Fig 3
Double chromosomal inversions. (a). Schematic representation of double inversion. A smaller chromosomal inversion (as found in EDL933 relative to Sakai and K-12) is shown first, illustrated by the smaller crossed lines. A second, larger inversion encompassing the smaller inversion is then shown. The double inversion shows (as found in EC502) the smaller initial inversion restored to its original orientation flanked by two separate inverted segments. Aligned chromosomes are shown in green, inverted segments are shown in yellow; however, the state of inversion depends on the pair of chromosomes aligned. These inversions are not drawn to scale. (b) Optical maps demonstrating double chromosomal inversions. The optical maps of EC502 aligned with the in silico map of Sakai and EC502 aligned with the in silico map of EDL933. The inversion within an inversion is apparent in the alignment with Sakai. Fragments within mini-optical maps of O-island prophages W and V flanking the larger inversion are highlighted in blue. A Mbp scale is shown at the bottom of the panel; the optical map inversions are to scale.
Source publication
Optical maps of 11 Escherichia coli O157 : H7 strains have been generated by the assembly of contiguous sets of restriction fragments across their entire 5.3 to 5.6 Mbp chromosomes. Each strain showed a distinct, highly individual configuration of 500-700 BamHI fragments, yielding a map resembling a DNA 'bar code'. The accuracy of optical mapping w...
Contexts in source publication
Context 1
... were four other inversions. The chromosomes of the four strains appear complex when their optical maps are aligned with Sakai, but appear simple when aligned with EDL933. The chromosome structures of these strains are the result of double inversions. This is shown schematically for EC502 in Fig. 3(a). The order of inversion is not critical, but the structures observed in the optical maps can be most simply explained by an inversion occurring within another inversion (Fig. 3a). Fig. 3 ...
Context 2
... but appear simple when aligned with EDL933. The chromosome structures of these strains are the result of double inversions. This is shown schematically for EC502 in Fig. 3(a). The order of inversion is not critical, but the structures observed in the optical maps can be most simply explained by an inversion occurring within another inversion (Fig. 3a). Fig. 3 ...
Context 3
... simple when aligned with EDL933. The chromosome structures of these strains are the result of double inversions. This is shown schematically for EC502 in Fig. 3(a). The order of inversion is not critical, but the structures observed in the optical maps can be most simply explained by an inversion occurring within another inversion (Fig. 3a). Fig. 3 ...
Citations
... These data are in agreement with a recent study showing that transcriptional activation of TA operons by multiple stressing conditions does not lead to a detectable phenotype (16), which leaves the question of their role(s) open. Interestingly, the genome of the Sp12 prophage, a homologue of CP-933P in the O157: H7 Sakai strain, was shown to be prone to inversions (60)(61)(62). In general, cryptic prophages are malleable and polymorphic, with variations largely due to recombination, duplication, and gene loss or acquisition events (5). ...
Transcriptional regulation of bacterial toxin-antitoxin (TA) systems allows compensation of toxin and antitoxin proteins to maintain a neutral state and avoid cell intoxication unless TA genes are lost. Such models have been primarily studied in plasmids, but TAs are equally present in other mobile genetic elements, such as transposons and prophages.
... Escherichia coli O157:H7 is a significant human zoonotic pathogen originating from ruminant hosts, especially cattle [8]. Over evolutionary time, numerous prophages (typically [16][17][18][19][20][21][22][23][24][25] have integrated into the genomes of E. coli O157:H7 strains with an integration bias towards the terminus (Ter) of replication [9]. Acquisition of these prophages, many of which are closely related λ-like phages, has driven the evolution of this pathogen by carriage of virulence genes including secreted effector proteins, sRNAs involved in virulence gene regulation and [7,10], importantly, these prophages include those that encode Shiga-toxin (Stx) subtypes. ...
... There is published evidence that E. coli O157:H7 type strain EDL933 can undergo large-chromosomal rearrangements (LCRs), mainly inversions [19,20], with these rearrangements being flanked by prophages. LCRs, such as inversions, duplications and translocations, occur by homologous recombination between repeat sequences on the same chromosome [21]. ...
The human zoonotic pathogen Escherichia coli O157:H7 is defined by its extensive prophage repertoire including those that encode Shiga toxin, the factor responsible for inducing life-threatening pathology in humans. As well as introducing genes that can contribute to the virulence of a strain, prophage can enable the generation of large-chromosomal rearrangements (LCRs) by homologous recombination. This work examines the types and frequencies of LCRs across the major lineages of the O157:H7 serotype. We demonstrate that LCRs are a major source of genomic variation across all lineages of E. coli O157:H7 and by using both optical mapping and Oxford Nanopore long-read sequencing prove that LCRs are generated in laboratory cultures started from a single colony and that these variants can be recovered from colonized cattle. LCRs are biased towards the terminus region of the genome and are bounded by specific prophages that share large regions of sequence homology associated with the recombinational activity. RNA transcriptional profiling and phenotyping of specific structural variants indicated that important virulence phenotypes such as Shiga-toxin production, type-3 secretion and motility can be affected by LCRs. In summary, E. coli O157:H7 has acquired multiple prophage regions over time that act to continually produce structural variants of the genome. These findings raise important questions about the significance of this prophage-mediated genome contingency to enhance adaptability between environments.
... Epidemiological investigations of EHEC outbreaks have noted REDP variations in strains isolated from implicated foods and clinical stool samples [45,46]. The presence of multiple cryptic prophage regions in the EHEC genome are thought to serve as recombination hotspots; however, a detailed understanding of the underlying molecular event(s) that lead to the observed chromosomal alterations is lacking, particularly in isolates from the bovine reservoir [47,48]. In this study, a precise examination of chromosome modifications in a chronological set of E. coli O157:H7 strains from a Wisconsin dairy farm (farm X) was conducted. ...
Background:
Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a significant foodborne pathogen that resides asymptomatically within cattle and other ruminants. The EHEC genome harbors an extensive collection of mobile genetic elements (MGE), including multiple prophage, prophage-like elements, plasmids, and insertion sequence (IS) elements.
Results:
A chronological collection of EHEC strains (FRIK804, FRIK1275, and FRIK1625) isolated from a Wisconsin dairy farm (farm X) comprised a closely related clade genetically differentiated by structural alterations to the chromosome. 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. Detailed analysis determined the inversion was due to homologous recombination between repeat sequences in prophage. The three farm X strains were distinguished by the presence or absence of indel 3 (61 kbp) and indel 4 (48 kbp); FRIK804 contained both of these regions, FRIK1275 lacked indel 4, and indels 3 and 4 were both absent in FRIK1625. Indel 3 was the stx2 prophage and indel 4 involved a deletion between two adjacent prophage with shared repeat sequences. Both FRIK804 and FRIK1275 produced functional phage while FRIK1625 did not, which is consistent with indel 3. Due to their involvement in recombination events, direct and inverted repeat sequences were identified, and their locations mapped to the chromosome. FRIK804 had a greater number and overall length of repeat sequences than E. coli K12 strain MG1655. Repeat sequences were most commonly associated with MGE.
Conclusions:
This research demonstrated that three EHEC strains from a Wisconsin dairy farm were closely related and distinguished by variability within prophage regions and other MGE. Chromosome alterations were associated with recombination events between repeat sequences. An inventory of direct and inverted repeat sequences found a greater abundance and total length of repeat sequences in the EHEC strains compared to E. coli strain MG1655. The locations of the repeat sequences were biased towards MGE. The findings from this study expand our understanding of the precise molecular events and elements that contributed to genetic diversification of wild-type EHEC in the bovine and farm environments.
... Optical mapping. Genomic DNA of Xoo XF89b and XM42 were isolated and digested with BamHI in optical mapping procedures as described by Kotewicz et al. 49 sequencer. The results yielded 323,730 trimmed reads with an average length of 741.72 bp (~50x). ...
Rice bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major rice disease. In Taiwan, the tropical indica type of Oryza sativa originally grown in this area is mix-cultivated with the temperate japonica type of O. sativa, and this might have led to adaptive changes of both rice host and Xoo isolates. In order to better understand how Xoo adapts to this unique environment, we collected and analyzed fifty-one Xoo isolates in Taiwan. Three different genetic marker systems consistently identified five groups. Among these groups, two of them had unique sequences in the last acquired ten spacers in the clustered regularly interspaced short palindromic repeats (CRISPR) region, and the other two had sequences that were similar to the Japanese isolate MAFF311018 and the Philippines isolate PXO563, respectively. The genomes of two Taiwanese isolates with unique CRISPR sequence features, XF89b and XM9, were further completely sequenced. Comparison of the genome sequences suggested that XF89b is phylogenetically close to MAFF311018, and XM9 is close to PXO563. Here, documentation of the diversity of groups of Xoo in Taiwan provides evidence of the populations from different sources and hitherto missing information regarding distribution of Xoo populations in East Asia.
... With hundreds of markers across the genome, optical maps differentiate strains and precisely describe genomic rearrangements, such as insertions, deletions, duplications, and inversions. WGM has been widely used in microbial genomic studies, with applications in areas of comparative genomics and strain typing (Kotewicz et al., 2007;Shukla et al., 2009;Schwan et al., 2010;Bosch et al., 2013) and whole genome sequence assembly (Aston et al., 1999;Zhou et al., 2004;Latreille et al., 2007;Wu et al., 2009). ...
... The comparison of optical maps of new isolates with in silico optical maps of characterized strains can identify potential functional, or phenotypic differences between strains, generating empirical markers to distinguish between identical isolates, providing a powerful tool for epidemiological studies (Kotewicz et al., 2007). In this work it is shown that optical maps are appropriate to distinguish closely-related B. suis strains based on the large number of restriction sites analyzed, allowing the discrimination between different strains of the same biovar and haplotype, making this method a very accurate and useful tool to investigate transmission events and outbreaks involving pathogens of this species. ...
... Additionally, sequencing and assembly errors at DNA sequences cannot be ignored as a potential source for further differences. However, previously published works have shown that the resolution and detail that optical maps provide underscore their usefulness for molecular epidemiological studies and its utility to complement genome sequencing projects (Sabirova et al., 2014;Schwan et al., 2010;Shukla et al., 2009;Wu et al., 2009;Kotewicz et al., 2007;Zhou et al., 2004). ...
Optical mapping is a technology able to quickly generate high resolution ordered whole-genome
restriction maps of bacteria, being a proven approach to search for diversity among bacterial isolates. In
this work, optical whole-genome maps were used to compare closely-related Brucella suis biovar 2
strains. This biovar is the unique isolated in domestic pigs and wild boars in Portugal and Spain and most
of the strains share specific molecular characteristics establishing an Iberian clonal lineage that can be
differentiated from another lineage mainly isolated in several Central European countries. We performed
the BamHI whole-genome optical maps of
five B. suis biovar 2
field strains, isolated from wild boars in
Portugal and Spain (three from the Iberian lineage and two from the Central European one) as well as of
the reference strain B. suis biovar 2 ATCC 23445 (Central European lineage, Denmark). Each strain showed
a distinct, highly individual configuration of 228–231 BamHI fragments. Nevertheless, a low divergence
was globally observed in chromosome II (1.6%) relatively to chromosome I (2.4%). Optical mapping also
disclosed genomic events associated with B. suis strains in chromosome I, namely one indel (3.5 kb) and
one large inversion (944 kb). By using targeted-PCR in a set of 176 B. suis strains, including all biovars and
haplotypes, the indel was found to be specific of the reference strain ATCC 23445 and the large inversion
was shown to be an exclusive genomic marker of the Iberian clonal lineage of biovar 2.
... Restriction length polymorphism (RFLP) X X X Coimbra et al., 2000;Beutin et al., 2005;Moreno et al., 2006;Abbadi and Strockbine, 2007 Luminex-based suspension assay X Lin et al., 2011;Clotilde et al., 2015 Amplified fragment length polymorphisms (AFLP) X Hahm et al., 2003;Leung et al., 2004 Optical mapping X X Kotewicz et al., 2007Kotewicz et al., , 2008Miller, 2013 Ribotyping X Martin et al., 1996;Carson et al., 2001 Multilocus variable number tandem repeat analysis (MLVA) Pulsed-field gel electrophoresis X X Krause et al., 1996;Ribot et al., 2006 Multilocus sequence typing (MLST) X Eichhorn et al., 2015;Manges et al., 2015 High throughput real-time PCR X X X Bugarel et al., 2010aBugarel et al., ,b, 2011aDelannoy et al., 2013;Tseng et al., 2014 Multiplex PCR X X X Fratamico and DebRoy, 2010;Botkin et al., 2012;Doumith et al., 2012;Fratamico et al., 2014;Iguchi et al., 2015b Whole genome sequencing and SNP analysis X X X X Zhang et al., 2006;Eppinger et al., 2011;Norman et al., 2012Norman et al., , 2015Joensen et al., 2014Joensen et al., , 2015Griffing et al., 2015;DebRoy et al., 2016;Ison et al., 2016 Virulence gene profiles used in 20 separate multiplex PCR assays with each assay containing 6-9 primer pairs that amplified products of different sizes so that they could be distinguished. A high-throughput PCR method based on the GeneDisc R array targeted virulence genes and O-and H-type-specific genes for identification of STEC associated with severe illness (Bugarel et al., 2010b). ...
E. coli plays an important role as a member of the gut microbiota; however, pathogenic strains also exist, including various diarrheagenic E. coli pathotypes and extraintestinal pathogenic E. coli that cause illness outside of the GI-tract. E. coli have traditionally been serotyped using antisera against the ca. 186 O-antigens and 53 H-flagellar antigens. Phenotypic methods, including bacteriophage typing and O- and H- serotyping for differentiating and characterizing E. coli have been used for many years; however, these methods are generally time consuming and not always accurate. Advances in next generation sequencing technologies have made it possible to develop genetic-based subtyping and molecular serotyping methods for E. coli, which are more discriminatory compared to phenotypic typing methods. Furthermore, whole genome sequencing (WGS) of E. coli is replacing established subtyping methods such as pulsed-field gel electrophoresis (PFGE), providing a major advancement in the ability to investigate food-borne disease outbreaks and for trace-back to sources. A variety of sequence analysis tools and bioinformatic pipelines are being developed to analyze the vast amount of data generated by WGS and to obtain specific information such as O- and H-group determination and the presence of virulence genes and other genetic markers.
... In a recent study, WGM successfully discriminated between isolates belonging to livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) and identified the transmission and persistence of this genetically homogeneous MRSA variant, where current typing techniques failed (17). Although WGM has been very successful for LA-MRSA, the number of reports in which WGM was applied for molecular typing of other bacterial pathogens is very limited (18,19). ...
Legionella is the causative agent for Legionnaires' disease (LD) and is held responsible for several large outbreaks in the world. More than 90% of LD cases are caused by Legionella pneumophila and studies on the origin and transmission routes of this pathogen rely on adequate molecular characterization of isolates. Current typing of L. pneumophila mainly depends on sequence based typing (SBT). However, studies have shown that in some outbreak situations, SBT has not the sufficient discriminatory power to distinguish between related and non-related L. pneumophila isolates. In this study, we used a novel high-resolution typing technique, named whole genome mapping (WGM), to differentiate between epidemiologically related and non-related L. pneumophila isolates. Assessment of the method by various validation experiments showed highly reproducible results and WGM was able to confirm two well-documented Dutch L. pneumophila outbreaks. Comparison of whole genome maps of both outbreaks together with WGMs of epidemiologically non-related L. pneumophila isolates showed major differences between the maps and WGM yielded a higher discriminatory power than SBT. In conclusion, WGM can be a valuable alternative to perform outbreak investigations of L. pneumophila in real time since the turnaround time from culture to comparison of the L. pneumophila maps is less than 24 hours.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
... As a direct complement to DNA sequencing, optical mapping can produce a scaffold to facilitate easier sequence assembly, and as an indirect complement, optical mapping promises a variety of applications for which low resolution maps will suffice. For example, it can be used to quickly identify large-scale structural variations, including duplications, deletions, insertions, inversions, and translocations, which are increasingly being linked to heritable traits of phenotypic significance [3,11,12], and it allows for the rapid identification of bacterial species and strains which could represent an important step against the growing problem of antibiotic resistance [13][14][15][16][17]. ...
Optical mapping by direct visualization of individual DNA molecules, stretched in nanochannels with sequence-specific fluorescent labeling, represents a promising tool for disease diagnostics and genomics. An important challenge for this technique is thermal motion of the DNA as it undergoes imaging; this blurs fluorescent patterns along the DNA and results in information loss. Correcting for this effect (a process referred to as kymograph alignment) is a common preprocessing step in nanochannel-based optical mapping workflows, and we present here a highly efficient algorithm to accomplish this via pattern recognition. We compare our method with the one previous approach, and we find that our method is orders of magnitude faster while producing data of similar quality. We demonstrate proof of principle of our approach on experimental data consisting of melt mapped bacteriophage DNA.
... WGM offers a relatively economical and rapid method of assessing rates of mutation and recombination, and may be used to determine the taxonomic position and the evolutionary history of a particular strain. The value of WGM as a strain-typing tool in E. coli outbreaks has been documented, and the method has recently been applied to subtyping analysis [9][10][11]. Furthermore, WGM allows classical genetic sequencing to better target particular genomic regions and alterations in genome structure [12]. ...
... Optical maps were prepared using Argus (OpGen) according to previously described methods [22]. Protoplasts of ZW T were prepared by enzymatic digestion of the cell wall [11]. High molecular weight DNA was prepared by embedding protoplasts in low melting temperature agarose plugs, followed by treatment with lysing solution. ...
A novel Gram-negative bacterium, designated ZWT, was isolated from a soil sample of the Western Desert of China, and its phenotypic properties and phylogenetic position were investigated using a polyphasic approach. Growth occurred on TGY medium at 5-42°C with an optimum of 30°C, and at pH 7.0-11.0 with an optimum of pH 9.0. The predominant cellular fatty acids were summed feature 3 (C16:1ω7c/C16:1ω6c or C16:1ω6c/C16:1ω7c) (39.22%), iso-C15:0 (27.91%), iso-C17:0 3OH (15.21%), C16:0 (4.98%), iso-C15:0 3OH (3.03%), C16:0 3OH (5.39%) and C14:0 (1.74%). The major polar lipid of strain ZWT is phosphatidylethanolamine. The only menaquinone observed was MK-7. The GC content of the DNA of strain ZWT is 44.9 mol%. rDNA phylogeny, genome relatedness and chemotaxonomic characteristics all indicate that strain ZWT represents a novel species of the genus Sphingobacterium. We propose the name S. deserti sp. nov., with ZWT (= KCTC 32092T = ACCC 05744T) as the type strain. Whole genome optical mapping and next-generation sequencing was used to derive a finished genome sequence for strain ZWT, consisting of a circular chromosome of 4,615,818 bp in size. The genome of strain ZWT features 3,391 protein-encoding and 48 tRNA-encoding genes. Comparison of the predicted proteome of ZWT with those of other sphingobacteria identified 925 species-unique proteins that may contribute to the adaptation of ZWT to its native, extremely arid and inhospitable environment. As the first finished genome sequence for any Sphingobacterium, our work will serve as a useful reference for subsequent sequencing and mapping efforts for additional strains and species within this genus.
... Despite the name, even whole-genome sequencing typically relies on sequencing short DNA fragment libraries and therefore cannot detect certain large-scale rearrangements, including inversions and other events involving long sequence repeats. To complement sequencing data, whole-genome mapping (referred to here as "optical mapping") techniques have been developed that produce a highresolution, physically ordered restriction map of bacterial genomes (39)(40)(41). We produced optical chromosomal maps for clones isolated from a long-term evolution experiment (LTEE) with E. coli. ...
... Most deletions in the P. aeruginosa study occurred through illegitimate and homologous recombination events, but IS elements were not involved. In contrast with that dramatic reduction, an analysis of 11 natural isolates of E. coli O157:H7 found only very limited genome reductions of up to 3.7 kbp, or~0.1%, and slight increases in chromosome size were detected in several of those isolates (39). Note, however, that these estimates exclude the effect of new insertions of IS elements, which are not detected by optical mapping. ...
... Owing to the large number of genes in these inversions, it is difficult to predict their effects, if any, on the fitness or other phenotypes of the evolved cells. Chromosomal inversions have been found in natural isolates of many bacterial species, including E. coli (39), Staphylococcus aureus (16,40), Enterococcus faecium (60), Francisella tularensis (13), and Bacillus anthracis (61). Some of these inversions have been related to phenotypic changes, including colony morphology (16) and virulence (61), but in most cases their effects are unknown. ...
Unlabelled:
Large-scale rearrangements may be important in evolution because they can alter chromosome organization and gene expression in ways not possible through point mutations. In a long-term evolution experiment, twelve Escherichia coli populations have been propagated in a glucose-limited environment for over 25 years. We used whole-genome mapping (optical mapping) combined with genome sequencing and PCR analysis to identify the large-scale chromosomal rearrangements in clones from each population after 40,000 generations. A total of 110 rearrangement events were detected, including 82 deletions, 19 inversions, and 9 duplications, with lineages having between 5 and 20 events. In three populations, successive rearrangements impacted particular regions. In five populations, rearrangements affected over a third of the chromosome. Most rearrangements involved recombination between insertion sequence (IS) elements, illustrating their importance in mediating genome plasticity. Two lines of evidence suggest that at least some of these rearrangements conferred higher fitness. First, parallel changes were observed across the independent populations, with ~65% of the rearrangements affecting the same loci in at least two populations. For example, the ribose-utilization operon and the manB-cpsG region were deleted in 12 and 10 populations, respectively, suggesting positive selection, and this inference was previously confirmed for the former case. Second, optical maps from clones sampled over time from one population showed that most rearrangements occurred early in the experiment, when fitness was increasing most rapidly. However, some rearrangements likely occur at high frequency and may have simply hitchhiked to fixation. In any case, large-scale rearrangements clearly influenced genomic evolution in these populations.
Importance:
Bacterial chromosomes are dynamic structures shaped by long histories of evolution. Among genomic changes, large-scale DNA rearrangements can have important effects on the presence, order, and expression of genes. Whole-genome sequencing that relies on short DNA reads cannot identify all large-scale rearrangements. Therefore, deciphering changes in the overall organization of genomes requires alternative methods, such as optical mapping. We analyzed the longest-running microbial evolution experiment (more than 25 years of evolution in the laboratory) by optical mapping, genome sequencing, and PCR analyses. We found multiple large genome rearrangements in all 12 independently evolving populations. In most cases, it is unclear whether these changes were beneficial themselves or, alternatively, hitchhiked to fixation with other beneficial mutations. In any case, many genome rearrangements accumulated over decades of evolution, providing these populations with genetic plasticity reminiscent of that observed in some pathogenic bacteria.
