Large-scale Recombination Events Research Articles

Comprehensive analysis of 66 complete molluscum contagiosum virus (MOCV) genomes: characterization and functional annotation of 47 novel complete MOCV genomes, including the first genome of MOCV genotype 3, and a proposal for harmonized MOCV genotyping indexing.

Four molluscum contagiosum virus (MOCV) genotypes (MOCV1-4) and four subtype variants were partially characterized using restriction enzyme profiling in the 1980s/1990s, but complete genome sequences of only MOCV1 and MOCV2 are available. The evolutionary pathways whereby genotypes/subtype variants with unavailable sequences emerged and whether all MOCVs can be detected using current diagnostic approaches remain unclear. We fully characterized 47 novel complete MOCV genomes, including the first complete MOCV3 genome, expanding the number of fully characterized genomes to 66. For reliably classifying the novel non-MOCV1/2 genomes, we developed and validated a framework for matching sequence-derived restriction maps with those defining MOCV subtypes in pioneering studies. Six phylogenetic subgroups (PG1-6) were identified, PG5 representing a novel MOCV2 subtype. The phylogenetic subgroups diverged from the prototype lineages following large-scale recombination events and hinted at partial sequence content of MOCV4 and direction of recombinant transfer in the events spawning PG5 and yet undetected MOCV1vb variant.

Diversity and Evolutionary History of Ti Plasmids of "tumorigenes" Clade of Rhizobium spp. and Their Differentiation from Other Ti and Ri Plasmids.

Agrobacteria are important plant pathogens responsible for crown/cane gall and hairy root diseases. Crown/cane gall disease is associated with strains carrying tumor-inducing (Ti) plasmids, while hairy root disease is caused by strains harboring root-inducing (Ri) plasmids. In this study, we analyzed the sequences of Ti plasmids of the novel "tumorigenes" clade of the family Rhizobiaceae ("tumorigenes" Ti plasmids), which includes two species, Rhizobium tumorigenes and Rhizobium rhododendri. The sequences of reference Ti/Ri plasmids were also included, which was followed by a comparative analysis of their backbone and accessory regions. The "tumorigenes" Ti plasmids have novel opine signatures compared with other Ti/Ri plasmids characterized so far. The first group exemplified by pTi1078 is associated with production of agrocinopine, nopaline, and ridéopine in plant tumors, while the second group comprising pTi6.2 is responsible for synthesis of leucinopine. Bioinformatic and chemical analyses, including opine utilization assays, indicated that leucinopine associated with pTi6.2 most likely has D,L stereochemistry, unlike the L,L-leucinopine produced in tumors induced by reference strains Chry5 and Bo542. Most of the "tumorigenes" Ti plasmids have conjugative transfer system genes that are unusual for Ti plasmids, composed of avhD4/avhB and traA/mobC/parA regions. Next, our results suggested that "tumorigenes" Ti plasmids have a common origin, but they diverged through large-scale recombination events, through recombination with single or multiple distinct Ti/Ri plasmids. Lastly, we showed that Ti/Ri plasmids could be differentiated based on pairwise Mash or average amino-acid identity distance clustering, and we supply a script to facilitate application of the former approach by other researchers.

Metabolomics analysis reveals metabolite changes during freeze-drying and oven-drying of Angelica dahurica

Angelica dahurica (Angelica dahurica Fisch. ex Hoffm.) is widely used as a traditional Chinese medicine and the secondary metabolites have significant pharmacological activities. Drying has been shown to be a key factor affecting the coumarin content of Angelica dahurica. However, the underlying mechanism of metabolism is unclear. This study sought to determine the key differential metabolites and metabolic pathways related to this phenomenon. Liquid chromatography with tandem mass spectrometry (LC–MS/MS) based targeted metabolomics analysis was performed on Angelica dahurica that were freeze-drying (− 80 °C/9 h) and oven-drying (60 °C/10 h). Furthermore, the common metabolic pathways of paired comparison groups were performed based on KEEG enrichment analysis. The results showed that 193 metabolites were identified as key differential metabolites, most of which were upregulated under oven drying. It also displayed that many significant contents of PAL pathways were changed. This study revealed the large-scale recombination events of metabolites in Angelica dahurica. First, we identified additional active secondary metabolites apart from coumarins, and volatile oil were significantly accumulated in Angelica dahurica. We further explored the specific metabolite changes and mechanism of the phenomenon of coumarin upregulation caused by temperature rise. These results provide a theoretical reference for future research on the composition and processing method of Angelica dahurica.

Polluted wetlands contain multidrug-resistance plasmids encoding CTX-M-type extended-spectrum β-lactamases

While most detailed analyses of antibiotic resistance plasmids focus on those found in clinical isolates, less is known about the vast environmental reservoir of mobile genetic elements and the resistance and virulence factors they encode. We selectively isolated three strains of cefotaxime-resistant Escherichia coli from a wastewater-impacted coastal wetland. The cefotaxime-resistant phenotype was transmissible to a lab strain of E. coli after one hour, with frequencies as high as 10−3 transconjugants per recipient. Two of the plasmids also transferred cefotaxime resistance to Pseudomonas putida, but these were unable to back-transfer this resistance from P. putida to E. coli. In addition to the cephalosporins, E. coli transconjugants inherited resistance to at least seven distinct classes of antibiotics. Complete nucleotide sequences revealed large IncF-type plasmids with globally distributed replicon sequence types F31:A4:B1 and F18:B1:C4 carrying diverse antibiotic resistance and virulence genes. The plasmids encoded extended-spectrum β-lactamases blaCTX-M-15 or blaCTX-M-55, each associated with the insertion sequence ISEc9, although in different local arrangements. Despite similar resistance profiles, the plasmids shared only one resistance gene in common, the aminoglycoside acetyltransferase aac(3)-IIe. Plasmid accessory cargo also included virulence factors involved in iron acquisition and defense against host immunity. Despite their sequence similarities, several large-scale recombination events were detected, including rearrangements and inversions. In conclusion, selection with a single antibiotic, cefotaxime, yielded conjugative plasmids conferring multiple resistance and virulence factors. Clearly, efforts to limit the spread of antibiotic resistance and virulence among bacteria must include a greater understanding of mobile elements in the natural and human-impacted environments.

Isolation and genomic analysis of a type IV pili-independent Thermus thermophilus phage, φMN1 from a Japanese hot spring.

A Thermus thermophilus lytic phage was isolated from a Japanese hot spring using a type IV pili-deficient strain as an indicator host, and designated as φMN1. Electron microscopic (EM) examination revealed that φMN1 had an icosahedral head and a contractile tail, suggesting that φMN1 belonged to Myoviridae. An EM analysis focused on φMN1 adsorption to the Thermus host cell showed that the receptor molecules for the phage were uniformly distributed on the outer surface of the cells. The circular double-stranded DNA of φMN1 was 76,659 base pairs in length, and the guanine and cytosine content was 61.8%. It was predicted to contain 99 open reading frames, and its putative distal tail fiber protein, which is essential for non-piliated host cell surface receptor recognition, was dissimilar in terms of sequence and length with its counterpart in the type IV pili-dependent φYS40. A phage proteomic tree revealed that φMN1 and φYS40 are in the same cluster, but many genes had low sequence similarities and some seemed to be derived from both mesophilic and thermophilic organisms. The gene organization suggested that φMN1 evolved from a non-Thermus phage through large-scale recombination events of the genes determining the host specificity, followed by gradual evolution by recombination of both the thermophilic and mesophilic DNAs assimilated by the host Thermus cells. This newly isolated phage will provide evolutionary insights into thermophilic phages.

New Insights into the Evolutionary and Genomic Landscape of Molluscum Contagiosum Virus (MCV) based on Nine MCV1 and Six MCV2 Complete Genome Sequences.

Molluscum contagiosum virus (MCV) is the sole member of the Molluscipoxvirus genus and the causative agent of molluscum contagiosum (MC), a common skin disease. Although it is an important and frequent human pathogen, its genetic landscape and evolutionary history remain largely unknown. In this study, ten novel complete MCV genome sequences of the two most common MCV genotypes were determined (five MCV1 and five MCV2 sequences) and analyzed together with all MCV complete genomes previously deposited in freely accessible sequence repositories (four MCV1 and a single MCV2). In comparison to MCV1, a higher degree of nucleotide sequence conservation was observed among MCV2 genomes. Large-scale recombination events were identified in two newly assembled MCV1 genomes and one MCV2 genome. One recombination event was located in a newly identified recombinant region of the viral genome, and all previously described recombinant regions were re-identified in at least one novel MCV genome. MCV genes comprising the identified recombinant segments have been previously associated with viral interference with host T-cell and NK-cell immune responses. In conclusion, the two most common MCV genotypes emerged along divergent evolutionary pathways from a common ancestor, and the differences in the heterogeneity of MCV1 and MCV2 populations may be attributed to the strictness of the constraints imposed by the host immune response.

Evolution via recombination: Cell-to-cell contact facilitates larger recombination events in Streptococcus pneumoniae.

Homologous recombination in the genetic transformation model organism Streptococcus pneumoniae is thought to be important in the adaptation and evolution of this pathogen. While competent pneumococci are able to scavenge DNA added to laboratory cultures, large-scale transfers of multiple kb are rare under these conditions. We used whole genome sequencing (WGS) to map transfers in recombinants arising from contact of competent cells with non-competent ‘target’ cells, using strains with known genomes, distinguished by a total of ~16,000 SNPs. Experiments designed to explore the effect of environment on large scale recombination events used saturating purified donor DNA, short-term cell assemblages on Millipore filters, and mature biofilm mixed cultures. WGS of 22 recombinants for each environment mapped all SNPs that were identical between the recombinant and the donor but not the recipient. The mean recombination event size was found to be significantly larger in cell-to-cell contact cultures (4051 bp in filter assemblage and 3938 bp in biofilm co-culture versus 1815 bp with saturating DNA). Up to 5.8% of the genome was transferred, through 20 recombination events, to a single recipient, with the largest single event incorporating 29,971 bp. We also found that some recombination events are clustered, that these clusters are more likely to occur in cell-to-cell contact environments, and that they cause significantly increased linkage of genes as far apart as 60,000 bp. We conclude that pneumococcal evolution through homologous recombination is more likely to occur on a larger scale in environments that permit cell-to-cell contact.

Capsular Switching and Other Large-Scale Recombination Events in Invasive Sequence Type 1 Group B Streptococcus.

We report several cases of recombination events leading to capsular switching among sequence type (ST) 1 group B Streptococcus strains. These strains otherwise shared a common genome backbone with serotype V ST1 strains. However, the genomes of ST1 serotype V strains and those of serotypes VI, VII, and VIII strains differed substantially.

Recombination-mediated remodelling of host-pathogen interactions during Staphylococcus aureus niche adaptation.

Large-scale recombination events have led to the emergence of epidemic clones of several major bacterial pathogens. However, the functional impact of the recombination on clonal success is not understood. Here, we identified a novel widespread hybrid clone (ST71) of livestock-associated Staphylococcus aureus that evolved from an ancestor belonging to the major bovine lineage CC97, through multiple large-scale recombination events with other S. aureus lineages occupying the same ruminant niche. The recombination events, affecting a 329 kb region of the chromosome spanning the origin of replication, resulted in allele replacement and loss or gain of an array of genes influencing host–pathogen interactions. Of note, molecular functional analyses revealed that the ST71 hybrid clone has acquired multiple novel pathogenic traits associated with acquired and innate immune evasion and bovine extracellular matrix adherence. These findings provide a paradigm for the impact of large-scale recombination events on the rapid evolution of bacterial pathogens within defined ecological niches.

Extensive Capsule Locus Variation and Large-Scale Genomic Recombination within the Klebsiella pneumoniae Clonal Group 258.

Klebsiella pneumoniae clonal group (CG) 258, comprising sequence types (STs) 258, 11, and closely related variants, is associated with dissemination of the K. pneumoniae carbapenemase (KPC). Hospital outbreaks of KPC CG258 infections have been observed globally and are very difficult to treat. As a consequence, there is renewed interest in alternative infection control measures such as vaccines and phage or depolymerase treatments targeting the K. pneumoniae polysaccharide capsule. To date, 78 immunologically distinct capsule variants have been described in K. pneumoniae. Previous investigations of ST258 and a small number of closely related strains suggested that capsular variation was limited within this clone; only two distinct ST258 capsule polysaccharide synthesis (cps) loci have been identified, both acquired through large-scale recombination events (>50 kb). In contrast to previous studies, we report a comparative genomic analysis of the broader K. pneumoniae CG258 (n = 39). We identified 11 different cps loci within CG258, indicating that capsular switching is actually common within the complex. We observed several insertion sequences (IS) within the cps loci, and show further intraclone diversification of two cps loci through IS activity. Our data also indicate that several large-scale recombination events have shaped the genomes of CG258, and that definition of the complex should be broadened to include ST395 (also reported to harbor KPC). As only the second report of extensive intraclonal cps variation among Gram-negative bacterial species, our findings alter our understanding of the evolution of these organisms and have key implications for the design of control measures targeting K. pneumoniae capsules.

Patterns of Sequence Divergence in 5′ Intergenic Spacers and Linked Coding Regions in 10 Species of Pathogenic Bacteria Reveal Distinct Recombinational Histories

We compared the pattern of nucleotide difference in 8034 genes and in their 5' intergenic spacers between conspecific pairs of genomes from 10 species of pathogenic bacteria. Certain genes or spacers showed much greater sequence divergence between the genotypes compared to others; such divergent regions plausibly originated by recombinational events by which a gene and/or spacers was donated from a divergent genome. Different patterns of divergence in genes and spacers identified different recombinational patterns. For example, in Chlamydophila pneumoniae, there were examples of both unusually divergent spacers and unusually divergent genes, but there were no cases in which a gene and its spacer were both unusually divergent. This pattern suggests that, in C. pneumoniae, recombination events have broken up the linkage between genes and 5' spacers. By contrast, in Streptococcus agalactiae, there were a number of cases in which both spacer and gene were unusually divergent, indicating that a number of large-scale recombination events that included both genes and 5' spacers have occurred; there was evidence of at least two large-scale recombination events in the genomic region including the pur genes in S. agalactiae.

A virulence-associated gene microarray: a tool for investigation of the evolution and pathogenic potential of Staphylococcus aureus

An oligonucleotide probe microarray for investigation of the evolution of epidemic Staphylococcus aureus strains has been constructed. The array comprises 383 probes based on virulence-associated genes present in four key strains. Twelve strains including seven for which the complete chromosomal nucleotide sequence was available were tested on the array. Twenty-six per cent of the probes were able to differentiate between strains to give a minimum of two gene differences between pairs. A gene difference distance tree based on the array data had approximately the same topology as one prepared using concatenated MLST sequences. Differences in the topologies of these trees were found to indicate that large-scale recombination events had occurred during the evolution of the species. One such occurrence appears to have been a key event in the genesis of the EMRSA-15 clone (ST22) that currently represents the most prevalent methicillin-resistant S. aureus (MRSA) in the UK.

Endogenous retroviruses related to jaagsiekte sheep retrovirus.

Ovine betaretroviruses consist of exogenous viruses [jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus, (ENTV)] associated with neoplastic diseases of the respiratory tract and 15-20 endogenous viruses (enJSRV) stably integrated in the ovine and caprine genome. Phylogenetic analysis of this group of retroviruses suggests that the enJSRV can be considered as 'modern' endogenous retroviruses with active, exogenous counterparts. Sequence analysis of JSRV, ENTV and enJSRV suggests that enJSRV do not directly contribute to the pathogenesis of ovine pulmonary adenocarcinoma (OPA) or enzootic nasal tumor through large-scale recombination events, but small-scale recombination or complementation of gene function cannot be excluded; experiments involving enJSRV-free sheep, which have not been found, would be needed to investigate this possibility. Evidence of expression of enJSRV structural proteins in tissues of the reproductive tract and lung implies that they do not have a primary role in disease. However, experimental exploitation of exogenous/endogenous retrovirus sequence differences by producing chimeras has been useful in establishing the determinants of JSRV Env-induced transformation. Even if enJSRV do not have a direct role in OPA, their expression during ontogeny or in neonatal life may impact the likelihood of exogenous JSRV infection and disease outcome via the induction of immunological tolerance. Aside from any role in disease, enJSRV loci may serve as useful genetic markers in the sheep and their frequent expression in the reproductive tract of the ewe may portend an important physiologic role in sheep.

Contrasting roles of interallelic recombination at the HLA-A and HLA-B loci.

A statistical study of DNA sequences of alleles at the highly polymorphic class I MHC loci of humans, HLA-A and HLA-B, showed evidence of both large-scale recombination events (involving recombination of exons 1-2 of one allele with exons 3-8 of another) and small-scale recombination events (involving apparent exchange of short DNA segments). The latter events occurred disproportionately in the region of the gene encoding the antigen recognition site (ARS) of the class I molecule. Furthermore, they involved the ARS codons which are under the strongest selection favoring allelic diversity at the amino acid level. Thus, the frequency of recombinant alleles appears to have been increased by some form of balancing selection (such as overdominant selection) favoring heterozygosity in the ARS. These analyses also revealed a striking difference between the A and B loci. Recombination events appear to have occurred about twice as frequently at the B locus, and recombinants at the B locus were significantly more likely to affect polymorphic sites in the ARS. At the A locus, there are well-defined allelic lineages that have persisted since prior to the human-chimpanzee divergence; but at the B locus, there is no evidence for such long-lasting allelic lineages. Thus, relatively frequent interallelic recombination has apparently been a feature of the long-term evolution of the B locus but not of the A locus.