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

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.

The Listeria monocytogenes persistence factor ClpL is a potent stand-alone disaggregase.

Heat stress can cause cell death by triggering the aggregation of essential proteins. In bacteria, aggregated proteins are rescued by the canonical Hsp70/AAA+ (ClpB) bi-chaperone disaggregase. Man-made, severe stress conditions applied during, e.g., food processing represent a novel threat for bacteria by exceeding the capacity of the Hsp70/ClpB system. Here, we report on the potent autonomous AAA+ disaggregase ClpL from Listeria monocytogenes that provides enhanced heat resistance to the food-borne pathogen enabling persistence in adverse environments. ClpL shows increased thermal stability and enhanced disaggregation power compared to Hsp70/ClpB, enabling it to withstand severe heat stress and to solubilize tight aggregates. ClpL binds to protein aggregates via aromatic residues present in its N-terminal domain (NTD) that adopts a partially folded and dynamic conformation. Target specificity is achieved by simultaneous interactions of multiple NTDs with the aggregate surface. ClpL shows remarkable structural plasticity by forming diverse higher assembly states through interacting ClpL rings. NTDs become largely sequestered upon ClpL ring interactions. Stabilizing ring assemblies by engineered disulfide bonds strongly reduces disaggregation activity, suggesting that they represent storage states.

ClpG Provides Increased Heat Resistance by Acting as Superior Disaggregase.

Elevation of temperature within and above the physiological limit causes the unfolding and aggregation of cellular proteins, which can ultimately lead to cell death. Bacteria are therefore equipped with Hsp100 disaggregation machines that revert the aggregation process and reactivate proteins otherwise lost by aggregation. In Gram-negative bacteria, two disaggregation systems have been described: the widespread ClpB disaggregase, which requires cooperation with an Hsp70 chaperone, and the standalone ClpG disaggregase. ClpG co-exists with ClpB in selected bacteria and provides superior heat resistance. Here, we compared the activities of both disaggregases towards diverse model substrates aggregated in vitro and in vivo at different temperatures. We show that ClpG exhibits robust activity towards all disordered aggregates, whereas ClpB acts poorly on the protein aggregates formed at very high temperatures. Extreme temperatures are expected not only to cause extended protein unfolding, but also to result in an accelerated formation of protein aggregates with potentially altered chemical and physical parameters, including increased stability. We show that ClpG exerts higher threading forces as compared to ClpB, likely enabling ClpG to process "tight" aggregates formed during severe heat stress. This defines ClpG as a more powerful disaggregase and mechanistically explains how ClpG provides increased heat resistance.