- Comparison of the FRIK804 genome with a reference EHEC strain Sakai found a unique prophage like element (PLE, indel 1) and an inversion (1.15 Mb) situated symmetrically with respect to the terminus region.. - Full list of author information is available at the end of the article. - The locations of the repeat sequences were biased towards MGE. - understanding of the precise molecular events and elements that contributed to genetic diversification of wild-type EHEC in the bovine and farm environments.. - By length, prophage account for 11% of the Sakai chromosome and a majority of MGE. - Most of the identified prophage ele- ments are considered incapable of excision or replication and are regarded as cryptic [21]. - Originally distin- guished on the basis of differing PFGE profiles, the dif- ferences between these strains included the insertional inactivation of stx2 by IS 629 in FRIK1275 and the ab- sence of the stx2 -prophage in FRIK1625. - de novo sequence assembly of the FRIK804 genome Sequence assembly using Illumina short-read data was hampered by an inability to resolve DNA sequence re- peats longer than read length. - A high-quality de novo assembly of the. - The gapless assembly of the FRIK804 genome was required to provide a refer- ence for the other strains analyzed in this study.. - Initial assembly of the FRIK804 genome used SPAdes and both SMRT and Illumina data [33]. - The extensive synteny of the two chromosomes was interrupted by a few structural differences. - Non-conserved regions consisted of Mu-like prophage with distinct strain-specific integration sites, an inverted segment of the chromosome that included the terminus, and two indels (Fig. - Alignment of the nucleotide se- quence of indel-1 from FRIK804 with the nucleotide sequences of PLE in the Sakai genome (SpLE1-SPLE6) identified common flanking regions shared with SpLE1 (Fig. - A majority of the ddp operon and dosP were within this region. - Comparison of the stx2 -prophage in FRIK804 (Φ804–. - Alignment of the prophage was interrupted at several locations. - An inversion measuring 1.15 Mbp disrupted the align- ment of the FRIK804 and Sakai chromosomes. - A 174 bp repeat se- quence precisely flanking the boundaries of the inversion in both Φ804–7/Φ804–15 and Sp6/Sp14 was identified.. - To confirm the precise boundaries of the inversion, two. - Alignment of the FRIK804 (outer) and Sakai (inner) chromosome found disruption of synteny by large-scale structural alterations. - Mapping of the chromosome provided a better. - understanding of the chromosome rearrangements that distinguished each strain. - Whole genome mapping was also valuable for verification of genome assembly of the FRIK804 chromosome. - Based on the sum of the length of the fragments, the. - 3 Alignment of prophage Φ 804 – 7 and Φ 804 – 15 and the locations of 16 inverted and direct repeat sequences, including the flanking ends of the inversion. - The inverted segment of the FRIK804 chromosome relative to the Sakai chromosome was flanked by a pair of repeats, the site of the crossover of the inversion is shown in red. - 4 a NcoI restriction site maps of the chromosomes from FRIK804 (outer), FRIK1275 (middle), and FRIK1625 (inner). - The location of indel-3 was consistent with the absence of the stx2 -prophage in FRIK1625. - b Hierarchical clustering and pairwise alignment scoring of NcoI chromosome restriction maps was used to assess relative similarity of the three farm X strains with 30 other E. - Guided by the nucleotide se- quence of FRIK804, the position of indel-3 in FRIK1625 was consistent with the absence of the stx2 -prophage.. - Pairwise alignment scoring of the ordered restriction maps of the three farm X strains and maps of 30 other E. - To precisely deter- mine the boundaries of the absent prophage region in FRIK1275 and FRIK1625, Illumina sequencing data from each strain (including FRIK804) were aligned to the nu- cleotide sequence of Φ804–9 and Φ804–10 using Bowtie (Fig. - An 822 bp direct repeat was situated at both ends of the region missing in FRIK1275 and FRIK1625. - The predicted location and function of the remaining Φ804–9 and Φ804–10 genes, in FRIK1275 and FRIK1625, aligned with those in FRIK804 (Fig. - PCR amplification of the region was performed using oligonucleotide primers spe- cific to sequences flanking the repeat sequence (ECs_. - Because of the excessive length, an amplicon was not observed using gDNA extracted from FRIK804 (>. - 5 Detection of the boundaries of inter-prophage deletion (indel-4) in Φ 804 – 9/ Φ 804 – 10 present in FRIK1275 and FRIK1625. - The difference in read coverage in Δ FRIK1275 and Δ FRIK1625 was below zero in the region of the deletion and terminated in direct repeats (shaded red) that flanked the deleted 47.7 kbp fragment. - Repeat sequence complexity was a measure of the re- peat copy number irrespective of orientation, i.e. - Measurement of the copy number of each 75-mer repeat sequence (and disregard- ing sequence orientation) in each strain found a greater number in FRIK804 compared to MG1655 (Fig. - Repetitive regions of the chromosome were defined as areas containing one or more repeat sequences. - A total of 417,747 bp (7.52%) of the FRIK804 chromosome consisted of repetitive re- gions. - The site of integration of the stx2 -prophage is specific in each EHEC lineage [41, 42], with the stx2 -prophage inte- grating into wrbA in LI and I/II strains. - Comparison of the nu- cleotide sequence of wrbA from the FRIK1625 with wrbA from a LI/II strain (without stx2 prophage) found 100% sequence identity (data not shown). - This shows that if the stx2 -prophage was present in FRIK1625, exci- sion was mediated by Int/Xis activity rather than hom- ologous recombination, and excision occurred without subsequent lysis of the host.. - Repeats located outside of the designated genetic elements were listed under chromosome. - d The length of repetitive regions, areas of the chromosome featuring one or more repeats, was calculated and classified by genetic element. - Amplification of a portion of the 16S rRNA gene (primers 16S-RT-F/R) was included as a control. - however, a detailed understanding of the underlying mo- lecular event(s) that lead to the observed chromosomal alterations is lacking, particularly in isolates from the bo- vine reservoir [47, 48]. - FRIK1625 was isolated from a single fecal sample in the last year of the study. - coli O157:H7 genomes as- sembled using short-read DNA sequence data (Illumina) was complicated by repeat sequences found in multiple regions of the chromosome. - The assembly of the FRIK804 genome was accomplished using SMRT long- read sequencing data and improved using short-read data. - Validation of the finished sequence assembly was conducted using whole-genome mapping data (optical mapping). - Pairwise alignment of the ordered restriction maps and hierarchical clustering determined the farm X strains comprised a single clade of strains (Fig. - This is important since inversion of the Ter (terminus of replication) region can stall or stop replica- tion forks and induce the SOS response in E. - Analysis of the three farm X strains determined that FRIK1275 and FRIK1625 shared a common plasmid pro- file with Sakai. - The IS 629 content of the farm X strains was similar. - FRIK1625 lacked the stx2 -prophage (indel 3) suggesting non-lethal excision of the stx2 -prophage. - Loss of the stx2 -prophage has been observed before during la- boratory passage [60, 61].. - Detailed analysis of the 47.7-kbp deletion in FRIK1275 and FRIK1625 was conducted by alignment of short- read sequence data to the intact sequence of adjacent prophage Φ804–9 and Φ804–10 from FRIK804. - A com- parison of the difference in read coverage between strains FRIK1275 and FRIK1625 with that of FRIK804 (no deletion) enabled demarcation of the deletion boundaries (Fig. - The propensity for deletions in this region may be due to the proximity of the two prophages.. - The frequency of recombination between homologous repeat sequences increases with the length of the repeat in a bi- phasic manner [63]. - We did not address approximate repeats in DNA se- quences because of the extensive number of homologous sequences present in the O157:H7 genome and the dra- matic decrease in the frequency of recombination when mismatches are present within the repeats [63].. - Analysis of areas of the chromosome containing one or more re- peats (repeat regions) found that most repeat regions were located within prophage/PLE. - Transcripts from genes upstream and down- stream of the stx2 ::IS 629 were detected by RT-PCR al- though Stx2 was not detected by Western blot [32].. - coli K12 strain MG1655 with a preponderance of the re- petitive sequences present in MGE. - This study contributes to our understanding of the precise molecular events contributing to genomic diversity in wild- type EHEC strains from the bovine and farm environments.. - The quality and quantity of the finished li- braries were assessed using an Agilent High Sensitivity DNA kit and Qubit® dsDNA HS Assay Kit, respectively.. - Improvement of the draft assemblies was iteratively performed until no sequence variants were found by Pilon. - Iterative improve- ment of the assembly was performed as previously outlined. - Circularization of the chromosome was per- formed manually using BLASTn [68–70] to identify overlapping regions. - Validation of the assembly was con- firmed by generating an in silico whole-genome map of NcoI restriction sites and comparing it to map generated. - The genome sequences of the E. - Alignment of the FRIK804 and Sakai chromosomes was performed using progressiveMauve [73] and BLASTn [68–70]. - The boundaries of the inversion present in strains of the farm X clade, with respect to Sakai, were verified using oligonucleotide primers ECs_2759-F, ECs_22760-R, ECs_1507-R, and ECs_1508-R. - PCR amplification of regions of inter-prophage deletions The boundaries of the inter-prophage region present in FRIK804 but absent in FRIK1275 and FRIK1625 was verified using oligonucleotide primers (Table S5). - Primers (Table S5) targeting regions immediately up- stream (stx2-US-RT-F/R) and downstream (stx2-DS-RT- F/R) of the IS 629 insertion in the FRIK1275 copy of stx2 were used. - PCR confirmation of inverted repeats present at the flanking ends of the inversion in farm X strains (FRIK804, FRIK1275, and FRIK1625) and control strain Sakai. - The portions of the two adjacent phage in all farm X strain has a shaded grey background. - Highlighted motifs (light orange) located within the segment of the FRIK804 chromosome that is inverted relative to strain Sakai.. - ES designed the study, preformed experiments, and prepared the draft of the manuscript. - Stanton was the recipient of the E. - https://doi.org/10.. - https://doi.org/10.1017/. - https://doi.org . - enterohemorrhagic Escherichia coli O157:H7. - Molecular analysis of the plasmid-encoded hemolysin of Escherichia coli O157:H7 strain EDL 933. - https://doi.org/10.1046/j . - https://doi.org/10.1073/. - Evolution of the Stx2-encoding Prophage in persistent bovine Escherichia coli O157:H7 strains. - https://doi.org/10.1128/. - https://doi.org/10.1017/S . - Insertion of inverted Ter sites into the terminus region of the Escherichia coli chromosome delays completion of DNA replication and disrupts the cell cycle. - https://doi.org/10.1111/. - https://doi.org/. - https://doi.org/. - Database resources of the National Center for biotechnology information. - https://doi.org
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