Characterizing Escherichia coli's transcriptional response to different styrene exposure modes reveals novel toxicity and tolerance insights

Abstract

The global transcriptional response of Escherichia coli to styrene and potential influence of exposure source was determined by performing RNA sequencing (RNA-seq) analysis on both styrene-producing and styrene-exposed cells. In both cases, styrene exposure appears to cause both cell envelope and DNA damage, to which cells respond by down-regulating key genes/pathways involved in DNA replication, protein production, and cell wall biogenesis. Among the most significantly up-regulated genes were those involved with phage shock protein response (e.g. pspABCDE/G), general stress regulators (e.g. marA, rpoH), and membrane-altering genes (notably, bhsA, ompR, ldtC), whereas efflux transporters were, surprisingly, unaffected. Subsequent studies with styrene addition demonstrate how strains lacking ompR [involved in controlling outer membrane (OM) composition/osmoregulation] or any of tolQ, tolA, or tolR (involved in OM constriction) each displayed over 40% reduced growth relative to wild-type. Conversely, despite reducing basal fitness, overexpression of plsX (involved in phospholipid biosynthesis) led to 70% greater growth when styrene exposed. These collective differences point to the likely importance of OM properties in controlling native styrene tolerance. Overall, the collective behaviours suggest that, regardless of source, prolonged exposure to inhibitory styrene levels causes cells to shift from'growth mode' to 'survival mode', redistributing cellular resources to fuel native tolerance mechanisms.

Keywords: RNA sequencing; Styrene; Toxicity.

Fig. 1.

(a) Total differentially expressed (DE) genes identified for styrene addition (A) and production (P), both relative to a no styrene control (C). Upper (red) and lower (blue) bars, along with associated inset values, show total DE genes up-regulated or down-regulated, respectively. (b) A Venn diagram (created with BioVenn; Hulsen et al., 2008) comparing DE genes unique to A (maroon, left) versus unique to P (gold, right), as well as DE genes common to both conditions (orange, middle). (c) For DE genes common to both A and P, the number of genes down-regulated under both conditions (blue), up-regulated under both conditions (red), and oppositely DE (green) are compared. Note: for (a)–(c), only DE genes with FDR adjusted p-value < .1 are included. Volcano plots of all DE genes (down-regulated, blue; up-regulated, red; no significant differential expression, black) identified in the case of (D) styrene addition (A) or (E) styrene production (P), with a significance threshold of FDR adjusted p-value < .1 (green dotted line).

Fig. 2.

Fold change of 3 986 E. coli genes for styrene production (P) versus styrene addition (A), both relative to the no styrene control (C). Each circle represents a different gene while the solid line represents y = x. The lower graph represents an inset of the complete dataset, the region of which is depicted via a dashed box in the upper graph.

Fig. 3.

Comparison of DE genes identified for styrene addition (A) and styrene production (P) across a selection of GO terms of notable significance and/or particular interest to this study. For each GO term, the total number of associated E. coli genes is listed in parenthesis. The y-axis indicates the percentage of total genes in each GO term that were significantly down-regulated (lower, blue) or up-regulated (upper, red) for A (light red or light blue with stripes) and P (solid red or solid blue). Superscripts ‘A’ and/or ‘P’ indicate that the entire GO term was significantly over-represented (p-value < .05) for that condition, as compared to the E. coli genome.

Fig. 4.

Comparison of DE genes identified for styrene addition (A) and styrene production (P) across a selection of KEGG pathways of notable significance and/or particular interest to this study. For each KEGG pathway, the total number of associated E. coli genes is listed in parenthesis. The y-axis indicates the percentage of total genes in each KEGG pathway that were significantly down-regulated (lower, blue) or up-regulated (upper, red) for A (light red or blue with stripes) and P (solid dark red or blue). Superscript ‘A’ and/or ‘P’ indicates that the entire KEGG pathway was significantly over-represented (p-value < .05) for that condition, as compared to the E. coli genome.

Fig. 5.

(a) Relative growth of single gene deletion mutants of interest comparing the final OD600 of deletion mutants following 6 hr of exposure to 100 mg/l styrene relative to no styrene control (green, solid) as well as the final OD600 of deletion mutants and wild-type E. coli BW25113 both with no styrene added (blue, striped). Error bars reported at one standard deviation (n = 4 for single gene deletion mutants, n = 8 for BW25113). (b) Relative growth of single gene overexpression strains comparing the final OD600 of strains induced with 10 µM IPTG following 6 hr of exposure to 100 mg/l styrene relative to no styrene control (green, solid) as well as the final OD600 of the single gene overexpression strains and wild-type E. coli BW25113 pCA24N control with no styrene added (blue, striped). Error bars reported at one standard deviation (n = 3). (c) Same conditions as (b) except using 100 µM IPTG for induction. Dashed lines indicate relative growth of 100% (i.e. no growth difference in the presence versus absence of styrene or no growth difference between strain of interest and the respective control both with no styrene). * indicates p < .001 and ** indicates p < .005 for two-tailed Student's t-test when compared to the respective control strain (BW25113 or BW25113 pCA24N).

Fig. 6.

(a) Relative growth of wild-type E. coli BW25113 (control; solid, light blue) and the BW25113 ΔompR mutant (red, striped). (b) Relative growth of BW25113 harbouring pCA24N plasmid (control; solid, light blue) BW25113 harbouring appropriate plasmids used to overexpress ompF (10 µM IPTG; diagonal striped, beige), plsX (10 and 100 µM IPTG; light green, checkered and dark green, dotted, respectively) and tolA (10 µM IPTG; horizontal striped, pink). For (a) and (b), OD600 were measured following 6 hr of exposure to 75, 125, or 175 mg/l styrene, each relative to a no styrene control. Error bars reported at one standard deviation (n = 3). Dashed lines indicate relative growth of 100% (i.e. no growth difference in the presence versus absence of styrene). * indicates p < .001 and ** indicates p < .005 for two-tailed Student's t-test when compared to the respective control strain (BW25113 or BW25113 pCA24N) at the equivalent concentrations of styrene.