A recently isolated human commensal Escherichia coli ST10 clone member mediates enhanced thermotolerance and tetrathionate respiration on a P1 phage‐derived IncY plasmid

Abstract The ubiquitous human commensal Escherichia coli has been well investigated through its model representative E. coli K‐12. In this work, we initially characterized E. coli Fec10, a recently isolated human commensal strain of phylogroup A/sequence type ST10. Compared to E. coli K‐12, the 4.88 Mbp Fec10 genome is characterized by distinct single‐nucleotide polymorphisms and acquisition of genomic islands. In addition, E. coli Fec10 possesses a 155.86 kbp IncY plasmid, a composite element based on phage P1. pFec10 harbours multiple cargo genes such as coding for a tetrathionate reductase and its corresponding regulatory two‐component system. Among the cargo genes is also the Transmissible Locus of Protein Quality Control (TLPQC), which mediates tolerance to lethal temperatures in bacteria. The disaggregase ClpGGI of TLPQC constitutes a major determinant of the thermotolerance of E. coli Fec10. We confirmed stand‐alone disaggregation activity, but observed distinct biochemical characteristics of ClpGGI‐Fec10 compared to the nearly identical Pseudomonas aeruginosa ClpGGI‐SG17M. Furthermore, we noted a unique contribution of ClpGGI‐Fec10 to the exquisite thermotolerance of E. coli Fec10, suggesting functional differences between both disaggregases in vivo. Detection of thermotolerance in 10% of human commensal E. coli isolates hints to the successful establishment of food‐borne heat‐resistant strains in the human gut.


Supplementary Figures
A Figure S1 Phylogenetic analysis, gene synteny and sequence homology of E. coli Fec10 and pFec10. A) Phylogenetic relationship of the genomes of selected closely related E. coli strains based on single nucleotide polymorphism (SNP) variant calling on 77.44% (3.269 Mbp) of the E. coli MG1655 core genome. In red=E. coli Fec10, sequence type ST10; orange=E. coli Fec6, sequence type ST399; blue=ancient E. coli reference strains. ECOR=E. coli strains from the E. coli reference collection. All strains are ST10, besides a=ST1721; b=ST93; c=ST3021; d=phylogroup B1, ST1079. Green=reference strain E. coli K-12 MG1655; outgroup E. coli Tob1 (phylogroup B2, ST2650). Bootstrap value=1, if not indicated otherwise. Tree visualized with iTOL https://itol.embl.de/ (Letunic and Bork 2016). B) Gene synteny of the E. coli Fec10 chromosome compared with E. coli K-12 MG1655 and E. coli NCTC86. Figure has been created with Easyfig (Sullivan, Petty and Beatson 2011). C) Homologous regions between pFec10 and most closely related phages P1 and SJ46. pFec10 has been normalized to the origin of replication. The graphical display of the nucleotide sequences aligned with tBLASTx using standard parameters was drawn with Circoletto (Darzentas 2010). D) Homologous regions between pFec10 and plasmid pSEE of Salmonella enterica subsp. enterica serovar Senftenberg ATCC43845. The graphical display of the nucleotide sequences aligned with tBLASTx using standard parameters was drawn with Circoletto (Darzentas 2010).

Figure S4
Heat shock tolerance of E. coli fecal isolates and derivatives. (A) Screening of a panel of E. coli fecal isolates for heat shock tolerance at 55 °C. (B) Selected fecal isolates and respective clpB and clpG GI mutants were subjected to treatment with the extreme lethal heat of 65 °C. The elevated temperature was subjected for the indicated time and, subsequently, 10-fold serial dilutions were spotted on LB agar plates and incubated for 16 h. Laboratory strain MC4100 and mutant MC4100 ∆clpB were included as references.

Figure S5
Phylogenetic analysis of ClpG GI proteins. ClpG GI protein sequences from various strains of diverse genera were analyzed together with other class I Clp family members (ClpA, B, C, D, E, L and V). Protein sequences were aligned with ClustalW using standard parameters and a maximum-likelihood tree was built using MEGA X. Branch lengths correspond to substitutions per site and branch support values are indicated in %. The robustness of the phylogenetic tree topologies was evaluated by bootstrap analysis with 500 replications in MEGA X.

Figure S6
Sequence variations within ClpG GI proteins. (A) Alignment of ClpG GI sequences. Residues on a dark blue background indicate 100% conservation and light blue background indicates 66% conservation. Sequences were aligned with ClustalW using default settings (Chenna et al. 2003) and displayed in JalView (Waterhouse et al. 2009). (B) Amino acid sequence variation in the N1-domain and ClpG specific C-terminal sequence between ClpG GI-Fec10 and ClpG GI-SG17M indicated in red. Variant amino acids in other domains as indicated. Domain designation as in (Lee et al. 2018)

Figure S9
Secondary structure analysis and melting temperature curves for ClpB K-12 , ClpG GI-SG17M and ClpG GI-Fec10 . (A) A reference signature spectrum for alpha and beta helices and random coils obtained from (Wei, Thyparambil and Latour 2014) with permission. Circular Dichroism Spectra (B, D) and melting temperature curves recorded at 222 nm (C, E) were recorded in the absence (B, C) and presence (D, E) of Mg 2+ /ATPγS. The difference in curve shape of ClpG GI-Fec10 (C) is explained by a lower starting mean residue ellipticity value. This does not impact T m calculation.

Supplementary experimental procedures Genome characterization and bioinformatic analyses
ResFinder 3.2 ( https://cge.cbs.dtu.dk/services/ResFinder/; (Zankari et al. 2012)) was used to identify antibiotic resistance genes. VirulenceFinder (Joensen et al. 2014) was applied to check for encoded virulence factors. Alternatively assessment of strain relationships was done with core genome multilocus sequence typing (cgMLST) using Ridom SeqSphere+ software (Junemann et al. 2013). Target genes were defined using E. coli K-12 MG1655 as seed. Missing values were an own category. SNP calling was performed with CSIPhylogeny1.4 (https://cge.cbs.dtu.dk/services/CSIPhylogeny/) and phylogeny was inferred by calculation of approximately-maximum-likelihood using an implemented modified version of FastTree2  To construct the unrooted phylogenetic tree, orthologous groups, amino acid sequences of all protein-coding genes were determined using ProteinOrtho V5.13 (Lechner et al. 2011) with parameters cov=67 (min. coverage of best BLAST alignments in per cent) and identity=50 (min. per cent identity of best BLAST alignments). The orthologous proteins from 521 complete and annotated genomes of E. coli available in GenBank in April 2019 (Coordinators 2018) and E. coli Fec10 and Fec6 were subsequently aligned. 238 common single-copied groups of orthologs were used for phylogenetic tree construction. Nucleotide sequences of genes in each orthologous group were aligned using MAFFT (Katoh and Standley 2013) in the linsi mode. Subsequently, RAxML (Stamatakis 2014) with the GTR+Gamma model with 100 bootstrap replicates for the concatenated gene alignment was used. Finally, the tree was visualised using the online tool iTOL (Letunic and Bork 2016). Conserved gene clusters (synteny groups) were searched for with bi-directional best hits or a BLASTP alignment threshold with an identity >35% on >80% protein length with a gap parameter of five against sequenced E. coli genomes including E. coli K-12 MG1655 (Vallenet et al. 2020). Variable and strain specific coding sequences were compared to E. coli K-12 strains (K-12, DH10B, BW2952, J53 and DH5α) defined as >80% amino acid identity over >80% coverage. Core genome and variable CDSs compared to 1080 strains of E. coli population was determined by PPanGGOLiN 0.1.4 using standard parameters (Vallenet et al. 2020). To estimate the fraction of vertically inherited genomes of closely related strains were aligned using PG-explorer (http://mouse.belozersky.msu.ru/tools/npge.html) (Nagaev 2015). For each pair of strains aligned regions were cut into windows of 1 bb to calculate the number of mismatches. The obtained histogram was decomposed into a weighted sum of the Poisson and Erlang distributions, the former reflecting vertical inheritance, and the latter, regions acquired