The Locus of Heat Resistance Confers Resistance to Chlorine and Other Oxidizing Chemicals in Escherichia coli

Abstract

Some chlorine-resistant Escherichia coli isolates harbor the locus of heat resistance (LHR), a genomic island conferring heat resistance. In this study, the protective effect of the LHR for cells challenged by chlorine and oxidative stress was quantified. Cloning of the LHR protected against NaClO (32 mM; 5 min), H2O2 (120 mM; 5 min), and peroxyacetic acid (105 mg/liter; 5 min) but not against 5.8 mM KIO4, 10 mM acrolein, or 75 mg/liter allyl isothiocyanate. The lethality of oxidizing treatments for LHR-negative strains of E. coli was about 2 log10 CFU/ml higher than that for LHR-positive strains of E. coli. The oxidation of cytoplasmic proteins and membrane lipids was quantified with the fusion probe roGFP2-Orp1 and the fluorescent probe BODIPY581/591, respectively. The fragment of the LHR coding for heat shock proteins protected cytoplasmic proteins but not membrane lipids against oxidation. The middle fragment of the LHR protected against the oxidation of membrane lipids but not of cytoplasmic proteins. The addition of H2O2, NaClO, and peroxyacetic acid also induced green fluorescent protein (GFP) expression in the oxidation-sensitive reporter strain E. coli O104:H4 Δstx2::gfp::amp. Cloning of pLHR reduced phage induction in E. coli O104:H4 Δstx2::gfp::amp after treatment with oxidizing chemicals. Screening of 160 strains of Shiga toxin-producing E. coli (STEC) revealed that none of them harbors the LHR, additionally suggesting that the LHR and Stx prophages are mutually exclusive. Taking our findings together, the contribution of the LHR to resistance to chlorine and oxidative stress is based on the protection of multiple cellular targets by different proteins encoded by the genetic island. IMPORTANCE Chlorine treatments are used in water and wastewater sanitation; the resistance of Escherichia coli to chlorine is thus of concern to public health. We show that a genetic island termed the locus of heat resistance (LHR) protects E. coli not only against heat but also against chlorine and other oxidizing chemicals, adding to our knowledge of the tools used by E. coli to resist stress. Specific detection of the oxidation of different cellular targets in combination with the cloning of fragments of the LHR provided insight into mechanisms of protection and demonstrated that different fragments of the LHR protect different cellular targets. In E. coli, the presence of the LHR virtually always excluded other virulence factors. It is tempting to speculate that the LHR is maintained by strains of E. coli with an environmental lifestyle but is excluded by pathogenic strains that adapted to interact with vertebrate hosts.

Heat and Pressure Resistance in Escherichia coli Relates to Protein Folding and Aggregation

“…Their high expression conforms to the observation that cloning of these proteins increases heat resistance of Pseudomonas, Klebsiella, and Cronobacter (Bojer et al, 2010;Gajdosova et al, 2011;Lee et al, 2018), and that their deletion from the LHR decreases the level of LHR-mediated heat resistance . The high level of expression of multiple LHR-encoded proteins also supports the conclusion that LHR-mediated heat resistance is a multifactorial phenotype Wang et al, 2020). High level protein expression in E. coli imposes a substantial metabolic burden on the cell and may lead to metabolic alterations and a reduced growth rate (Pasini et al, 2016).…”

“…Peroxiredoxin OsmC mitigates oxidative stress caused by organic hydroperoxides by detoxification with cysteine thiol groups (Lesniak et al, 2003). The differential composition of inclusion bodies in E. coli MG1655 ibpA-yfp (pLHR) and (pRK767) suggests that the LHR reduces aggregation of metabolic proteins and stressrelated proteins, and conforms to the reduced oxidation of cytoplasmic proteins in strains of E. coli carrying the LHR (Wang et al, 2020).…”

“…It is flanked by mobile genetic elements (Mercer et al, 2015) and is transmitted by lateral gene transfer (Boll et al, 2017). The LHR is present in approximately 2% of strains of E. coli but is only rarely present in Shiga-toxin producing strains of E. coli (Mercer et al, 2015;Ma and Chui, 2017;Wang et al, 2020). Several anthropogenic environments select for LHR-positive Enterobacteriaceae.…”

“…A high frequency of heat resistant Enterobacteriaceae was reported in beef (Dlusskaya et al, 2011;Mercer et al, 2015;), in raw milk cheese after thermization at 60 • C, and in Daqu, a solid state cereal fermentation that reaches temperatures of up to 60 • C (Marti et al, 2016;Boll et al, 2017;Wang et al, 2018). The high frequency of LHR-positive strains of E. coli in chlorine-treated waste-water (Zhi et al, 2016) relates to the contribution of the LHR to chlorine resistance (Wang et al, 2020). The presence of the transmissible LHR in pathogenic organisms associated with the food and water supply may present significant challenges to public health.…”

“…These include genes coding for YfdX family proteins with unknown function, and genes which encode for functions predicted to relate to intracellular oxidative stress and accumulation of compatible solutes . Collectively, LHR-encoded proteins prevent oxidation of cytoplasmic proteins, and oxidation of membrane lipids in E. coli (Wang et al, 2020).…”