Escherichia coli O157:H7 converts plant-derived choline to glycine betaine for osmoprotection during pre- and post-harvest colonization of injured lettuce leaves

Authors: Scott, Russell; Thilmony, Roger; Harden, Leslie - Les; Zhou, Yaguang; Brandl, Maria

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/23/2017
Publication Date: 12/8/2017
Citation: Scott, R.A., Thilmony, R.L., Harden, L.A., Zhou, Y., Brandl, M. 2017. Escherichia coli O157:H7 converts plant-derived choline to glycine betaine for osmoprotection during pre- and post-harvest colonization of injured lettuce leaves. Frontiers in Microbiology. 8:2436. https://doi.org/10.3389/fmicb.2017.02436.
DOI: https://doi.org/10.3389/fmicb.2017.02436

Interpretive Summary: The human pathogen E. coli O157:H7 multiplies to high densities in damaged plant tissue. A lettuce-associated outbreak strain of E. coli O157:H7, strain TW14588 (EcO157) responds to conditions in lettuce lysates and shredded lettuce in Modified Atmosphere Packaging via upregulation of the betTIBA operons, which functions in the synthesis of the osmoprotectant glycine betaine from choline as a substrate. Mass spectrometry analysis revealed abundant choline in lettuce leaf lysates and wound washes. We present several lines of evidence to show that EcO157 experiences osmotic stress in damaged lettuce leaf tissue: 1) Hypertonic lettuce lysates inhibited the wild type strain significantly less than a BetTIBA deletion mutant, which correlated with accumulation of glycine betaine in the wild type but not in the betTIBA mutant cells, indicating that lettuce-derived choline was available for import and conversion to glycine betaine as an osmoprotectant in EcO157; 2) The population size of a BetTIBA mutant increased more slowly in shredded lettuce than that of the wild type strain, and a double BetTIBA-OtsBA mutant was competitively less fit than the wild type strain in shredded lettuce and in leaf wounds of whole lettuce plants; and 3) Mass spectrometry data showed that D9-choline was converted to D9-glycine betaine in the wild type but not in the BetTIBA mutant during growth in cut lettuce, providing further evidence of the role of the glycine betaine biosynthetic pathway in the osmotic stress response of EcO157 in wounded leaf tissue. We propose a model in which EcO157 exploits choline released from plant cells for osmoprotection and enhanced colonization of injured leaf tissue.

Technical Abstract: The opportunistic colonization of damaged plant tissue by human enteric pathogens may contribute to the occurrence of outbreaks of foodborne illness linked to produce. E. coli O157:H7 (EcO157) responds to physicochemical stresses in cut lettuce and lettuce lysates by upregulation of several stress response pathways. We investigated the tolerance of EcO157 to osmotic stress imposed by the leakage of osmolytes from injured lettuce leaf tissue. An abundant pool of choline, but sparse quantities of glycine betaine and proline were detected by MS analysis of these bacterial osmoprotectants in lettuce leaf lysates and wound washes. An EcO157 mutant in betTIBA, which are required for glycine betaine biosynthesis from imported choline, achieved population sizes two-fold lower than the parental strain (P<0.05) on cut lettuce in Modified Atmosphere Packaging (MAP). To demonstrate the utilization of available choline by EcO157 for osmoadaptation in injured leaf tissue, deuterated (D-9) choline was introduced to wound sites in MAP lettuce; LC-MS analysis revealed the conversion of D9-choline to D-9 glycine betaine in the parental strain, but no significant amounts were observed in the BetTIBA mutant. The EcO157 BetTIBA-OtsBA double mutant, which is additionally deficient in de novo synthesis of the osmoprotectant trehalose, was significantly less fit than the parental strain after their co-inoculation onto injured lettuce leaves and MAP cut lettuce. However, its competitive fitness followed a different time-dependent trend in MAP lettuce, likely due to differences in O2 content, which modulates betTIBA activity. Our study demonstrates that damaged lettuce leaf tissue does not