is 16,946 bp in length and contains a putative origin of light strand replication (OL), control region, 22 tRNA, 2 rRNA, and 13 protein-coding genes. Its content and organization are similar to other Squamata. Phylogenetic analysis of P. blainvillii resolves it in a clade with P. sherbrookei Nieto-Montes de Oca, Arenas-Moreno, Beltrán-Sánchez & Leaché, sister in position to Uma notata Baird. Mitochondrial marker analysis of P. blainvillii from Marina shows that it belongs to a coastal Santa Lucia Mountain Range haplogroup that is distinct from other populations of P. blainvillii in California.

Characterization of Two Cryptic Plasmids Isolated in Haiti from Clinical Vibrio cholerae Non-O1/Non-O139.

We report the complete sequence of two novel plasmids, pSDH-1 and pSDH-2, isolated from clinical Vibrio cholerae non-O1/non-O139 during the early phase of the 2010 Haitian cholera epidemic. Plasmids were revealed by employing single-cell genomics and their genome content suggests self-mobilization and, for pSDH-2, a toxin-antitoxin (TA) system for plasmid stabilization was identified. The putative origin of replication of pSDH-2 was mapped suggesting it replicates following the ColE1 model of plasmid replication. pSDH-1 and pSDH-2 were widespread among environmental V. cholerae non-O1/non-O139 with variable prevalence in four Haitian Departments. pSDH-2 was the most common element, either alone or with pSDH-1. The two plasmids detection adds to the composite scenario of mobile genetic elements (MGEs) observed in V. cholerae in Haiti. The role these small cryptic plasmids circulating in Vibrio spp. play in bacterial fitness or pathogenicity merits further investigation.

The complete mitochondrial genome of Pseudocrossocheilus bamaensis (Cypriniformes: Cyprinidae: Labeoninae)

Pseudocrossocheilus bamaensis is endemic to the upstream of Xijiang River, Guangxi, China and its natural population has been declining due to overfishing and habitat destruction. Here, we first report the complete mitogenome of this species, which is 16,594 bp in length, containing 13 protein-coding genes, 22 tRNA genes, two rRNA genes, as well as a putative light strand origin of replication (OL) and a putative control region (D-loop). The overall base composition is 31.8% of A, 15.7% of G, 26.8% of T and 25.7% of C, with a high A + T content (58.6%). Phylogenetic analysis suggests that P. bamaensis is closely related to Ptychidio jordani and Rectoris posehensis. These results will contribute to the molecular evolution and phylogenetic research of Labeoninae and further protection of its fish species.

Identification and characterization of ARS-like sequences as putative origin(s) of replication in human malaria parasite Plasmodium falciparum.

DNA replication is a fundamental process in genome maintenance, and initiates from several genomic sites (origins) in eukaryotes. In Saccharomyces cerevisiae, conserved sequences known as autonomously replicating sequences (ARSs) provide a landing pad for the origin recognition complex (ORC), leading to replication initiation. Although origins from higher eukaryotes share some common sequence features, the definitive genomic organization of these sites remains elusive. The human malaria parasite Plasmodium falciparum undergoes multiple rounds of DNA replication; therefore, control of initiation events is crucial to ensure proper replication. However, the sites of DNA replication initiation and the mechanism by which replication is initiated are poorly understood. Here, we have identified and characterized putative origins in P. falciparum by bioinformatics analyses and experimental approaches. An autocorrelation measure method was initially used to search for regions with marked fluctuation (dips) in the chromosome, which we hypothesized might contain potential origins. Indeed, S. cerevisiae ARS consensus sequences were found in dip regions. Several of these P. falciparum sequences were validated with chromatin immunoprecipitation-quantitative PCR, nascent strand abundance and a plasmid stability assay. Subsequently, the same sequences were used in yeast to confirm their potential as origins in vivo. Our results identify the presence of functional ARSs in P. falciparum and provide meaningful insights into replication origins in these deadly parasites. These data could be useful in designing transgenic vectors with improved stability for transfection in P. falciparum.

The genome sequence of Agrotis segetum granulovirus, isolate AgseGV-DA, reveals a new Betabaculovirus species of a slow killing granulovirus

The European isolate Agrotis segetum granulovirus DA (AgseGV-DA) is a slow killing, type I granulovirus due to low dose-mortality responses within seven days post infection and a tissue tropism of infection restricted solely to the fat body of infected Agrotis segetum host larvae. The genome of AgseGV-DA was completely sequenced and compared to the whole genome sequences of the Chinese isolates AgseGV-XJ and AgseGV-L1. All three isolates share highly conserved genomes. The AgseGV-DA genome is 131,557bp in length and encodes for 149 putative open reading frames, including 37 baculovirus core genes and the per os infectivity factor ac110. Comprehensive investigations of repeat regions identified one putative non-hr like origin of replication in AgseGV-DA. Phylogenetic analysis based on concatenated amino acid alignments of 37 baculovirus core genes as well as pairwise distances based on the nucleotide alignments of partial granulin, lef-8 and lef-9 sequences with deposited betabaculoviruses confirmed AgseGV-DA, AgseGV-XJ and AgseGV-L1 as representative isolates of the same Betabaculovirus species. AgseGV encodes for a distinct putative enhancin, distantly related to enhancins from other granuloviruses.