Functional Differences between E. coli and ESKAPE Pathogen GroES/GroEL

Only K. pneumoniae, A. baumannii, and E. cloacae GroES/GroEL rescue GroES/GroEL-deficient E. coli. Shown is the LG6 colony number from antibiotic selection plate reported after transformation with individual ESKAPE pBAD-promoted groESL (Km^r) plasmid, E. coli pBAD groESL (Km^r) plasmid, or pBAD (Km^r) empty vector. LG6 (Cm^r) did not grow on kanamycin plates. (A) With 0.2% arabinose/kanamycin. (B) With kanamycin only. (C) With 0.5% dextrose–kanamycin. (D) With 500 μM IPTG–0.2% arabinose–kanamycin. (E) With 500 μM IPTG–0.5% dextrose–kanamycin. (F) With 500 μM IPTG–kanamycin. Data represent at least three independent experiments and are reported as mean with standard deviation (SD). EF, E. faecium; SA, S. aureus; KP, K. pneumoniae; AB, A. baumannii; PA, P. aeruginosa; EC, E. cloacae; Coli, E. coli; EV, empty vector.

All pBAD groESL plasmids from Gram-negative ESKAPE pathogens rescue transformed AI90 after the sacB pACYC E. coli groESL plasmid is counterselected. (A) Scheme of ESKAPE groESL plasmid shuffle into the E. coli groEL-null background AI90 strain. (B) AI90 colony number from 5% sucrose–0.2% arabinose–ampicillin selection plate reported after transformation with individual ESKAPE pBAD groESL (Amp^r) plasmid, E. coli pBAD groESL (Amp^r) plasmid, or pBAD (Amp^r) empty vector. The symbol “#” indicates colonies were visualized on these plates but retained mutant sacB groEL plasmid. Results represent three independent experiments and are reported as mean with SD. (C) All Gram-negative ESKAPE pathogens rescued groEL-deficient AI90 after sacB pACYC groEL (Cm^r) plasmid shuffle. Plasmids from surviving colonies after shuffle were isolated and run on 0.5% DNA gel. Ladder, DNA ladder; sacB, sacB pACYC E. coli groESL plasmid; Coli, pBAD E. coli groESL; EC, pBAD E. cloacae groESL; AB, pBAD A. baumannii groESL; KP, pBAD K. pneumoniae groESL plasmid; PA, pBAD P. aeruginosa groESL plasmid.

Viable ESKAPE groESL knock-ins were generated by λ-red recombineering in MG1655. (A) Due to high sequence identity between ESKAPE pathogens and E. coli groESL, only MGΔgroESL::EF groESL (Cam^r) could be obtained from knock-in (lower groESL sequence homology compared to Gram-negative pathogens). From this knock-in, K. pneumoniae, A. baumannii, P. aeruginosa, and E. cloacae groESL knock-ins were generated. Full S. aureus groESL knock-in could not be obtained. (B) PCR products for MG1655 and knock-ins for all ESKAPE pathogens using primers flanking the groESL gene visualized on agarose gel. Coli, E. coli MG1655 WT groESL; EF, MGΔgroESL::EF groESL (Cam^r); SA, MGΔgroESL::SA groESL (Cam^r) partial knock-in; KP, MGΔgroESL::KP groESL (Cam^r); AB, MGΔgroESL::AB groESL (Cam^r); PA, MGΔgroESL::PA groESL (Cam^r); EC, MGΔgroESL::EC groESL (Cam^r).

Coexpression of GroEL^ESKAPE and E. coli GroEL^D473C/532Δ forms nonfunctional-tetradecameric GroEL hetero-oligomers. (A) GroEL^ESKAPE was expressed in its respective knock-in strain (Fig. 4), purified by Q Sepharose FF (FFQ), and incubated with thiopropyl Sepharose (TPS) resin, but does not bind to resin. (B) GroEL^D473C/532Δ (cysteine and truncation mutant) was expressed in BL21, purified by FFQ, captured on TPS resin, and then eluted with increasing concentrations of DTT. (C) GroEL^ESKAPE was coexpressed with GroEL^D473C/532Δ in BL21, purified by FFQ, captured on TPS resin, and then eluted with increasing concentrations of DTT. (D) GroEL^D473C/532Δ runs at a lower molecular weight than GroEL^ESKAPE by SDS-PAGE. Captured hetero-oligomer DTT elution fractions, made up of GroEL^ESKAPE and GroEL^D473C/532Δ displays two bands, representing a mixed-GroEL ring. (E) Denatured (DTT, heat, and SDS treated) or nondenatured samples were run on a 4 to 10% native gradient gel and visualized by Coomassie brilliant blue staining. The fractions analyzed were non-DTT fraction GroEL^{P. aeruginosa} (PA), DTT fraction GroEL^D473C/532Δ (532Δ), and DTT fraction GroEL^{P. aeruginosa/D473C/532Δ} mixed complex (PA/Δ). (F) Denatured (DTT, heat, and SDS treated) or native samples were run on a 4 to 10% native gradient gel and visualized by Coomassie brilliant blue staining. The fractions analyzed were non-DTT fraction GroEL^{E. faecium} (EF), DTT fraction GroEL^D473C/532Δ (532Δ), and DTT fraction GroEL^{E. faecium/D473C/532Δ} mixed complex (EF/Δ). (G) Malachite green ATPase assay using 50 nM GroEL and 100 μM ATP measured at 660 nm over time. Black, GroEL^D473C/532Δ; red, GroEL^{P. aeruginosa}; blue, GroEL^{E. faecium}; pink, GroEL^{P. aeruginosa/D473C/532Δ}; green, GroEL^{E. faecium/D473C/532Δ}; gold, ATP only (spontaneous ATP hydrolysis).

ESKAPE GroEL domain replacement by E. coli GroEL domains produces functional chimeras capable of rescuing GroES/GroEL-deficient E. coli. Chimeras were tested for their ability to rescue LG6 in cases where ESKAPE GroEL formed a dominant-negative phenotype. All plasmids contain E. coli groES upstream of the groEL chimera. (A) E. coli GroEL tetradecamers and monomer (PDB 1SX3) with labeled apical (gray), intermediate (teal), and equatorial (forest green) domains. (B) Outline of GroEL domains from N to C terminus. Equatorial (EQ; forest green), intermediate (I; teal), and apical (A; gray) domains. (C) Replacing the P. aeruginosa (PA) equatorial domain with the E. coli (Coli) equatorial domain and replacing the E. faecium (EF) equatorial and apical domains with E. coli equatorial and apical domains produced viable (green checkmark) LG6 colonies when these chimeras were expressed from pBAD-promoted plasmids. All other chimeras could not rescue LG6 (red X mark).

ESKAPE groESL knock-ins present with an elongated phenotype. MGΔgroESL::ESKAPEgroESL (Cm^r) cells show an elongated phenotype at various temperatures compared to the parent strain, MG1655, between 24 and 42°C. The 400× images were captured for each strain after growth to mid-log phase in LB medium without antibiotic at stated temperatures. Coli, E. coli MG1655 at 24°C (left) and 42°C (right); EF, MGΔgroESL::EF groESL at 24°C (left) and 42°C (right); KP, MGΔgroESL::KP groESL at 24°C (left) and 42°C (right); AB, MGΔgroESL::AB groESL at 24°C (left) and 42°C (right); PA, MGΔgroESL::PA groESL at 24°C (left) and 42°C (right); EC, MGΔgroESL::EC groESL at 24°C (left) and 42°C (right). The scale bar represents 80.5 μm.

ESKAPE groESL knock-ins display similar growth kinetics and GroES/GroEL induction at various temperatures compared to the parent strain. MG1655ΔgroESL::ESKAPE groESL (Cm^r) shows similar growth kinetics and GroEL/ES induction compared to the parent strain, MG1655, between 24 and 42°C. In three independent experiments and reported as mean with SD, growth in LB medium without antibiotic at the stated temperature was measured by OD₆₀₀ over time to determine growth rate of each individual strain. (A) Growth at 24°C. (B) Growth at 30°C. (C) Growth at 37°C. (D) Growth at 42°C. Black, MG1655; red, MGΔgroESL::EF groESL (Cm^r); blue, MGΔgroESL::AB groESL (Cm^r); green, MGΔgroESL::KP groESL (Cm^r); pink, MGΔgroESL::PA groESL (Cm^r); open/white, MGΔgroESL::EC groESL (Cm^r). (E) Whole-cell lysates from E. coli and ESKAPE pathogens from MG1655 or knock-in strains expressing the respective GroES/GroEL were analyzed via SDS-PAGE. The black arrows indicate the positions of GroEL (upper) and GroES (lower). The lane numbers 24 and 42 represent 24 and 42°C for 5 min, respectively. Coli, E. coli; EF, MGΔgroESL::EF groESL; KP, MGΔgroESL::KP groESL; AB, MGΔgroESL::AB groESL; PA, MGΔgroESL::PA groESL; EC, MGΔgroESL::EC groESL.