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Escherichia coli O157:H7 strains harbor at least three distinct sequence types of Shiga toxin 2a-converting phages.

Yin S, Rusconi B, Sanjar F, Goswami K, Xiaoli L, Eppinger M, Dudley EG - BMC Genomics (2015)

Bottom Line: The PST2 cluster, identified in two clade 8 strains, was related to stx2a-converting phages previously identified in non-O157 Shiga-toxin producing E. coli (STEC) strains associated with a high incidence of HUS.Diversification within a phage type is mainly driven by IS629 and by a small number of SNPs.Polymorphisms between phage genomes may help explain differences in Stx2a production between strains, however our data indicates that genes encoded external to the phage affect toxin production as well.

View Article: PubMed Central - PubMed

Affiliation: Department of Food Science, The Pennsylvania State University, University Park, PA, 16802, USA.

ABSTRACT

Background: Shiga toxin-producing Escherichia coli O157:H7 is a foodborne pathogen that causes severe human diseases including hemolytic uremic syndrome (HUS). The virulence factor that mediates HUS, Shiga toxin (Stx), is encoded within the genome of a lambdoid prophage. Although draft sequences are publicly available for a large number of E. coli O157:H7 strains, the high sequence similarity of stx-converting bacteriophages with other lambdoid prophages poses challenges to accurately assess the organization and plasticity among stx-converting phages due to assembly difficulties.

Methods: To further explore genome plasticity of stx-converting prophages, we enriched phage DNA from 45 ciprofloxacin-induced cultures for subsequent 454 pyrosequencing to facilitate assembly of the complete phage genomes. In total, 22 stx2a-converting phage genomes were closed.

Results: Comparison of the genomes distinguished nine distinct phage sequence types (PSTs) delineated by variation in obtained sequences, such as single nucleotide polymorphisms (SNPs) and insertion sequence element prevalence and location. These nine PSTs formed three distinct clusters, designated as PST1, PST2 and PST3. The PST2 cluster, identified in two clade 8 strains, was related to stx2a-converting phages previously identified in non-O157 Shiga-toxin producing E. coli (STEC) strains associated with a high incidence of HUS. The PST1 cluster contained phages related to those from E. coli O157:H7 strain Sakai (lineage I, clade 1), and PST3 contained a single phage that was distinct from the rest but most related to the phage from E. coli O157:H7 strain EC4115 (lineage I/II, clade 8). Five strains carried identical stx2a-converting phages (PST1-1) integrated at the same chromosomal locus, but these strains produced different levels of Stx2.

Conclusion: The stx2a-converting phages of E. coli O157:H7 can be categorized into at least three phage types. Diversification within a phage type is mainly driven by IS629 and by a small number of SNPs. Polymorphisms between phage genomes may help explain differences in Stx2a production between strains, however our data indicates that genes encoded external to the phage affect toxin production as well.

No MeSH data available.


Related in: MedlinePlus

IS629 is responsible for most diversity observed within (a) PST1 and (b) PST2 clusters. BLAST comparisons of phage genomes were visualized using Easyfig [77]. Blue shaded regions connect homologous sequences and orange shaded regions connect reverse complemented homologous sequences. Arrows represent ORFs. Dark pink and green arrows designate the location of stx2a and IS629, respectively
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Fig2: IS629 is responsible for most diversity observed within (a) PST1 and (b) PST2 clusters. BLAST comparisons of phage genomes were visualized using Easyfig [77]. Blue shaded regions connect homologous sequences and orange shaded regions connect reverse complemented homologous sequences. Arrows represent ORFs. Dark pink and green arrows designate the location of stx2a and IS629, respectively

Mentions: Predicted phage proteomes were compared with Large Scale Blast Score Ratio (LS-BSR) [31]. Applying hierarchical average linkage clustering algorithm, which is part of MeV v.4.8 [32], on this proteomic matrix (Pearson Correlation) [33] identified three distinct phage clusters, designated as Phage Sequence type 1 (PST1), PST2 and PST3 (Fig. 1). These three clusters were also apparent within a whole genome alignment (Additional file 2: Figure S1). The 19 genomes classified as PST1 were further divided into six subtypes (PST1-1 to PST1-6) by assessing IS629 copy number and insertion sites (Fig. 2a). PST1-1 contained 13 phage genomes that each have IS629 inserted at the same position (nucleotides 24,350–25,659), splitting a predicted phage protein into two predicted proteins (as an example, those with protein_id AKI86024 and AKI86027 in the PA4 phage GenBank entry, KP682372). This insertion sequence is located downstream of the stx2 gene. Genome sequences of the 62 kb PST1-1 phage differed at most by five SNPs, and these SNP level differences were observed within the predicted OR2 operator, portal protein, tail fiber, and several hypothetical proteins (Additional file 3: Table S2). All PST1-1 isolates carry the stx2a-converting prophage found in the prototypical strain E. coli O157:H7 Sakai [30]. The two phages in PST1-2 were 100 % identical on the nucleotide level, and PSTs 1–3, −4, −5, and −6 each represent single phage found in strains PA27, PA36, PA45, and PA52, respectively.Fig. 1


Escherichia coli O157:H7 strains harbor at least three distinct sequence types of Shiga toxin 2a-converting phages.

Yin S, Rusconi B, Sanjar F, Goswami K, Xiaoli L, Eppinger M, Dudley EG - BMC Genomics (2015)

IS629 is responsible for most diversity observed within (a) PST1 and (b) PST2 clusters. BLAST comparisons of phage genomes were visualized using Easyfig [77]. Blue shaded regions connect homologous sequences and orange shaded regions connect reverse complemented homologous sequences. Arrows represent ORFs. Dark pink and green arrows designate the location of stx2a and IS629, respectively
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4587872&req=5

Fig2: IS629 is responsible for most diversity observed within (a) PST1 and (b) PST2 clusters. BLAST comparisons of phage genomes were visualized using Easyfig [77]. Blue shaded regions connect homologous sequences and orange shaded regions connect reverse complemented homologous sequences. Arrows represent ORFs. Dark pink and green arrows designate the location of stx2a and IS629, respectively
Mentions: Predicted phage proteomes were compared with Large Scale Blast Score Ratio (LS-BSR) [31]. Applying hierarchical average linkage clustering algorithm, which is part of MeV v.4.8 [32], on this proteomic matrix (Pearson Correlation) [33] identified three distinct phage clusters, designated as Phage Sequence type 1 (PST1), PST2 and PST3 (Fig. 1). These three clusters were also apparent within a whole genome alignment (Additional file 2: Figure S1). The 19 genomes classified as PST1 were further divided into six subtypes (PST1-1 to PST1-6) by assessing IS629 copy number and insertion sites (Fig. 2a). PST1-1 contained 13 phage genomes that each have IS629 inserted at the same position (nucleotides 24,350–25,659), splitting a predicted phage protein into two predicted proteins (as an example, those with protein_id AKI86024 and AKI86027 in the PA4 phage GenBank entry, KP682372). This insertion sequence is located downstream of the stx2 gene. Genome sequences of the 62 kb PST1-1 phage differed at most by five SNPs, and these SNP level differences were observed within the predicted OR2 operator, portal protein, tail fiber, and several hypothetical proteins (Additional file 3: Table S2). All PST1-1 isolates carry the stx2a-converting prophage found in the prototypical strain E. coli O157:H7 Sakai [30]. The two phages in PST1-2 were 100 % identical on the nucleotide level, and PSTs 1–3, −4, −5, and −6 each represent single phage found in strains PA27, PA36, PA45, and PA52, respectively.Fig. 1

Bottom Line: The PST2 cluster, identified in two clade 8 strains, was related to stx2a-converting phages previously identified in non-O157 Shiga-toxin producing E. coli (STEC) strains associated with a high incidence of HUS.Diversification within a phage type is mainly driven by IS629 and by a small number of SNPs.Polymorphisms between phage genomes may help explain differences in Stx2a production between strains, however our data indicates that genes encoded external to the phage affect toxin production as well.

View Article: PubMed Central - PubMed

Affiliation: Department of Food Science, The Pennsylvania State University, University Park, PA, 16802, USA.

ABSTRACT

Background: Shiga toxin-producing Escherichia coli O157:H7 is a foodborne pathogen that causes severe human diseases including hemolytic uremic syndrome (HUS). The virulence factor that mediates HUS, Shiga toxin (Stx), is encoded within the genome of a lambdoid prophage. Although draft sequences are publicly available for a large number of E. coli O157:H7 strains, the high sequence similarity of stx-converting bacteriophages with other lambdoid prophages poses challenges to accurately assess the organization and plasticity among stx-converting phages due to assembly difficulties.

Methods: To further explore genome plasticity of stx-converting prophages, we enriched phage DNA from 45 ciprofloxacin-induced cultures for subsequent 454 pyrosequencing to facilitate assembly of the complete phage genomes. In total, 22 stx2a-converting phage genomes were closed.

Results: Comparison of the genomes distinguished nine distinct phage sequence types (PSTs) delineated by variation in obtained sequences, such as single nucleotide polymorphisms (SNPs) and insertion sequence element prevalence and location. These nine PSTs formed three distinct clusters, designated as PST1, PST2 and PST3. The PST2 cluster, identified in two clade 8 strains, was related to stx2a-converting phages previously identified in non-O157 Shiga-toxin producing E. coli (STEC) strains associated with a high incidence of HUS. The PST1 cluster contained phages related to those from E. coli O157:H7 strain Sakai (lineage I, clade 1), and PST3 contained a single phage that was distinct from the rest but most related to the phage from E. coli O157:H7 strain EC4115 (lineage I/II, clade 8). Five strains carried identical stx2a-converting phages (PST1-1) integrated at the same chromosomal locus, but these strains produced different levels of Stx2.

Conclusion: The stx2a-converting phages of E. coli O157:H7 can be categorized into at least three phage types. Diversification within a phage type is mainly driven by IS629 and by a small number of SNPs. Polymorphisms between phage genomes may help explain differences in Stx2a production between strains, however our data indicates that genes encoded external to the phage affect toxin production as well.

No MeSH data available.


Related in: MedlinePlus