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. 2022 Oct 13;17(10):e0276046.
doi: 10.1371/journal.pone.0276046. eCollection 2022.

Allelic variation of Escherichia coli outer membrane protein A: Impact on cell surface properties, stress tolerance and allele distribution

Chunyu Liao  1 , Miguel C Santoscoy  2 , Julia Craft  3 , Chiron Anderson  3 , Michelle L Soupir  4 , Laura R Jarboe  1   2
Affiliations

Allelic variation of Escherichia coli outer membrane protein A: Impact on cell surface properties, stress tolerance and allele distribution

Chunyu Liao et al. PLoS One. .

Abstract

Outer membrane protein A (OmpA) is one of the most abundant outer membrane proteins of Gram-negative bacteria and is known to have patterns of sequence variations at certain amino acids-allelic variation-in Escherichia coli. Here we subjected seven exemplar OmpA alleles expressed in a K-12 (MG1655) ΔompA background to further characterization. These alleles were observed to significantly impact cell surface charge (zeta potential), cell surface hydrophobicity, biofilm formation, sensitivity to killing by neutrophil elastase, and specific growth rate at 42°C and in the presence of acetate, demonstrating that OmpA is an attractive target for engineering cell surface properties and industrial phenotypes. It was also observed that cell surface charge and biofilm formation both significantly correlate with cell surface hydrophobicity, a cell property that is increasingly intriguing for bioproduction. While there was poor alignment between the observed experimental values relative to the known sequence variation, differences in hydrophobicity and biofilm formation did correspond to the identity of residue 203 (N vs T), located within the proposed dimerization domain. The relative abundance of the (I, δ) allele was increased in extraintestinal pathogenic E. coli (ExPEC) isolates relative to environmental isolates, with a corresponding decrease in (I, α) alleles in ExPEC relative to environmental isolates. The (I, α) and (I, δ) alleles differ at positions 203 and 251. Variations in distribution were also observed among ExPEC types and phylotypes. Thus, OmpA allelic variation and its influence on OmpA function warrant further investigation.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Overview of OmpA structure and points of allelic variation as previously described [46].
Residues 93, 129 and 161 (shown in gray) are not considered as part of the allele classification system used here. (A) The 8-strand conformation, based on [39]. (B) The 16-strand ‘large pore’ conformation, based on [38]. Inset, naming convention for OmpA alleles characterized here. Colors indicate amino acid chemistry, with polar residues shown in green, neutral residues in purple, basic residues in blue, acidic residues in red and hydrophobic residues in black.
Fig 2
Fig 2. E. coli cell surface properties vary (one-way ANOVA, p < 0.005) according to OmpA allele.
OmpA variants were expressed in MG1655 ΔompA from the pGEN-MCS plasmid using the MG1655 ompA promoter, as previously described [46]. Statistically distinct groups are designated with different lowercase letters (two-tailed students t-test, p < 0.005). Error bars indicate the standard deviation. K-12 E. coli expresses the (I, α) allele and this allele is indicated in each figure. (A) Cell surface charge, measured in CaCO3 solution pH 8.0 with 10 mM ionic strength. (B) Cell surface hydrophobicity, assessed by partitioning cells between aqueous and organic phases. (C) Propensity for biofilm formation in defined media containing glucose and casamino acids at 30°C. (D) Sensitivity to killing by neutrophil elastase (NE), assessed by quantification of CFUs four hours after treatment relative to a sham control.
Fig 3
Fig 3. Correlation between cell surface properties, judged by the 95% confidence interval (CI) of the estimated slope.
(A) Association between cell surface charge (zeta potential) and hydrophobicity. (B) Association between propensity for biofilm formation and hydrophobicity. The (I, α) allele, encoded by K12 E. coli strains, is bolded for reference.
Fig 4
Fig 4. Expression of various OmpA alleles is associated with changes in log-phase specific growth rate during growth in glucose minimal media at (A) 42°C; and (B) 37°C with 100 mM acetate (pH 7.0).
Other inhibitors did not meet the criteria of statistical significance. The (I, α) allele is indicated in each figure–this is the allele encoded in K-12 E. coli such as MG1655. Lowercase letters indicate statistically distinct values (two-tailed t-test, p<0.005).
Fig 5
Fig 5. The OmpA (I, δ) allele is enriched in ExPEC relative to environmental isolates.
Terms in parentheses below the x-axis labels are the naming system used by [51]. The previously-described distribution of OmpA among 78 environmental isolates [46] is compared to 412 ExPEC isolates [50,51]. The (I, α) allele is indicated with shading, as this is the allele encoded in K-12 E. coli such as MG1655.
Fig 6
Fig 6. Phylogenetic distribution of the amino acid sequence of the seven exemplar OmpA alleles characterized here amongst representative members of the Enterobacteriaceae family.
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Grants and funding

Funding for this work was provided by the National Science Foundation (https://www.nsf.gov/) grants CBET-1236510 (MLS) and CBET-1604576 (LRJ), and the Unites States Department of Agriculture National Institute of Food and Agriculture (https://www.nifa.usda.gov/), award number 2017-6702-26137 (LRJ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.