Diversity,taxonomy, and evolution of archaeal viruses of the class Caudoviricetes

The archaeal tailed viruses (arTV), evolutionarily related to tailed double-stranded DNA (dsDNA) bacteriophages of the class Caudoviricetes, represent the most common isolates infecting halophilic archaea. Only a handful of these viruses have been genomically characterized, limiting our appreciation of their ecological impacts and evolution. Here, we present 37 new genomes of haloarchaeal tailed virus isolates, more than doubling the current number of sequenced arTVs. Analysis of all 63 available complete genomes of arTVs, which we propose to classify into 14 new families and 3 orders, suggests ancient divergence of archaeal and bacterial tailed viruses and points to an extensive sharing of genes involved in DNA metabolism and counterdefense mechanisms, illuminating common strategies of virus–host interactions with tailed bacteriophages. Coupling of the comparative genomics with the host range analysis on a broad panel of haloarchaeal species uncovered 4 distinct groups of viral tail fiber adhesins controlling the host range expansion. The survey of metagenomes using viral hallmark genes suggests that the global architecture of the arTV community is shaped through recurrent transfers between different biomes, including hypersaline, marine, and anoxic environments.

Comparative genomics and host range analysis reveals the remarkable diversity and evolution of tailed archaeal viruses of the order Caudoviricetes, which together with their bacterial relatives arguably represent the most abundant and widespread virus group on our planet.     

Comparative Analysis of the Mosaic Genomes of Tailed Archaeal Viruses and Proviruses Suggests Common Themes for Virion Architecture and Assembly with Tailed Viruses of Bacteria

Tailed double-stranded DNA viruses (order Caudovirales) represent the dominant morphotype among viruses infecting bacteria. Analysis and comparison of complete genome sequences of tailed bacterial viruses provided insights into their origin and evolution. Structural and genomic studies have unexpectedly revealed that tailed bacterial viruses are evolutionarily related to eukaryotic herpesviruses. Organisms from the third domain of life, Archaea, are also infected by viruses that, in their overall morphology, resemble tailed viruses of bacteria. However, high-resolution structural information is currently unavailable for any of these viruses, and only a few complete genomes have been sequenced so far. Here we identified nine proviruses that are clearly related to tailed bacterial viruses and integrated into chromosomes of species belonging to four different taxonomic orders of the Archaea. This more than doubled the number of genome sequences available for comparative studies. Our analyses indicate that highly mosaic tailed archaeal virus genomes evolve by homologous and illegitimate recombination with genomes of other viruses, by diversification, and by acquisition of cellular genes. Comparative genomics of these viruses and related proviruses revealed a set of conserved genes encoding putative proteins similar to virion assembly and maturation, as well as genome packaging proteins of tailed bacterial viruses and herpesviruses. Furthermore, fold prediction and structural modeling experiments suggest that the major capsid proteins of tailed archaeal viruses adopt the same topology as the corresponding proteins of tailed bacterial viruses and eukaryotic herpesviruses. Data presented in this study strongly support the hypothesis that tailed viruses infecting archaea share a common ancestry with tailed bacterial viruses and herpesviruses.     

Recent Advances in the Identification of Replication Origins Based on the Z-curve Method

Precise DNA replication is critical for the maintenance of genetic integrity in all organisms. In all three domains of life, DNA replication starts at a specialized locus, termed as the replication origin, oriC or ORI, and its identification is vital to understanding the complex replication process. In bacteria and eukaryotes, replication initiates from single and multiple origins, respectively, while archaea can adopt either of the two modes. The Z-curve method has been successfully used to identify replication origins in genomes of various species, including multiple oriCs in some archaea. Based on the Z-curve method and comparative genomics analysis, we have developed a web-based system, Ori-Finder, for finding oriCs in bacterial genomes with high accuracy. Predicted oriC regions in bacterial genomes are organized into an online database, DoriC. Recently, archaeal oriC regions identified by both in vivo and in silico methods have also been included in the database. Here, we summarize the recent advances of in silico prediction of oriCs in bacterial and archaeal genomes using the Z-curve based method.     

Genomics of bacteria and archaea: the emerging dynamic view of the prokaryotic world

The first bacterial genome was sequenced in 1995, and the first archaeal genome in 1996. Soon after these breakthroughs, an exponential rate of genome sequencing was established, with a doubling time of approximately 20 months for bacteria and approximately 34 months for archaea. Comparative analysis of the hundreds of sequenced bacterial and dozens of archaeal genomes leads to several generalizations on the principles of genome organization and evolution. A crucial finding that enables functional characterization of the sequenced genomes and evolutionary reconstruction is that the majority of archaeal and bacterial genes have conserved orthologs in other, often, distant organisms. However, comparative genomics also shows that horizontal gene transfer (HGT) is a dominant force of prokaryotic evolution, along with the loss of genetic material resulting in genome contraction. A crucial component of the prokaryotic world is the mobilome, the enormous collection of viruses, plasmids and other selfish elements, which are in constant exchange with more stable chromosomes and serve as HGT vehicles. Thus, the prokaryotic genome space is a tightly connected, although compartmentalized, network, a novel notion that undermines the ‘Tree of Life’ model of evolution and requires a new conceptual framework and tools for the study of prokaryotic evolution.     

Gene decay in archaea

The gene-dense chromosomes of archaea and bacteria were long thought to be devoid of pseudogenes, but with the massive increase in available genome sequences, whole genome comparisons between closely related species have identified mutations that have rendered numerous genes inactive. Comparative analyses of sequenced archaeal genomes revealed numerous pseudogenes, which can constitute up to 8.6% of the annotated coding sequences in some genomes. The largest proportion of pseudogenes is created by gene truncations, followed by frameshift mutations. Within archaeal genomes, large numbers of pseudogenes contain more than one inactivating mutation, suggesting that pseudogenes are deleted from the genome more slowly in archaea than in bacteria. Although archaea seem to retain pseudogenes longer than do bacteria, most archaeal genomes have unique repertoires of pseudogenes.     

Class I defective herpes simplex virus DNA as a molecular cloning vehicle in eucaryotic cells

Defective herpes simplex virus type 1 genomes are composed of head-to-tail tandem repeats of small regions of the nondefective genome. Monomeric repeat units of class I defective herpes simplex virus genomes were cloned into bacterial plasmids. The repeat units functioned as replicons since both viral and convalently linked bacterial plasmid DNA replicated (with the help of DNA from nondefective virus) when transfected into rabbit skin cells. Recombinant plasmids were packaged into virions and were propagated from culture to culture by infection with progeny virus. Replication was evidently by a rolling circle mechanism since plasmid DNA was present in a high-molecular-weight form in transfected cells. Circular recombinant plasmid DNA replicated with a high degree of fidelity. In contrast, linear plasmid DNA underwent extensive deletions of both viral and bacterial sequences when transfected into rabbit skin cells. Derivative plasmids, a fraction of the size of the parental plasmid, were rescued by transforming Escherichia coli with DNA from the transfected rabbit skin cells. These plasmids functioned as shuttle vectors since they replicated faithfully in both eucaryotic and procaryotic cells.     

Molecular cloning of EcoRII endonuclease and methylase genes

Summary The genes for restriction-modification system EcoRII have been cloned from plasmid N3 DNA using RSF2124 as a vector plasmid. The hybrid plasmids designated pFK321 and pFK322 contained a 5.8 megadaltons EcoRI — fragment derived from N3 DNA including the genes for restriction-modification system EcoRII and a gene for resistance to sulfanilamide.     

The Z curve database: a graphic representation of genome sequences

MOTIVATION: Genome projects for many prokaryotic and eukaryotic species have been completed and more new genome projects are being underway currently. The availability of a large number of genomic sequences for researchers creates a need to find graphic tools to study genomes in a perceivable form. The Z curve is one of such tools available for visualizing genomes. The Z curve is a unique three-dimensional curve representation for a given DNA sequence in the sense that each can be uniquely reconstructed given the other. The Z curve database for more than 1000 genomes have been established here. RESULTS: The database contains the Z curves for archaea, bacteria, eukaryota, organelles, phages, plasmids, viroids and viruses, whose genomic sequences are currently available. All the 3-dimensional Z curves and their three component curves are stored in the database. The applications of the Z curve database on comparative genomics, gene prediction, computation of G+C content with a windowless technique, prediction of replication origins and terminations of bacterial and archaeal genomes and study of local deviations from the Chargaff Parity Rule 2 etc. are presented in detail. The Z curve database reported here is a treasure trove in which biologists could find useful biological knowledge.     

Similar Gene Estimates from Circular and Linear Standards in Quantitative PCR Analyses Using the Prokaryotic 16S rRNA Gene as a Model

Quantitative PCR (qPCR) is one of the most widely used tools for quantifying absolute numbers of microbial gene copies in test samples. A recent publication showed that circular plasmid DNA standards grossly overestimated numbers of a target gene by as much as 8-fold in a eukaryotic system using quantitative PCR (qPCR) analysis. Overestimation of microbial numbers is a serious concern in industrial settings where qPCR estimates form the basis for quality control or mitigation decisions. Unlike eukaryotes, bacteria and archaea most commonly have circular genomes and plasmids and therefore may not be subject to the same levels of overestimation. Therefore, the feasibility of using circular DNA plasmids as standards for 16S rRNA gene estimates was assayed using these two prokaryotic systems, with the practical advantage being rapid standard preparation for ongoing qPCR analyses. Full-length 16S rRNA gene sequences from Thermovirga lienii and Archaeoglobus fulgidus were cloned and used to generate standards for bacterial and archaeal qPCR reactions, respectively. Estimates of 16S rRNA gene copies were made based on circular and linearized DNA conformations using two genomes from each domain: Desulfovibrio vulgaris, Pseudomonas aeruginosa, Archaeoglobus fulgidus, and Methanocaldocococcus jannaschii. The ratio of estimated to predicted 16S rRNA gene copies ranged from 0.5 to 2.2-fold in bacterial systems and 0.5 to 1.0-fold in archaeal systems, demonstrating that circular plasmid standards did not lead to the gross over-estimates previously reported for eukaryotic systems.     

Thermal Adaptation of the Small Subunit Ribosomal RNA Gene: A Comparative Study

We carried out a comprehensive survey of small subunit ribosomal RNA sequences from archaeal, bacterial, and eukaryotic lineages in order to understand the general patterns of thermal adaptation in the rRNA genes. Within each lineage, we compared sequences from mesophilic, moderately thermophilic, and hyperthermophilic species. We carried out a more detailed study of the archaea, because of the wide range of growth temperatures within this group. Our results confirmed that there is a clear correlation between the GC content of the paired stem regions of the 16S rRNA genes and the optimal growth temperature, and we show that this correlation cannot be explained simply by phylogenetic relatedness among the thermophilic archaeal species. In addition, we found a significant, positive relationship between rRNA stem length and growth temperature. These correlations are found in both bacterial and archaeal rRNA genes. Finally, we compared rRNA sequences from warm-blooded and cold-blooded vertebrates. We found that, while rRNA sequences from the warm-blooded vertebrates have a higher overall GC content than those from the cold-blooded vertebrates, this difference is not concentrated in the paired regions of the molecule, suggesting that thermal adaptation is not the cause of the nucleotide differences between the vertebrate lineages. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Nicolas Galtier]     

Metagenomic analyses of novel viruses and plasmids from a cultured environmental sample of hyperthermophilic neutrophiles

Two novel viral genomes and four plasmids were assembled from an environmental sample collected from a hot spring at Yellowstone National Park, USA, and maintained anaerobically in a bioreactor at 85°C and pH 6. The double‐stranded DNA viral genomes are linear (22.7 kb) and circular (17.7 kb), and derive apparently from archaeal viruses HAV1 and HAV2. Genomic DNA was obtained from samples enriched in filamentous and tadpole‐shaped virus‐like particles respectively. They yielded few significant matches in public sequence databases reinforcing, further, the wide diversity of archaeal viruses. Several variants of HAV1 exhibit major genomic alterations, presumed to arise from viral adaptation to different hosts. They include insertions up to 350 bp, deletions up to 1.5 kb, and genes with extensively altered sequences. Some result from recombination events occurring at low complexity direct repeats distributed along the genome. In addition, a 33.8 kb archaeal plasmid pHA1 was characterized, encoding a possible conjugative apparatus, as well as three cryptic plasmids of thermophilic bacterial origin, pHB1 of 2.1 kb and two closely related variants pHB2a and pHB2b, of 5.2 and 4.8 kb respectively. Strategies are considered for assembling genomes of smaller genetic elements from complex environmental samples, and for establishing possible host identities on the basis of sequence similarity to host CRISPR immune systems.     

Engineering large viral DNA genomes using the CRISPR‐Cas9 system

Manipulation of viral genomes is essential for studying viral gene function and utilizing viruses for therapy. Several techniques for viral genome engineering have been developed. Homologous recombination in virus‐infected cells has traditionally been used to edit viral genomes; however, the frequency of the expected recombination is quite low. Alternatively, large viral genomes have been edited using a bacterial artificial chromosome (BAC) plasmid system. However, cloning of large viral genomes into BAC plasmids is both laborious and time‐consuming. In addition, because it is possible for insertion into the viral genome of drug selection markers or parts of BAC plasmids to affect viral function, artificial genes sometimes need to be removed from edited viruses. Herpes simplex virus (HSV), a common DNA virus with a genome length of 152 kbp, causes labialis, genital herpes and encephalitis. Mutant HSV is a candidate for oncotherapy, in which HSV is used to kill tumor cells. In this study, the clustered regularly interspaced short palindromic repeat‐Cas9 system was used to very efficiently engineer HSV without inserting artificial genes into viral genomes. Not only gene‐ablated HSV but also gene knock‐in HSV were generated using this method. Furthermore, selection with phenotypes of edited genes promotes the isolation efficiencies of expectedly mutated viral clones. Because our method can be applied to other DNA viruses such as Epstein–Barr virus, cytomegaloviruses, vaccinia virus and baculovirus, our system will be useful for studying various types of viruses, including clinical isolates.     

Prokaryotic genomes: the emerging paradigm of genome-based microbiology

Comparative analysis of the complete sequences of seven bacterial and three archaeal genomes leads to the first generalizations of emerging genome-based microbiology. Protein sequences are, generally, highly conserved, with ∼70% of the gene products in bacteria and archaea containing ancient conserved regions. In contrast, there is little conservation of genome organization, except for a few essential operons. The most striking conclusions derived by comparison of multiple genomes from phylogenetically distant species are that the number of universally conserved gene families is very small and that multiple events of horizontal gene transfer and genome fusion are major forces in evolution.     

Erythromycin resistance-conferring plasmid pRSB105, isolated from a sewage treatment plant, harbors a new macrolide resistance determinant, an integron-containing Tn402-like element, and a large region of unknown function

The erythromycin resistance plasmid pRSB105 was previously isolated from an activated sludge bacterial community of a municipal wastewater treatment plant. Compilation of the complete pRSB105 nucleotide sequence revealed that the plasmid is 57,137 bp in size and has a mean G+C content of 56.66 mol%. The pRSB105 backbone is composed of two different replication and/or partitioning modules and a functional mobilization region encoding the mobilization genes mobCDE and mobBA. The first replicon (Rep1) is nearly identical to the corresponding replication module of the multiresistance plasmid pRSB101 isolated from an unknown activated sludge bacterium. Accordingly, pRSB101 and pRSB105 are sister plasmids belonging to a new plasmid family. The second replicon (Rep2) of pRSB105 was classified as a member of the IncP-6 group. While Rep1 confers replication ability only in gamma-proteobacteria, Rep2 extents the host range of the plasmid since it is also functional in the beta-proteobacterium Ralstonia eutropha. Plasmid pRSB105 harbors the macrolide resistance genes mel and mph, encoding, respectively, a predicted ABC-type efflux permease and a macrolide-2'-phosphotransferase. Erythromycin resistance is mainly attributed to mel, whereas mph contributes to erythromycin resistance to a lesser extent. The second resistance region, represented by an integron-containing Tn402-like element, includes a beta-lactam (oxa10) and a trimethoprim (dfrB2) resistance gene cassette. In addition to antibiotic resistance modules, pRSB105 encodes a functional restriction/modification system and two nonresistance regions of unknown function. The presence of different mobile genetic elements that flank resistance and nonresistance modules on pRSB105 indicates that these elements were involved in acquisition of accessory plasmid modules. Comparative genomics of pRSB105 and related plasmids elucidated that pRSB105 evolved by integration of distinct modules from different plasmid sources, including Pseudomonas aeruginosa plasmids, and thus represents a mosaic plasmid.     

5’UTR中AUG的分布及其对翻译起始的影响

以细菌和古菌基因组5′ UTR序列作为研究对象,分析在5′ UTR 的3个不同阅读框架中三联体AUG的分布,发现无论是细菌还是古菌基因组都在阅读框1中有非常明显的AUG缺失(depletion)。AUG的缺失表明在起始密码子上游的AUG很可能会对基因的翻译起始产生影响。分析得知：绝大部分的AUG都是以uORF(upstream open reading frame)的形式出现的,uAUG(upstream AUG)的数量很少,特别是在阅读框1中,而且在细菌基因组的阅读框1中uAUG较多地出现在了含有SD序列的基因上游。比较发现,uAUG引导的序列在同义密码子使用上的偏好性较真正的编码序列差,这可能表明细菌和古菌在同义密码子使用上的偏好性也是决定基因准确地翻译起始的重要因素之一。     

Identification and distribution of sequences having similarity to mitochondrial plasmids in mitochondrial genomes of filamentous fungi

Mitochondrial plasmids are autonomously replicating genetic elements commonly associated with fungal and plant species. Analysis of several plant and fungal mitochondrial genomes has revealed regions that show significant homology to mitochondrial plasmids, suggesting that plasmids have had a long-term association with their mitochondrial hosts. To assess the degree to which plasmids have invaded fungal mitochondrial genomes, BLAST search parameters were modified to identify plasmid sequences within highly AT-rich mtDNAs, and output data were parsed by E value, score, and sequence complexity. High scoring hits were evaluated for the presence of shared repetitive elements and location within plasmids and mtDNAs. Our searches revealed multiple sites of sequence similarity to four distinct plasmids in the wild-type mtDNA of Neurospora crassa, which collectively comprise more than 2% of the mitochondrial genome. Regions of plasmid similarity were not restricted to plasmids known to be associated with senescence, indicating that all mt plasmids can potentially integrate into mitochondrial DNA. Unexpectedly, plasmid-related sequences were found to be clustered in regions that have disproportionately low numbers of PstI palindromic sequences, suggesting that these repetitive elements may play a role in eliminating foreign DNA. A separate class of GC-rich palindromes was identified that appear to be mobile, as indicated by their occurrence within regions of plasmid homology. Sites of sequence similarity to mitochondrial plasmids were also detected in other filamentous fungi, but to a lesser degree. The tools developed here will be useful in assessing the contribution plasmids have made to mitochondrial function and in understanding the co-evolution of mitochondrial plasmids and their hosts.Electronic Supplementary Material Supplementary material is available for this article at     

Presence of a family of plasmids (29 to 65 kilobases) with a 26-kilobase common region in different strains of the sulfur-oxidizing bacterium Acidithiobacillus caldus

Three large cryptic plasmids from different isolates of Acidithiobacillus caldus were rescued by using an in vitro transposition system that delivers a kanamycin-selectable marker and an Escherichia coli plasmid origin of replication. The largest of the plasmids, the 65-kb plasmid pTcM1, was isolated from a South African A. caldus strain, MNG. This plasmid was sequenced and compared to that of pTcF1 (39 kb, from strain "f," South Africa) and pC-SH12 (29 kb, from strain C-SH12, Australia). With the exception of a 2.7-kb insertion sequence, pC-SH12 appears to represent the DNA common to all three plasmids and includes a number of accessory genes plus the plasmid "backbone" containing the replication region. The two larger plasmids carry, in addition, a number of insertion sequences of the ISL3 family and a composite transposon related to the Tn21 subfamily containing a highly mosaic region within the borders of the inverted repeats. Genes coding for arsenic resistance, plasmid mobilization, plasmid stability, and a putative restriction-modification system occur within these mosaic regions.     

极端嗜盐古菌中CRISPR结构的生物信息学分析

摘要：【目的】利用生物信息学方法了解目前拥有全基因组序列的极端嗜盐古菌中CRISPR结构的特征。【方法】通过比对,保守性分析,GC含量分析,RNA结构预测等方法对已有全基因组序列的嗜盐古菌基因组进行研究。【结果】在5株嗜盐古菌基因组中发现CRISPR结构,在leader序列内得到具有回文性质的保守motif。发现在大CRISPR结构内repeat序列具有很强的保守性。同时根据第四位碱基的不同,repeat序列可形成两类不同的RNA二级结构。【结论】leader序列中回文结构的发现对其可能为蛋白结合位点的假     

Bioinformatic and partial functional analysis of pEspA and pEspB, two plasmids from Exiguobacterium arabatum sp. nov. RFL1109

The complete nucleotide sequences of two plasmids from Exiguobacterium arabatum sp. nov. RFL1109, pEspA (4563bp) and pEspB (38,945bp), have been determined. Five ORFs were identified in the pEspA plasmid, and putative functions were assigned to two of them. Using deletion mapping approach, the Rep-independent replication region of pEspA, which functions in Bacillus subtilis, was localized within a 0.6kb DNA region. Analysis of the pEspB sequence revealed 42 ORFs. From these, function of two genes encoding enzymes of the Lsp1109I restriction-modification system was confirmed experimentally, while putative functions of another 18 ORFs were suggested based on comparative analysis. Three functional regions have been proposed for the pEspB plasmid: the putative conjugative transfer region, the region involved in plasmid replication and maintenance, and the region responsible for transposition of the IS21 family-like transposable elements.     

Microbial diversity with dominance of 16S rRNA gene sequences with high GC contents at 74 and 98 °C subsurface crude oil deposits in Japan

We prepared DNA from the production waters of oil deposits and wellheads of the high- and hypertemperature Japanese oil wells #AR39 (depth, 1230 m; temperature, 74 °C; pressure, 2.92 MPa) and #SR123 (depth, 1687 m; temperature, 98 °C; pressure, 11.3 MPa) to detect indigenous bacterial and archaeal microorganisms. We used PCR to amplify the 16S rRNA genes of microbial communities and characterized them based on their sequences. A few species of microorganisms with high GC contents were detected in samples from oil deposits, whereas the microbial constituents and their GC contents were diverse in wellhead samples. A comparison of the composition of the microbial communities found that the predominant indigenous populations in the #SR123 oil deposit were Thermotoga hypogea-, Thermotoga petrophila- and Thermodesulfobacterium commune-like bacteria with a 61-63% GC content in their 16S rRNA gene sequences, and Archaeoglobus fulgidus-like archaea with a 65% GC content, whereas the major population in #AR39 comprised Thermacetogenium phaeum- and Fervidobacterium pennavorans-like bacteria and Methanothermobacter thermautotrophicus-like archaea with a 60%, 60% and 61% GC content, respectively.