Pacbio sequencing of copper-tolerant Xanthomonas citri reveals presence of a chimeric plasmid structure and provides insights into reassortment and shuffling of transcription activator-like effectors among X. citri strains.
ABSTRACT: Xanthomonas citri, a causal agent of citrus canker, has been a well-studied model system due to recent availability of whole genome sequences of multiple strains from different geographical regions. Major limitations in our understanding of the evolution of pathogenicity factors in X. citri strains sequenced by short-read sequencing methods have been tracking plasmid reshuffling among strains due to inability to accurately assign reads to plasmids, and analyzing repeat regions among strains. X. citri harbors major pathogenicity determinants, including variable DNA-binding repeat region containing Transcription Activator-like Effectors (TALEs) on plasmids. The long-read sequencing method, PacBio, has allowed the ability to obtain complete and accurate sequences of TALEs in xanthomonads. We recently sequenced Xanthomonas citri str. Xc-03-1638-1-1, a copper tolerant A group strain isolated from grapefruit in 2003 from Argentina using PacBio RS II chemistry. We analyzed plasmid profiles, copy number and location of TALEs in complete genome sequences of X. citri strains.We utilized the power of long reads obtained by PacBio sequencing to enable assembly of a complete genome sequence of strain Xc-03-1638-1-1, including sequences of two plasmids, 249 kb (plasmid harboring copper resistance genes) and 99 kb (pathogenicity plasmid containing TALEs). The pathogenicity plasmid in this strain is a hybrid plasmid containing four TALEs. Due to the intriguing nature of this pathogenicity plasmid with Tn3-like transposon association, repetitive elements and multiple putative sites for origins of replication, we might expect alternative structures of this plasmid in nature, illustrating the strong adaptive potential of X. citri strains. Analysis of the pathogenicity plasmid among completely sequenced X. citri strains, coupled with Southern hybridization of the pathogenicity plasmids, revealed clues to rearrangements of plasmids and resulting reshuffling of TALEs among strains.We demonstrate in this study the importance of long-read sequencing for obtaining intact sequences of TALEs and plasmids, as well as for identifying rearrangement events including plasmid reshuffling. Rearrangement events, such as the hybrid plasmid in this case, could be a frequent phenomenon in the evolution of X. citri strains, although so far it is undetected due to the inability to obtain complete plasmid sequences with short-read sequencing methods.
Project description:Xanthomonas citri pv. vignicola strains cause bacterial blight of the legume crop cowpea. We report whole-genome sequences of three X. citri pv. vignicola strains obtained using PacBio single-molecule real-time sequencing. Such genomic data provide new information on pathogenicity factors, such as transcription activator-like effectors.
Project description:Xanthomonas citri ssp. citri (X. citri), causal agent of citrus canker, uses transcription activator-like effectors (TALEs) as major pathogenicity factors. TALEs, which are delivered into plant cells through the type III secretion system (T3SS), interact with effector binding elements (EBEs) in host genomes to activate the expression of downstream susceptibility genes to promote disease. Predictably, TALEs bind EBEs in host promoters via known combinations of TALE amino acids to DNA bases, known as the TALE code. We introduced 14 EBEs, matching distinct X. citri TALEs, into the promoter of the pepper Bs3 gene (ProBs31EBE ), and fused this engineered promoter with multiple EBEs (ProBs314EBE ) to either the ?-glucuronidase (GUS) reporter gene or the coding sequence (cds) of the pepper gene, Bs3. TALE-induced expression of the Bs3 cds in citrus leaves resulted in no visible hypersensitive response (HR). Therefore, we utilized a different approach in which ProBs31EBE and ProBs314EBE were fused to the Xanthomonas gene, avrGf1, which encodes a bacterial effector that elicits an HR in grapefruit and sweet orange. We demonstrated, in transient assays, that activation of ProBs314EBE by X. citri TALEs is T3SS dependent, and that the expression of AvrGf1 triggers HR and correlates with reduced bacterial growth. We further demonstrated that all tested virulent X. citri strains from diverse geographical locations activate ProBs314EBE . TALEs are essential for the virulence of X. citri strains and, because the engineered promoter traps are activated by multiple TALEs, this concept has the potential to confer broad-spectrum, durable resistance to citrus canker in stably transformed plants.
Project description:This study aimed to assess the feasibility of using the Oxford Nanopore Technologies (ONT) MinION long-read sequencer in reconstructing fully closed plasmid sequences from eight Enterobacteriaceae isolates of six different species with plasmid populations of varying complexity. Species represented were Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Enterobacter cloacae, Serratia marcescens and Klebsiella oxytoca, with plasmid populations ranging from 1-11 plasmids with sizes of 2-330?kb. Isolates were sequenced using Illumina (short-read) and ONT's MinION (long-read) platforms, and compared with fully resolved PacBio (long-read) sequence assemblies for the same isolates. We compared the performance of different assembly approaches including SPAdes, plasmidSPAdes, hybridSPAdes, Canu, Canu+Pilon (canuPilon) and npScarf in recovering the plasmid structures of these isolates by comparing with the gold-standard PacBio reference sequences. Overall, canuPilon provided consistently good quality assemblies both in terms of assembly statistics (N50, number of contigs) and assembly accuracy [presence of single nucleotide polymorphisms (SNPs)/indels with respect to the reference sequence]. For plasmid reconstruction, Canu recovered 70?% of the plasmids in complete contigs, and combining three assembly approaches (Canu or canuPilon, hybridSPAdes and plasmidSPAdes) resulted in a total 78?% recovery rate for all the plasmids. The analysis demonstrated the potential of using MinION sequencing technology to resolve important plasmid structures in Enterobacteriaceae species independent of and in conjunction with Illumina sequencing data. A consensus assembly derived from several assembly approaches could present significant benefit in accurately resolving the greatest number of plasmid structures.
Project description:BACKGROUND: Spiroplasma citri BR3-3X and S. kunkelii CR2-3X cause serious diseases worldwide on citrus and maize species, respectively. S. citri BR3-3X harbors a plasmid, pBJS-Original (pBJS-O), that encodes the spiroplasma adhesion related protein 1 (SARP1), a protein implicated in binding of the pathogen to cells of its leafhopper vector, Circulifer tenellus. The S. kunkelii CR2-3X plasmid, pSKU146, encodes a homolog of SARP1, Sk-ARP1. Due to the close phylogenetic relationship of the two pathogens, we hypothesized that the two plasmids are closely related as well. RESULTS: The nucleotide sequence of pBJS-O was determined and compared to the sequences of a plasmid from BR3-T (pBJS-T), which is a multiply passaged leafhopper transmissible derivative of BR3-3X, and to known plasmid sequences including that of pSKU146. In addition to arp1, the 13,374 bp pBJS-O sequence putatively contains nine genes, recognized as open reading frames (ORFs). Several pBJS-O ORFs have homologs on pSKU146. However, the sequences flanking soj-like genes on both plasmids were found to be more distant from one another than sequences in any other region. Further, unlike pSKU146, pBJS-O lacks the conserved oriT region characteristic of the IncP group of bacterial plasmids. We were unable to identify a region in pBJS-O resembling a known plasmid origin of transfer. In regions where sequence was available for the plasmid from both BR3-3X and BR3-T, the pBJS-T sequence had a 0.4 kb deletion relative to its progenitor, pBJS-O. Southern blot hybridization of extrachromosomal DNA from various S. citri strains and spiroplasma species to an arp-specific probe and a probe made from the entire plasmid DNA of BR3-3X revealed limited conservation of both sequences in the genus Spiroplasma. Finally, we also report the presence on the BR3-3X chromosome of arp2, an S. citri homolog of arp1 that encodes the predicted protein SARP2. The C-terminal domain of SARP2 is homologous to that of SARP1, but its N-terminal domain is distinct. CONCLUSION: Our data suggest that pBJS is a novel S. citri plasmid that does not belong to any known plasmid incompatibility group. The differences between pBJS-O and pSKU146 suggest that one or more events of recombination have contributed to the divergence of the plasmids of the two sister Spiroplasma species; the plasmid from S. citri itself has diverged slightly during the derivation of S. citri BR3-T from BR3-3X. Our data also show that pBJS-O encodes the putative adhesin SARP1. The presence of traE and mob on pBJS-O suggests a role for the plasmid in spiroplasmal conjugation.
Project description:Spiroplasma citri GII3 contains highly related low-copy-number plasmids pSci1 to -6. Despite the strong similarities between their replication regions, these plasmids coexist in the spiroplasma cells, indicating that they are mutually compatible. The pSci1 to -6 plasmids encode the membrane proteins known as S. citri adhesion-related proteins (ScARPs) (pSci1 to -5) and the hydrophilic protein P32 (pSci6), which had been tentatively associated with insect transmission, as they were not detected in non-insect-transmissible strains. With the aim of further investigating the role of plasmid-encoded determinants in insect transmission, we have constructed S. citri mutant strains that differ in their plasmid contents by developing a plasmid curing/replacement strategy based on the incompatibility of plasmids having identical replication regions. Experimental transmission of these S. citri plasmid mutants through injection into the leafhopper vector Circulifer haematoceps revealed that pSci6, more precisely, the pSci6_06 coding sequence, encoding a protein of unknown function, was essential for transmission. In contrast, ScARPs and P32 were dispensable for both acquisition and transmission of the spiroplasmas by the leafhopper vector, even though S. citri mutants lacking pSci1 to -5 (encoding ScARPs) were acquired and transmitted at lower efficiencies than the wild-type strain GII3.
Project description:Borrelia (B.) burgdorferi sensu lato, including the tick-transmitted agents of human Lyme borreliosis, have particularly complex genomes, consisting of a linear main chromosome and numerous linear and circular plasmids. The number and structure of plasmids is variable even in strains within a single genospecies. Genes on these plasmids are known to play essential roles in virulence and pathogenicity as well as host and vector associations. For this reason, it is essential to explore methods for rapid and reliable characterisation of molecular level changes on plasmids. In this study we used three strains: a low passage isolate of B. burgdorferi sensu stricto strain B31(-NRZ) and two closely related strains (PAli and PAbe) that were isolated from human patients. Sequences of these strains were compared to the previously sequenced reference strain B31 (available in GenBank) to obtain proof-of-principle information on the suitability of next generation sequencing (NGS) library construction and sequencing methods on the assembly of bacterial plasmids. We tested the effectiveness of different short read assemblers on Illumina sequences, and of long read generation methods on sequence data from Pacific Bioscience single-molecule real-time (SMRT) and nanopore (Oxford Nanopore Technologies) sequencing technology.Inclusion of mate pair library reads improved the assembly in some plasmids as did prior enrichment of plasmids. While cp32 plasmids remained refractory to assembly using only short reads they were effectively assembled by long read sequencing methods. The long read SMRT and nanopore sequences came, however, at the cost of indels (insertions or deletions) appearing in an unpredictable manner. Using long and short read technologies together allowed us to show that the three B. burgdorferi s.s. strains investigated here, whilst having similar plasmid structures to each other (apart from fusion of cp32 plasmids), differed significantly from the reference strain B31-GB, especially in the case of cp32 plasmids.Short read methods are sufficient to assemble the main chromosome and many of the plasmids in B. burgdorferi. However, a combination of short and long read sequencing methods is essential for proper assembly of all plasmids including cp32 and thus, for gaining an understanding of host- or vector adaptations. An important conclusion from our work is that the evolution of Borrelia plasmids appears to be dynamic. This has important implications for the development of useful research strategies to monitor the risk of Lyme disease occurrence and how to medically manage it.
Project description:Transcription activator-like effectors (TALEs) are virulence factors of Xanthomonas that induce the expression of host susceptibility (S) genes by specifically binding to effector binding elements (EBEs) in their promoter regions. The DNA binding specificity of TALEs is dictated by their tandem repeat regions, which are highly variable between different TALEs. Mutation of the EBEs of S genes is being utilized as a key strategy to generate resistant crops against TALE-dependent pathogens. However, TALE adaptations through rearrangement of their repeat regions is a potential obstacle for successful implementation of this strategy. We investigated the consequences of TALE adaptations in the citrus pathogen Xanthomonas citri subsp. citri (Xcc), in which PthA4 is the TALE required for pathogenicity, whereas CsLOB1 is the corresponding susceptibility gene, on host resistance. Seven TALEs, containing two-to-nine mismatching-repeats to the EBEPthA4 that were unable to induce CsLOB1 expression, were introduced into Xcc pthA4:Tn5 and adaptation was simulated by repeated inoculations into and isolations from sweet orange for a duration of 30 cycles. While initially all strains failed to promote disease, symptoms started to appear between 9-28 passages in four TALEs, which originally harbored two-to-five mismatches. Sequence analysis of adapted TALEs identified deletions and mutations within the TALE repeat regions which enhanced putative affinity to the CsLOB1 promoter. Sequence analyses suggest that TALEs adaptations result from recombinations between repeats of the TALEs. Reintroduction of these adapted TALEs into Xcc pthA4:Tn5 restored the ability to induce the expression of CsLOB1, promote disease symptoms and colonize host plants. TALEs harboring seven-to-nine mismatches were unable to adapt to overcome the incompatible interaction. Our study experimentally documented TALE adaptations to incompatible EBE and provided strategic guidance for generation of disease resistant crops against TALE-dependent pathogens.
Project description:Whole genome sequencing can provide essential public health information. However, it is now known that widely used short-read methods have the potential to miss some randomly-distributed segments of genomes. This can prevent phages, plasmids, and virulence factors from being detected or properly identified. Here, we compared assemblies of three complete Shiga toxin-producing Escherichia coli (STEC) O26:H11/H- genomes from two different sequence types (ST21 and 29), each acquired using the Nextera XT MiSeq, MinION nanopore-based sequencing, and Pacific Biosciences (PacBio) sequencing. Each closed genome consisted of a single chromosome, approximately 5.7 Mb for CFSAN027343, 5.6 Mb for CFSAN027346, and 5.4 MB for CFSAN027350. However, short-read whole genome sequencing (WGS) using Nextera XT MiSeq failed to identify some virulence genes in plasmids and on the chromosome, both of which were detected using the long-read platforms. Results from long-read MinION and PacBio allowed us to identify differences in plasmid content: a single 88 kb plasmid in CFSAN027343; a 157kb plasmid in CFSAN027350; and two plasmids in CFSAN027346 (one 95 Kb, one 72 Kb). These data enabled rapid characterization of the virulome, detection of antimicrobial genes, and composition/location of Stx phages. Taken together, positive correlations between the two long-read methods for determining plasmids, virulome, antimicrobial resistance genes, and phage composition support MinION sequencing as one accurate and economical option for closing STEC genomes and identifying specific virulence markers.
Project description:Enteric redmouth disease caused by the pathogen Yersinia ruckeri is a significant problem for fish farming around the world. Despite its importance, only a few virulence factors of Y. ruckeri have been identified and studied in detail. Here, we report and analyze the complete DNA sequence of pYR4, a plasmid from a highly pathogenic Norwegian Y. ruckeri isolate, sequenced using PacBio SMRT technology. Like the well-known pYV plasmid of human pathogenic Yersiniae, pYR4 is a member of the IncFII family. Thirty-one percent of the pYR4 sequence is unique compared to other Y. ruckeri plasmids. The unique regions contain, among others genes, a large number of mobile genetic elements and two partitioning systems. The G+C content of pYR4 is higher than that of the Y. ruckeri NVH_3758 genome, indicating its relatively recent horizontal acquisition. pYR4, as well as the related plasmid pYR3, comprises operons that encode for type IV pili and for a conjugation system (tra). In contrast to other Yersinia plasmids, pYR4 cannot be cured at elevated temperatures. Our study highlights the power of PacBio sequencing technology for identifying mis-assembled segments of genomic sequences. Comparative analysis of pYR4 and other Y. ruckeri plasmids and genomes, which were sequenced by second and the third generation sequencing technologies, showed errors in second generation sequencing assemblies. Specifically, in the Y. ruckeri 150 and Y. ruckeri ATCC29473 genome assemblies, we mapped the entire pYR3 plasmid sequence. Placing plasmid sequences on the chromosome can result in erroneous biological conclusions. Thus, PacBio sequencing or similar long-read methods should always be preferred for de novo genome sequencing. As the tra operons of pYR3, although misplaced on the chromosome during the genome assembly process, were demonstrated to have an effect on virulence, and type IV pili are virulence factors in many bacteria, we suggest that pYR4 directly contributes to Y. ruckeri virulence.
Project description:BACKGROUND:Elucidating the ecological and biological identity of extrachromosomal mobile genetic elements (eMGEs), such as plasmids and bacteriophages, in the human gut remains challenging due to their high complexity and diversity. RESULTS:Here, we show efficient identification of eMGEs as complete circular or linear contigs from PacBio long-read metagenomic data. De novo assembly of PacBio long reads from 12 faecal samples generated 82 eMGE contigs (2.5~666.7-kb), which were classified as 71 plasmids and 11 bacteriophages, including 58 novel plasmids and six bacteriophages, and complete genomes of five diverse crAssphages with terminal direct repeats. In a dataset of 413 gut metagenomes from five countries, many of the identified plasmids were highly abundant and prevalent. The ratio of gut plasmids by our plasmid data is more than twice that in the public database. Plasmids outnumbered bacterial chromosomes three to one on average in this metagenomic dataset. Host prediction suggested that Bacteroidetes-associated plasmids predominated, regardless of microbial abundance. The analysis found several plasmid-enriched functions, such as inorganic ion transport, while antibiotic resistance genes were harboured mostly in low-abundance Proteobacteria-associated plasmids. CONCLUSIONS:Overall, long-read metagenomics provided an efficient approach for unravelling the complete structure of human gut eMGEs, particularly plasmids.