Analysis of the Campylobacter jejuni genome by SMRT DNA sequencing identifies restriction-modification motifs.
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ABSTRACT:
SUBMITTER: O'Loughlin JL
PROVIDER: S-EPMC4335053 | biostudies-literature | 2015
REPOSITORIES: biostudies-literature
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Analysis of the Campylobacter jejuni genome by SMRT DNA sequencing identifies restriction-modification motifs.
O'Loughlin Jason L JL Eucker Tyson P TP Chavez Juan D JD Samuelson Derrick R DR Neal-McKinney Jason J Gourley Christopher R CR Bruce James E JE Konkel Michael E ME
PloS one 20150219 2
Campylobacter jejuni is a leading bacterial cause of human gastroenteritis. The goal of this study was to analyze the C. jejuni F38011 strain, recovered from an individual with severe enteritis, at a genomic and proteomic level to gain insight into microbial processes. The C. jejuni F38011 genome is comprised of 1,691,939 bp, with a mol.% (G+C) content of 30.5%. PacBio sequencing coupled with REBASE analysis was used to predict C. jejuni F38011 genomic sites and enzymes that may be involved in D ...[more]
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Project description:Campylobacter species are the most prevalent bacterial pathogen causing acute enteritis worldwide. In contrast to Campylobacter jejuni, about 5 % of Campylobacter coli strains exhibit susceptibility to restriction endonuclease digestion by DpnI cutting specifically 5'-G(m)ATC-3' motifs. This indicates significant differences in DNA methylation between both microbial species. The goal of the study was to analyze the methylome of a C. coli strain susceptible to DpnI digestion, to identify its methylation motifs and restriction modification systems (RM-systems), and compare them to related organisms like C. jejuni and Helicobacter pylori.Using one SMRT cell and the PacBio RS sequencing technology followed by PacBio Modification and Motif Analysis the complete genome of the DpnI susceptible strain C. coli BfR-CA-9557 was sequenced to 500-fold coverage and assembled into a single contig of 1.7 Mbp. The genome contains a CJIE1-like element prophage and is phylogenetically closer to C. coli clade 1 isolates than clade 3. 45,881 6-methylated adenines (ca. 2.7 % of genome positions) that are predominantly arranged in eight different methylation motifs and 1,788 4-methylated cytosines (ca. 0.1 %) have been detected. Only two of these motifs correspond to known restriction modification motifs. Characteristic for this methylome was the very high fraction of methylation of motifs with mostly above 99 %.Only five dominant methylation motifs have been identified in C. jejuni, which have been associated with known RM-systems. C. coli BFR-CA-9557 shares one (RAATTY) of these, but four ORFs could be assigned to putative Type I RM-systems, seven ORFs to Type II RM-systems and three ORFs to Type IV RM-systems. In accordance with DpnI prescreening RM-system IIP, methylation of GATC motifs was detected in C. coli BfR-CA-9557. A homologous IIP RM-system has been described for H. pylori. The remaining methylation motifs are specific for C. coli BfR-CA-9557 and have been neither detected in C. jejuni nor in H. pylori. The results of this study give us new insights into epigenetics of Campylobacteraceae and provide the groundwork to resolve the function of RM-systems in C. coli.
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Project description:Conjugation is an important mechanism for horizontal gene transfer in Campylobacter jejuni, the leading cause of human bacterial gastroenteritis in developed countries. However, to date, the factors that significantly influence conjugation efficiency in Campylobacter spp. are still largely unknown. Given that multiple recombinant loci could independently occur within one recipient cell during natural transformation, the genetic materials from a high-frequency conjugation (HFC) C. jejuni strain may be cotransformed with a selection marker into a low-frequency conjugation (LFC) recipient strain, creating new HFC transformants suitable for the identification of conjugation factors using a comparative genomics approach. To test this, an erythromycin resistance selection marker was created in an HFC C. jejuni strain; subsequently, the DNA of this strain was naturally transformed into NCTC 11168, an LFC C. jejuni strain, leading to the isolation of NCTC 11168-derived HFC transformants. Whole-genome sequencing analysis and subsequent site-directed mutagenesis identified Cj1051c, a putative restriction-modification enzyme (aka CjeI) that could drastically reduce the conjugation efficiency of NCTC 11168 (>5,000-fold). Chromosomal complementation of three diverse HFC C. jejuni strains with CjeI also led to a dramatic reduction in conjugation efficiency (?1,000-fold). The purified recombinant CjeI could effectively digest the Escherichia coli-derived shuttle vector pRY107. The endonuclease activity of CjeI was abolished upon short heat shock treatment at 50°C, which is consistent with our previous observation that heat shock enhanced conjugation efficiency in C. jejuni Together, in this study, we successfully developed and utilized a unique cotransformation strategy to identify a restriction-modification enzyme that significantly influences conjugation efficiency in C. jejuni IMPORTANCE Conjugation is an important horizontal gene transfer mechanism contributing to the evolution of bacterial pathogenesis and antimicrobial resistance. Campylobacter jejuni, the leading foodborne bacterial organism, displays significant strain diversity due to horizontal gene transfer; however, the molecular components influencing conjugation efficiency in C. jejuni are still largely unknown. In this study, we developed a cotransformation strategy for comparative genomics analysis and successfully identified a restriction-modification enzyme that significantly influences conjugation efficiency in C. jejuni The new cotransformation strategy developed in this study is also expected to be broadly applied in other naturally competent bacteria for functional comparative genomics research.
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Project description:BackgroundRestriction and modification enzymes typically recognise short DNA sequences of between two and eight bases in length. Understanding the mechanism of this recognition represents a significant challenge that we begin to address for the BsaHI restriction-modification system, which recognises the six base sequence GRCGYC.ResultsThe DNA sequences of the genes for the BsaHI methyltransferase, bsaHIM, and restriction endonuclease, bsaHIR, have been determined (GenBank accession #EU386360), cloned and expressed in E. coli. Both the restriction endonuclease and methyltransferase enzymes share significant similarity with a group of 6 other enzymes comprising the restriction-modification systems HgiDI and HgiGI and the putative HindVP, NlaCORFDP, NpuORFC228P and SplZORFNP restriction-modification systems. A sequence alignment of these homologues shows that their amino acid sequences are largely conserved and highlights several motifs of interest. We target one such conserved motif, reading SPERRFD, at the C-terminal end of the bsaHIR gene. A mutational analysis of these amino acids indicates that the motif is crucial for enzymatic activity. Sequence alignment of the methyltransferase gene reveals a short motif within the target recognition domain that is conserved among enzymes recognising the same sequences. Thus, this motif may be used as a diagnostic tool to define the recognition sequences of the cytosine C5 methyltransferases.ConclusionWe have cloned and sequenced the BsaHI restriction and modification enzymes. We have identified a region of the R. BsaHI enzyme that is crucial for its activity. Analysis of the amino acid sequence of the BsaHI methyltransferase enzyme led us to propose two new motifs that can be used in the diagnosis of the recognition sequence of the cytosine C5-methyltransferases.
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Project description:BackgroundCampylobacter species are a leading cause of bacterial foodborne illness worldwide. Despite the global efforts to curb them, Campylobacter infections have increased continuously in both developed and developing countries. The development of effective strategies to control the infection by this pathogen is warranted. The essential genes of bacteria are the most prominent targets for this purpose. In this study, we used transposon sequencing (Tn-seq) of a genome-saturating library of Tn5 insertion mutants to define the essential genome of C. jejuni at a high resolution.ResultWe constructed a Tn5 mutant library of unprecedented complexity in C. jejuni NCTC 11168 with 95,929 unique insertions throughout the genome and used the genomic DNA of the library for the reconstruction of Tn5 libraries in the same (C. jejuni NCTC 11168) and different strain background (C. jejuni 81-176) through natural transformation. We identified 166 essential protein-coding genes and 20 essential transfer RNAs (tRNA) in C. jejuni NCTC 11168 which were intolerant to Tn5 insertions during in vitro growth. The reconstructed C. jejuni 81-176 library had 384 protein coding genes with no Tn5 insertions. Essential genes in both strain backgrounds were highly enriched in the cluster of orthologous group (COG) categories of 'Translation, ribosomal structure and biogenesis (J)', 'Energy production and conversion (C)', and 'Coenzyme transport and metabolism (H)'.ConclusionComparative analysis among this and previous studies identified 50 core essential genes of C. jejuni, which can be further investigated for the development of novel strategies to control the spread of this notorious foodborne bacterial pathogen.
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Project description:Comparison of the entire coding sequence of flaA (1,764 nucleotides) from 15 isolates of Campylobacter jejuni showed two regions of high variability, one region approximately from base positions 700 to 1,450 and a short variable region (SVR) from base positions 450 to 600. Parsimony analysis of the SVR sequences yielded a dendrogram similar to that which was derived by analysis of the entire gene. PCR was used to generate templates, and the SVR was sequenced with primers constructed to hybridize to conserved flanking sequences. The SVRs of 22 isolates of C. jejuni from four outbreaks that have been well characterized and a larger panel of isolates from three additional outbreaks were sequenced. Analysis of the nucleotide sequences produced results that grouped the isolates very similarly to other subtyping techniques. Sequence data were also generated for isolates from three additional outbreaks. Categorizing the isolates by fla SVR DNA sequence placed them in epidemiologically relevant groups. Sequence analysis of the C. jejuni flaA SVR may be a useful tool for epidemiologic investigations and could complement or replace serotyping and other subtyping methods.
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Project description:The sequencing, assembly, and analysis of bacterial genomes is central to tracking and characterizing foodborne pathogens. The bulk of bacterial genome sequencing at the US Food and Drug Administration is performed using short-read Illumina MiSeq technology, resulting in highly accurate but fragmented genomic sequences. The MinION sequencer from Oxford Nanopore is an evolving technology that produces long-read sequencing data with low equipment cost. The goal of this study was to compare Campylobacter genome assemblies generated from MiSeq and MinION data independently, as well as hybrid genome assemblies combining both data types. Two reference strains and two field isolates of C. jejuni were sequenced using MiSeq and MinION, and the sequence data were assembled using the software programs SPAdes and Canu, respectively. Hybrid genome assembly was performed using the program Unicycler. Comparison of the C. jejuni 81-176 and RM1221 genome assemblies to the PacBio reference genomes revealed that the SPAdes assemblies had the most accurate nucleotide identity, while the hybrid assemblies were the most contiguous. Assemblies generated only from MinION data using Canu were the least accurate, containing many indels and substitutions that affected downstream analyses. The hybrid sequencing approach was the most useful for detecting plasmids, large genome rearrangements, and repetitive elements such as rRNA and tRNA genes. The full genomes of both C. jejuni field isolates were completed and circularized using hybrid sequencing, and a plasmid was detected in one isolate. Continued development of nanopore sequencing technologies will likely enhance the accuracy of hybrid genome assemblies and enable public health laboratories to routinely generate complete circularized bacterial genome sequences.
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Project description:Campylobacter jejuni is recognised as the leading cause of bacterial gastroenteritis in industrialised countries. Although the majority of Campylobacter infections are self-limiting, antimicrobial treatment is necessary in severe cases. Therefore, the development of antimicrobial resistance (AMR) in Campylobacter is a growing public health challenge and surveillance of AMR is important for bacterial disease control. The aim of this study was to predict antimicrobial resistance in C. jejuni from whole-genome sequencing data. A total of 516 clinical C. jejuni isolates collected between 2014 and 2017 were subjected to WGS. Resistance phenotypes were determined by standard broth dilution, categorising isolates as either susceptible or resistant based on epidemiological cutoffs for six antimicrobials: ciprofloxacin, nalidixic acid, erythromycin, gentamicin, streptomycin, and tetracycline. Resistance genotypes were identified using an in-house database containing reference genes with known point mutations and the presence of resistance genes was determined using the ResFinder database and four bioinformatical methods (modified KMA, ABRicate, ARIBA, and ResFinder Batch Upload). We identified seven resistance genes including tet(O), tet(O/32/O), ant(6)-Ia, aph(2″)-If, blaOXA, aph(3')-III, and cat as well as mutations in three genes: gyrA, 23S rRNA, and rpsL. There was a high correlation between phenotypic resistance and the presence of known resistance genes and/or point mutations. A correlation above 98% was seen for all antimicrobials except streptomycin with a correlation of 92%. In conclusion, we found that WGS can predict antimicrobial resistance with a high degree of accuracy and have the potential to be a powerful tool for AMR surveillance.
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Project description:BackgroundWhole genome sequencing (WGS) is useful for determining clusters of human cases, investigating outbreaks, and defining the population genetics of bacteria. It also provides information about other aspects of bacterial biology, including classical typing results, virulence, and adaptive strategies of the organism. Cell culture invasion and protein expression patterns of four related multilocus sequence type 21 (ST21) C. jejuni isolates from a significant Canadian water-borne outbreak were previously associated with the presence of a CJIE1 prophage. Whole genome sequencing was used to examine the genetic diversity among these isolates and confirm that previous observations could be attributed to differential prophage carriage. Moreover, we sought to determine the presence of genome sequences that could be used as surrogate markers to delineate outbreak-associated isolates.ResultsDifferential carriage of the CJIE1 prophage was identified as the major genetic difference among the four outbreak isolates. High quality single-nucleotide variant (hqSNV) and core genome multilocus sequence typing (cgMLST) clustered these isolates within expanded datasets consisting of additional C. jejuni strains. The number and location of homopolymeric tract regions was identical in all four outbreak isolates but differed from all other C. jejuni examined. Comparative genomics and PCR amplification enabled the identification of large chromosomal inversions of approximately 93 kb and 388 kb within the outbreak isolates associated with transducer-like proteins containing long nucleotide repeat sequences. The 93-kb inversion was characteristic of the outbreak-associated isolates, and the gene content of this inverted region displayed high synteny with the reference strain.ConclusionsThe four outbreak isolates were clonally derived and differed mainly in the presence of the CJIE1 prophage, validating earlier findings linking the prophage to phenotypic differences in virulence assays and protein expression. The identification of large, genetically syntenous chromosomal inversions in the genomes of outbreak-associated isolates provided a unique method for discriminating outbreak isolates from the background population. Transducer-like proteins appear to be associated with the chromosomal inversions. CgMLST and hqSNV analysis also effectively delineated the outbreak isolates within the larger C. jejuni population structure.
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