Sequence-variable mosaics: composites of recurrent transposition characterizing the genomes of phylogenetically diverse phytoplasmas

Phytoplasmas are cell wall-less prokaryotes characterized by small, AT-rich genomes that encode capabilities for obligate, transkingdom parasitism and pathogenicity in plants and insect vectors. Inability to isolate and characterize phytoplasmas in pure culture has led to adoption of the 'Candidatus species' convention to refer to distinct phytoplasma lineages. In this study, we provide evidence that multiple, sequence-variable mosaics (SVMs) of clustered genes and repetitive extragenic palindromes are characteristic features of phytoplasma genome architecture in phylogenetically diverse species. The findings suggest that the origin of SVMs was an ancient event in evolution of the phytoplasma clade, while current forms of SVMs are results of dramatic and more recent events. Sequence diversity of hypervariable regions indicated rapid evolution possibly involving capture of mobile elements recurrently targeted to SVMs. Multiple events of targeted mobile element attack, recombination, and rearrangement conceivably account for the composite structure of SVMs. Proteins encoded by the highly variable region included a lysophospholipase and other putatively secreted and/or transmembrane, cell surface-interacting proteins potentially significant in phytoplasma-host interactions.     

Folate biosynthesis pseudogenes, PsifolP and PsifolK, and an O-sialoglycoprotein endopeptidase gene homolog in the phytoplasma genome

Phytoplasmas are wall-less phytopathogenic prokaryotes of small genome sizes that are obligate parasites of insect vectors and plant hosts. We have cloned a clover phyllody (CPh) phytoplasma DNA locus containing five potential coding sequences. Two were identified as pseudogenes (PsifolP and PsifolK) homologous to folP and folK genes, which encode dihydropteroate synthase (DHPS) and 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), respectively, in other bacteria. Evolution of the phytoplasma presumably involved loss of functions through the formation of these and other pseudogenes during adaptation to obligate parasitism. The findings suggest that the phytoplasma lacks capacity for de novo folate biosynthesis and possesses a transport system for absorption of preformed folate from host cells. The PsifolP-PsifolK region was flanked by three open reading frames (ORFs) encoding a DegV family protein, a hypothetical protein with a P60-like lipoprotein domain homologous with the P60-like Mycoplasma hominis protein, and a glycoprotease (Gcp) protein that possibly functions as a host adaptation or virulence factor.     

Living with genome instability: the adaptation of phytoplasmas to diverse environments of their insect and plant hosts

Phytoplasmas ("Candidatus Phytoplasma," class Mollicutes) cause disease in hundreds of economically important plants and are obligately transmitted by sap-feeding insects of the order Hemiptera, mainly leafhoppers and psyllids. The 706,569-bp chromosome and four plasmids of aster yellows phytoplasma strain witches' broom (AY-WB) were sequenced and compared to the onion yellows phytoplasma strain M (OY-M) genome. The phytoplasmas have small repeat-rich genomes. This comparative analysis revealed that the repeated DNAs are organized into large clusters of potential mobile units (PMUs), which contain tra5 insertion sequences (ISs) and genes for specialized sigma factors and membrane proteins. So far, these PMUs appear to be unique to phytoplasmas. Compared to mycoplasmas, phytoplasmas lack several recombination and DNA modification functions, and therefore, phytoplasmas may use different mechanisms of recombination, likely involving PMUs, for the creation of variability, allowing phytoplasmas to adjust to the diverse environments of plants and insects. The irregular GC skews and the presence of ISs and large repeated sequences in the AY-WB and OY-M genomes are indicative of high genomic plasticity. Nevertheless, segments of approximately 250 kb located between the lplA and glnQ genes are syntenic between the two phytoplasmas and contain the majority of the metabolic genes and no ISs. AY-WB appears to be further along in the reductive evolution process than OY-M. The AY-WB genome is approximately 154 kb smaller than the OY-M genome, primarily as a result of fewer multicopy sequences, including PMUs. Furthermore, AY-WB lacks genes that are truncated and are part of incomplete pathways in OY-M.     

Homologous recombination mediated by two palindromic repeated sequences in the mitochondrial genome of Oryza

Palindromic repeated sequences (PRSs) are distributed in at least ten regions of the mitochondrial (mt) genome of rice and are, apparently, mobile. In the present study, we examined the possibility of homologous recombination via some PRSs during the course of evolution of Oryza. We first performed Southern hybridization of the DNA from 11 species (18 strains) of Oryza in order to identify the distribution of PRSs in the mitochondrial genome of Oryza. The hybridization patterns revealed genome type-specific and/or species-specific variations. We speculated that homologous recombination via some PRSs might have made a contribution to such variations. After subsequent polymerase chain reaction, Southern hybridization and sequencing, we concluded that homologous recombination mediated by two PRSs occurred in the mtDNA of Oryza after divergence of the BB genome type and the other genome types of Oryza. Evidence was obtained that some PRSs were involved in both insertion and recombination events during the evolution of Oryza. Our results indicate, therefore, that PRSs have contributed considerably to the polymorphism of Oryza mtDNAs.     

The genome of ‘Candidatus Phytoplasma solani’ strain SA-1 is highly dynamic and prone to adopting foreign sequences

Bacteria of the genus ‘Candidatus Phytoplasma’ are uncultivated intracellular plant pathogens transmitted by phloem-feeding insects. They have small genomes lacking genes for essential metabolites, which they acquire from either plant or insect hosts. Nonetheless, some phytoplasmas, such as ‘Ca. P. solani’, have broad plant host range and are transmitted by several polyphagous insect species. To understand better how these obligate symbionts can colonize such a wide range of hosts, the genome of ‘Ca. P. solani’ strain SA-1 was sequenced from infected periwinkle via a metagenomics approach. The de novo assembly generated a draft genome with 19 contigs totalling 821,322 bp, which corresponded to more than 80% of the estimated genome size. Further completion of the genome was challenging due to the high occurrence of repetitive sequences. The majority of repeats consisted of gene arrangements characteristic of phytoplasma potential mobile units (PMUs). These regions showed variation in gene orders intermixed with genes of unknown functions and lack of similarity to other phytoplasma genes, suggesting that they were prone to rearrangements and acquisition of new sequences via recombination. The availability of this high-quality draft genome also provided a foundation for genome-scale genotypic analysis (e.g., average nucleotide identity and average amino acid identity) and molecular phylogenetic analysis. Phylogenetic analyses provided evidence of horizontal transfer for PMU-like elements from various phytoplasmas, including distantly related ones. The ‘Ca. P. solani’ SA-1 genome also contained putative secreted protein/effector genes, including a homologue of SAP11, found in many other phytoplasma species.     

The repertoire of minimal mobile elements in the Neisseria species and evidence that these are involved in horizontal gene transfer in other bacteria

In the Neisseria spp., natural competence for transformation and homologous recombination generate antigenic variants through creation of mosaic genes (such as opas) and through recombination with silent cassettes (such as pilE/pilS) and gene-complement diversity through the horizontal exchange of whole genes or groups of genes, in minimal mobile elements (MMEs). An MME is a region encompassing 2 conserved genes between which different whole-gene cassettes are found in different strains, which are chromosomally incorporated solely through the action of homologous recombination. Comparative analyses of the neisserial genome sequences identified 39 potential MME sites, the contents of which were investigated in 11 neisserial strains. One hundred and eight different MME regions were identified, 20 of which contain novel sequences and these contain 12 newly identified neisserial coding sequences. Neisserial uptake signal sequences are associated with 38 of the 40 MMEs studied. In some sites, divergent dinucleotide signatures of the sequences between the flanking genes suggest relatively recent horizontal acquisition of some cassettes. The neisserial MMEs were used to interrogate all of the other available bacterial genome sequences, revealing frequent conservation of the flanking genes combined with the presence of different gene cassettes between them. In some cases, these sites can definitively be classified as MMEs in these other genera. These findings provide additional evidence for the MME model, indicate that MME-directed investigations are a good basis for the identification of novel strain-specific genes and differences within bacterial populations and demonstrate that these elements are probably ubiquitously involved in genetic exchange, particularly in naturally competent bacteria.     

Efficient repair of genomic double-strand breaks by homologous recombination between directly repeated sequences in the plant genome

Previous studies demonstrated that in somatic plant cells, homologous recombination (HR) is several orders of magnitude less efficient than nonhomologous end joining and that HR is little used for genomic double-strand break (DSB) repair. Here, we provide evidence that if genomic DSBs are induced in close proximity to homologous repeats, they can be repaired in up to one-third of cases by HR in transgenic tobacco. Our findings are relevant for the evolution of plant genomes because they indicate that sequences containing direct repeats such as retroelements might be less stable in plants that harbor active mobile elements than anticipated previously. Furthermore, our experimental setup enabled us to demonstrate that transgenic sequences flanked by sites of a rare cutting restriction enzyme can be excised efficiently from the genome of a higher eukaryote by HR as well as by nonhomologous end joining. This makes DSB-induced recombination an attractive alternative to the currently applied sequence-specific recombination systems used for genome manipulations, such as marker gene excision.     

Pangenome Analysis of Burkholderia pseudomallei: Genome Evolution Preserves Gene Order despite High Recombination Rates

The pangenomic diversity in Burkholderia pseudomallei is high, with approximately 5.8% of the genome consisting of genomic islands. Genomic islands are known hotspots for recombination driven primarily by site-specific recombination associated with tRNAs. However, recombination rates in other portions of the genome are also high, a feature we expected to disrupt gene order. We analyzed the pangenome of 37 isolates of B. pseudomallei and demonstrate that the pangenome is ‘open’, with approximately 136 new genes identified with each new genome sequenced, and that the global core genome consists of 4568±16 homologs. Genes associated with metabolism were statistically overrepresented in the core genome, and genes associated with mobile elements, disease, and motility were primarily associated with accessory portions of the pangenome. The frequency distribution of genes present in between 1 and 37 of the genomes analyzed matches well with a model of genome evolution in which 96% of the genome has very low recombination rates but 4% of the genome recombines readily. Using homologous genes among pairs of genomes, we found that gene order was highly conserved among strains, despite the high recombination rates previously observed. High rates of gene transfer and recombination are incompatible with retaining gene order unless these processes are either highly localized to specific sites within the genome, or are characterized by symmetrical gene gain and loss. Our results demonstrate that both processes occur: localized recombination introduces many new genes at relatively few sites, and recombination throughout the genome generates the novel multi-locus sequence types previously observed while preserving gene order.     

Comparative Genome Analysis of Wheat Blue Dwarf Phytoplasma,an Obligate Pathogen That Causes Wheat Blue Dwarf Disease in China

Wheat blue dwarf (WBD) disease is an important disease that has caused heavy losses in wheat production in northwestern China. This disease is caused by WBD phytoplasma, which is transmitted by Psammotettix striatus. Until now, no genome information about WBD phytoplasma has been published, seriously restricting research on this obligate pathogen. In this paper, we report a new sequencing and assembling strategy for phytoplasma genome projects. This strategy involves differential centrifugation, pulsed-field gel electrophoresis, whole genome amplification, shotgun sequencing, de novo assembly, screening of contigs from phytoplasma and the connection of phytoplasma contigs. Using this scheme, the WBD phytoplasma draft genome was obtained. It was comprised of six contigs with a total size of 611,462 bp, covering ∼94% of the chromosome. Five-hundred-twenty-five protein-coding genes, two operons for rRNA genes and 32 tRNA genes were identified. Comparative genome analyses between WBD phytoplasma and other phytoplasmas were subsequently carried out. The results showed that extensive arrangements and inversions existed among the WBD, OY-M and AY-WB phytoplasma genomes. Most protein-coding genes in WBD phytoplasma were found to be homologous to genes from other phytoplasmas; only 22 WBD-specific genes were identified. KEGG pathway analysis indicated that WBD phytoplasma had strongly reduced metabolic capabilities. However, 46 transporters were identified, which were involved with dipeptides/oligopeptides, spermidine/putrescine, cobalt and Mn/Zn transport, and so on. A total of 37 secreted proteins were encoded in the WBD phytoplasma chromosome and plasmids. Of these, three secreted proteins were similar to the reported phytoplasma virulence factors TENGU, SAP11 and SAP54. In addition, WBD phytoplasma possessed several proteins that were predicted to play a role in its adaptation to diverse environments. These results will provide clues for research on the pathogenic mechanisms of WBD phytoplasma and will also provide a perspective about the genome sequencing of other phytoplasmas and obligate organisms.     

基因组编辑技术中供体DNA类型及选择

基因组编辑技术能够实现基因组的精确修饰和改造,是后基因组时代研究基因功能和遗传信息的主要手段。传统的基因打靶技术通过低效率的细胞自发同源重组实现目的基因的定点修饰。真核细胞中DNA双链断裂介导的同源重组效率远高于自发同源重组,利用人工核酸内切酶特异性地在基因组靶序列处引入双链断裂,通过提供适当形式的、含有一定长度同源臂的供体DNA,能够实现相对高效的基因组靶向编辑。本文系统总结了环状质粒、线性化质粒、聚合酶链式反应产物及单链寡聚脱氧核苷酸4种类型的供体DNA在基因组精确编辑研究中的应用及候选原则,以期为以后相关研究中供体DNA的选择、设计提供参考和借鉴。     

Regulation of DNA strand exchange in homologous recombination

Homologous recombination, the exchange of DNA strands between homologous DNA molecules, is involved in repair of many structural diverse DNA lesions. This versatility stems from multiple ways in which homologous DNA strands can be rearranged. At the core of homologous recombination are recombinase proteins such as RecA and RAD51 that mediate homology recognition and DNA strand exchange through formation of a dynamic nucleoprotein filament. Four stages in the life cycle of nucleoprotein filaments are filament nucleation, filament growth, homologous DNA pairing and strand exchange, and filament dissociation. Progression through this cycle requires a sequence of recombinase-DNA and recombinase protein-protein interactions coupled to ATP binding and hydrolysis. The function of recombinases is controlled by accessory proteins that allow coordination of strand exchange with other steps of homologous recombination and that tailor to the needs of specific aberrant DNA structures undergoing recombination. Accessory proteins are also able to reverse filament formation thereby guarding against inappropriate DNA rearrangements. The dynamic instability of the recombinase-DNA interactions allows both positive and negative action of accessory proteins thereby ensuring that genome maintenance by homologous recombination is not only flexible and versatile, but also accurate.     

Analysis of homologous gene clusters in Caenorhabditis elegans reveals striking regional cluster domains

An algorithm for detecting local clusters of homologous genes was applied to the genome of Caenorhabditis elegans. Clusters of two or more homologous genes are abundant, totaling 1391 clusters containing 4607 genes, over one-fifth of all genes in C. elegans. Cluster genes are distributed unevenly in the genome, with the large majority located on autosomal chromosome arms, regions characterized by higher genetic recombination and more repeat sequences than autosomal centers and the X chromosome. Cluster genes are transcribed at much lower levels than average and very few have gross phenotypes as assayed by RNAi-mediated reduction of function. The molecular identity of cluster genes is unusual, with a preponderance of nematode-specific gene families that encode putative secreted and transmembrane proteins, and enrichment for genes implicated in xenobiotic detoxification and innate immunity. Gene clustering in Drosophila melanogaster is also substantial and the molecular identity of clustered genes follows a similar pattern. I hypothesize that autosomal chromosome arms in C. elegans undergo frequent local gene duplication and that these duplications support gene diversification and rapid evolution in response to environmental challenges. Although specific gene clusters have been documented in C. elegans, their abundance, genomic distribution, and unusual molecular identities were previously unrecognized.     

A genome-wide study of recombination rate variation in Bartonella henselae

ABSTRACT: BACKGROUND: Rates of recombination vary by three orders of magnitude in bacteria but the reasons for this variation is unclear. We performed a genome-wide study of recombination rate variation among genes in the intracellular bacterium Bartonella henselae, which has among the lowest estimated ratio of recombination relative to mutation in prokaryotes. RESULTS: The 1.9 Mb genomes of B. henselae strains IC11, UGA10 and Houston-1 genomes showed only minor gene content variation. Nucleotide sequence divergence levels were less than 1% and the relative rate of recombination to mutation was estimated to 1.1 for the genome overall. Four to eight segments per genome presented significantly enhanced divergences, the most pronounced of which were the virB and trw gene clusters for type IV secretion systems that play essential roles in the infection process. Consistently, multiple recombination events were identified inside these gene clusters. High recombination frequencies were also observed for a gene putatively involved in iron metabolism. A phylogenetic study of this gene in 80 strains of Bartonella quintana, B. henselae and B. grahamii indicated different population structures for each species and revealed horizontal gene transfers across Bartonella species with different host preferences. CONCLUSIONS: Our analysis has shown little novel gene acquisition in B. henselae, indicative of a closed pan-genome, but higher recombination frequencies within the population than previously estimated. We propose that the dramatically increased fixation rate for recombination events at gene clusters for type IV secretion systems is driven by selection for sequence variability.     

Cosmid cloning and sample sequencing of the genome of the uncultivable mollicute,Western X-disease phytoplasma,using DNA purified by pulsed-field gel electrophoresis

Western X-disease (WX) phytoplasma is an uncultivable, intracellular pathogen of plants and insects with an AT-rich genome of 670 kb. As part of the genome sequencing project of WX phytoplasma, we have cloned approximately 50% of its genome into the pcosRW2 cosmid vector using DNA purified from pulsed-field gels. One of the cosmid clones with an insert of 24.6 kb was sequenced, which along with the cosmid end sequences, represents 60 kb of unique sequence from the WX phytoplasma genome. The putative genes identified in this sequence represent a wide variety of functions and many had not been previously identified in a phytoplasma.     

The structure-specific endonuclease Ercc1-Xpf is required for targeted gene replacement in embryonic stem cells.

The Ercc1-Xpf heterodimer, a highly conserved structure-specific endonuclease, functions in multiple DNA repair pathways that are pivotal for maintaining genome stability, including nucleotide excision repair, interstrand crosslink repair and homologous recombination. Ercc1-Xpf incises double-stranded DNA at double-strand/single-strand junctions, making it an ideal enzyme for processing DNA structures that contain partially unwound strands. Here we demonstrate that although Ercc1 is dispensable for recombination between sister chromatids, it is essential for targeted gene replacement in mouse embryonic stem cells. Surprisingly, the role of Ercc1-Xpf in gene targeting is distinct from its previously identified role in removing nonhomologous termini from recombination intermediates because it was required irrespective of whether the ends of the DNA targeting constructs were heterologous or homologous to the genomic locus. Our observations have implications for the mechanism of gene targeting in mammalian cells and define a new role for Ercc1-Xpf in mammalian homologous recombination. We propose a model for the mechanism of targeted gene replacement that invokes a role for Ercc1-Xpf in making the recipient genomic locus receptive for gene replacement.     

Unveiling novel RecO distant orthologues involved in homologous recombination

The generation of a RecA filament on single-stranded DNA is a critical step in homologous recombination. Two main pathways leading to the formation of the nucleofilament have been identified in bacteria, based on the protein complexes mediating RecA loading: RecBCD (AddAB) and RecFOR. Many bacterial species seem to lack some of the components involved in these complexes. The current annotation of the Helicobacter pylori genome suggests that this highly diverse bacterial pathogen has a reduced set of recombination mediator proteins. While it is now clear that homologous recombination plays a critical role in generating H. pylori diversity by allowing genomic DNA rearrangements and integration through transformation of exogenous DNA into the chromosome, no complete mediator complex is deduced from the sequence of its genome. Here we show by bioinformatics analysis the presence of a RecO remote orthologue that allowed the identification of a new set of RecO proteins present in all bacterial species where a RecR but not RecO was previously identified. HpRecO shares less than 15% identity with previously characterized homologues. Genetic dissection of recombination pathways shows that this novel RecO and the remote RecB homologue present in H. pylori are functional in repair and in RecA-dependent intrachromosomal recombination, defining two initiation pathways with little overlap. We found, however, that neither RecOR nor RecB contributes to transformation, suggesting the presence of a third, specialized, RecA-dependent pathway responsible for the integration of transforming DNA into the chromosome of this naturally competent bacteria. These results provide insight into the mechanisms that this successful pathogen uses to generate genetic diversity and adapt to changing environments and new hosts.     

基因敲除技术在微生物中的应用

随着分子生物学技术的发展,基因敲除技术越来越广泛地应用于动植物、微生物领域,成为研究生物基因功能最有力的工具之一。基因敲除技术在改造动植物、微生物基因组、研究发育生物学、鉴定新基因新功能、育种以及医疗领域都有应用价值。针对微生物方面,对实现基因敲除的各种原理方法,RecA系统同源重组法, Red系统同源重组法,基于自杀载体的同源重组法,基于温敏型质粒的同源重组法, CRISPR/Cas系统介导的基因敲除方法进行了总结,比较各自的优缺点,并提供一些成功案例以及各种方法相关的发明专利,以期对了解基因敲除技术的方法与发展提供参考。     

Alteration of location of dimer linkage sequence in retroviral RNA: little effect on replication or homologous recombination.

Retrovirus particles contain a dimer of retroviral genomic RNA. A defined region of the retrovirus genome has previously been shown to be important for both dimerization and encapsidation. To study the importance of the position of this encapsidation and dimerization signal for retroviral replication and homologous recombination, we used a previously described spleen necrosis virus-based helper cell system. This system allows retroviral vectors with multiple genetic markers to be studied after a single cycle of retroviral replication. The sequence responsible for dimerization, the encapsidation/dimer linkage sequence (E/DLS), was moved from its normal location near the 5' end of the retroviral genome to a location near the 3' end of the genome. We characterized four pairs of retroviral vectors: (i) with both E/DLSs at the 5' ends of the genomes, (ii) with both E/DLSs at the 3' ends of the genomes, and (iii) two with one E/DLS at the 5' end of the genome and one at the 3' end of the genome. We found that moving the E/DLS to the 3' end of the genome resulted in at most an approximately factor of 5 reduction in virus titer in a single cycle of retroviral replication. Furthermore, we found no changes that were attributable to the alteration of the position of the E/DLS in the minus-strand DNA primer transfers or the plus-strand DNA primer transfers, the rate of homologous recombination, or the number of internal template switches in recombinant proviruses. These results indicate that any alignment or conformation necessary for retroviral replication or recombination is not the result of the position of the E/DLS.