Article Figures & Data
Figures
- FIG 1
Phylogenomic analysis of representative EPEC isolates. The genome sequences of representative EPEC isolates were compared with those of a reference collection of diverse E. coli and Shigella isolates that had been sequenced previously and are available in the public domain. The genomes were aligned using Mugsy (95) as previously described (31, 94). A 1.9-Mb aligned region was used to generate a maximum-likelihood phylogeny with 100 bootstrap replicates using RAxML v.7.2.8 (97), and the results were visualized using FigTree v.1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/ ). The representative EPEC isolates that were analyzed using RNA-Seq are indicated in bold.
- FIG 2
Principal-component analysis and hierarchical cluster analysis of the RNA-Seq samples examined in this study. (A) Principal-component analysis of the expression of gene clusters identified in the EPEC isolates, comparing all RNA-Seq samples analyzed. The first (PC1) and second (PC2) principal components were visualized in a scatterplot to demonstrate the clustering of the strains by gene content and gene expression. The comparative matrix is composed of the maximum normalized expression values for each gene cluster in each of the isolates and under each of the conditions examined for the gene clusters that were present among all isolates. The first component contains 36.75% of the variation, and the second component is responsible for 12.7% of the variation. The samples are colored by isolate, and symbols represent LB or DMEM as indicated in the legend. (B) A heatmap with clustering analysis of the expression values was constructed for the 674 LS-BSR gene clusters that were present in all of the EPEC isolates and had the greatest standard deviations of expression values. The normalized gene expression values were used to compute the standard deviation for each LS-BSR gene cluster across all samples. The heatmap was constructed using the R package gplots v2.11.0. A red label at the top of the heatmap designates the DMEM samples, while a blue label designates the LB samples.
- FIG 3
Comparison of the global transcriptomes of nine EPEC isolates. A circular plot of the log2-fold-change (LFC) values for genes that exhibited significant differential expression during exponential growth in DMEM compared to LB is shown. The outermost track contains all of the significant LFC values for each of the indicated nine EPEC isolates. The inner tracks are numbered to correspond to the same number of each EPEC isolate in the outermost track. The phylogroup that each EPEC isolate belongs to is indicated in parentheses following the isolate designation. For example, 5-B171 (B1) indicates that data tracks containing B171 data are labeled track 5 in all of the comparisons, while B171 is the isolate designation and B1 the phylogroup designation. The inner tracks contain the LFC values of genes of another of the EPEC isolates belonging to the same LS-BSR gene cluster as the genes in the outermost reference track. The genes that were not identified in the other EPEC isolates or did not exhibit significant differential expression are absent from the inner tracks.
- FIG 4
Comparison of the global transcriptomes of multiple EPEC isolates of the EPEC8 phylogenomic lineage. (A) Circular plot of the log2-fold-change (LFC) values for genes that exhibited significant differential expression during exponential growth in DMEM compared to LB. The outermost track contains all of the significant LFC values for one of the three EPEC8 isolates. The inner tracks contain the LFC values of genes of another of the EPEC8 isolates belonging to the same LS-BSR gene cluster as the genes in the outermost reference track. The genes that were not identified in the other EPEC8 isolates and/or did not exhibit significant differential expression are absent from the inner tracks. The number of genes that had significant differential expression is indicated in parentheses next to each isolate name. (B) The number of genes that were highly conserved (LS-BSR ≥ 0.8) in all four of the EPEC isolates is indicated in the center. The number of genes that were identified with significant similarity (LS-BSR ≥ 0.8) that also exhibited significant differential expression in two or three EPEC isolates is also designated. The number of isolate-specific genes indicates the genes that were identified with significant similarity in one EPEC isolate and that were divergent or absent from the other two EPEC8 isolates. (C) Venn diagram showing the number of genes differentially expressed for each of the EPEC8 isolates analyzed in this study grown to an OD600 of 0.5 in DMEM compared to LB. The number of core genes that were highly conserved (LS-BSR ≥ 0.8) in all three of the EPEC8 isolates that also exhibited significant differential expression in all of the EPEC8 isolates is indicated in the center. There were no genes that were present with significant similarity and also differentially expressed in only two of the three EPEC8 isolates. The number of isolate-specific genes indicates those genes that exhibited significant similarity (LS-BSR ≥ 0.8) that were divergent or absent (LS-BSR < 0.8) from the other two EPEC8 isolates and also exhibited significant differential expression during growth to an OD600 of 0.5 in DMEM compared to LB.
- FIG 5
Differential expression analysis of genes carried by the LEE and BFP regions in each of the representative EPEC isolates analyzed. (A) Diagram of the genetic structure and results of differential expression analysis of LEE genes for the nine representative EPEC isolates examined (E2348/69, B171, C581-05, 401588, 401140, 402290, 302053, 100329, and E110019) during exponential growth (OD600 = 0.5) in DMEM compared to LB. The values in the heatmap are the significant log2-fold-change (LFC) values for LEE genes. (B) Diagram of the gene organization and the RNA-Seq LFC values of genes carried by the BFP operon of the eight EPEC isolates that contained BFP (E2348/69, B171, C581-05, 401588, 401140, 402290, 302053, and 100329). The expression values in the heatmap are significant LFC values for BFP genes.
- FIG 6
Differential expression analysis of known virulence genes of EPEC. The phylogroup that each EPEC isolate belongs to is indicated in parentheses. (A) Heatmap of LS-BSR values indicating the presence or absence of known EPEC virulence genes in the genomes of each of the EPEC isolates analyzed. Genes present with significant similarity are indicated by yellow, genes with divergent similarity are indicated by black, and genes that are absent are indicated by blue. (B) Heatmap of the log2-fold-change (LFC) values for known virulence genes of EPEC that exhibited significant differential expression during exponential growth in DMEM compared to LB. The color gradient indicates decreased expression (green) or increased expression (red) of the virulence genes, while white indicates a gene that either was not present in the isolate or did not exhibit significant differential expression.
Tables
- TABLE 1
Number of shared or unique genes identified using LS-BSR analysisa
Isolate ID Location of
isolationDate of
isolationPhylogenomic
lineagebPhylogroupc No. of isolate-specific
gene clustersdE2348/69 England 1969 EPEC1 B2 206 B171 United States 1983 EPEC2 B1 131 C581-05 Africa NK EPEC4 B2 141 401140 Kenya 2008 EPEC5 A 90 402290 Kenya 2009 EPEC7 B1 169 401588 Kenya 2008 EPEC8 B2 212 302053 Mozambique 2009 EPEC9 B2 53 100329 The Gambia 2008 EPEC10 A 62 E110019 Finland 1987 None B1 164 ↵a ID, identifier; NK, date of isolation not known.
↵b The total number of core gene clusters (LS-BSR ≥ 0.8) in all EPEC isolates.
↵c The numbers of gene clusters with significant similarity (LS-BSR ≥ 0.8) in all genomes of one phylogroup that were divergent (LS-BSR < 0.8, ≥ 0.4) or absent (LS-BSR < 0.4) from genomes of other phylogroups were 44 (phylogroup A), 62 (phylogroup B1), and 128 (phylogroup B2).
↵d The isolate-specific genes are those identified in one genome with an LS-BSR ≥ 0.8 and in the other genomes with an LS-BSR < 0.4.
- TABLE 2
Number of genes that were differentially expressed in the EPEC isolates examined in this study
Isolate ID Phylogenomic
lineageaPhylogroupa LFC ≥ 2b LFC ≤ −2b Total DE
genescNo. of DE
genes of
core
clustersdNo. of DE
genes of
phylogroup-
specific clusterseNo. of
DE genes
of isolate-
specific clustersfTotal DE
sRNAsgE2348/69 EPEC1 B2 180 251 431 253 18 10 6 B171 EPEC2 B1 220 255 475 249 2 4 10 C581-05 EPEC4 B2 162 235 397 291 12 1 17 401140 EPEC5 A 145 189 334 242 1 3 22 402290 EPEC7 B1 243 268 511 354 1 2 7 401588 EPEC8 B2 267 134 401 253 11 4 12 302053 EPEC9 B2 228 315 543 392 15 0 21 100329 EPEC10 A 278 294 572 394 1 2 30 E110019 None B1 172 246 418 280 0 7 9 ↵a The phylogenomic lineage and phylogroup are those that have been previously described (Hazen et al. [32], Jaureguy et al. [41], Tenaillon et al. [42]).
↵b LFC, log2-fold change of the genes that exhibited significant (LFC ≥ 2 or ≤ −2 and FDR ≤ 0.05) differential expression (DE).
↵c The total number of genes that exhibited significant (LFC ≥ 2 or ≤ −2 and FDR ≤ 0.05) DE.
↵d The total number of core gene clusters (LS-BSR ≥ 0.8 in all genomes) was 2,989.
↵e The number of clusters in all EPEC genomes of one phylogroup that were divergent or absent (LS-BSR < 0.8) from EPEC genomes of the other phylogroups were 44 (phylogroup A), 62 (phylogroup B1), and 128 (phylogroup B2).
↵f The isolate-specific genes are those that were in one genome with an LS-BSR ≥ 0.8 and in the other genomes with an LS-BSR < 0.4.
↵g The total number of sRNA that were previously investigated in E. coli by Raghavan et al. (60) and exhibited significant (LFC ≥ 2 or ≤ −2 and FDR ≤ 0.05) DE in each of the nine EPEC isolates.
TABLE S1
Primers used in this study. Download TABLE S1, PDF file, 0.1 MB.
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TABLE S2
Characteristics of the RNA-Seq generated in this study. Download TABLE S2, PDF file, 0.05 MB.
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TABLE S3
Differential expression of core, phylogroup-specific, and isolate-specific genes. Download TABLE S3, PDF file, 0.1 MB.
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FIG S1
qRT-PCR verification of RNA-Seq expression trends. The expression trends of selected genes that were determined by the use of RNA-Seq to have significant differential expression levels were analyzed using quantitative reverse transcription-PCR (qRT-PCR). The values represent the log2 of the relative fold differences (2−ΔΔCT) between the results determined with DMEM samples and those determined with LB samples for the nine EPEC reference isolates (Table 1). The fold change value was calculated using two biological replicates for each gene and three technical replicates for each medium type. The error bars denote the standard deviations of the ΔΔCT values. The expression trends observed using qRT-PCR were consistent with those determined using RNA-Seq. Data are not shown for the isolates that did not contain a particular gene(s) (cdtA, hmuV chuU, and shuA chuA). Download FIG S1, PDF file, 0.1 MB.
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TABLE S4
Differential expression of core, phylogroup-specific, and unique genes from EPEC8 isolates. Download TABLE S4, PDF file, 0.1 MB.
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DATA SET S1
Fasta file of nucleotide sequences of each LS-BSR gene cluster. Download DATA SET S1, PDF file, 5.6 MB.
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DATA SET S2
Differentially expressed genes of all the isolates (each represented on a different sheet). Download DATA SET S2, XLSX file, 0.7 MB.
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TABLE S5
Differential expression of select nonvirulence genes. Download TABLE S5, PDF file, 0.1 MB.
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TABLE S6
Differential expression of the LS-BSR gene clusters that are only in EPEC and encode proteins with predicted secreted or surface-associated domains. Download TABLE S6, PDF file, 0.05 MB.
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TABLE S7
Differential expression of known E. coli sRNAs. Download TABLE S7, PDF file, 0.05 MB.
Copyright © 2017 Hazen et al.
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