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

The GroES/GroEL chaperonin from E. coli has long served as the model system for other chaperonins. This assumption seemed valid because of the high conservation between the chaperonins.

MG1655 K-12 GroES/GroEL argues for conserved structure and function ( Fig. 1; see Table S1, Table S2, and Fig. S1 in the supplemental material). Amino acid identity is higher between the Gram-negative pathogens and E. coli (60.2 to 93.8% for GroES and 75.4 to 96.4% for GroEL) compared to Gram-positive pathogens and E. coli (43.3 to 46.4% for GroES and 57.1 to 60.6% for GroEL) ( Fig. 1A and C). This trend is also observed for amino acid similarity in the Gram-negative bacteria (78.4 to 97.9% for GroES and 86.7 to 98.0% for GroEL) and Gram-positive bacteria (62.9% for GroES and 75.8 to 77.4% for GroEL) ( Fig. 1B and D). Furthermore, predicted ESKAPE GroES and GroEL isoelectric points (4.87 to 5.38 and 4.56 to 5.04, respectively) are congruent with those of E. coli GroES and GroEL (5.15 and 4.85, respectively) ( Table S2). The isoelectric points of the Gram-positive ESKAPE pathogens are least like those of E. coli. Overall, E. faecium and S. aureus GroES/GroEL contain fewer residues than E. coli GroES/GroEL and lack the GGM C-terminal repeat found in E. coli GroES/GroEL (24) and other Gram-negative ESKAPE pathogens.
LG6 contains a lac-promoted groESL operon that, in the absence of lactose or IPTG (isopropyl-b-D-thiogalactopyranoside) fails to produce sufficient endogenous GroES/GroEL to sustain viable colonies (25). When LG6 cells were transformed with a plasmid containing pBAD-driven groESL from the ESKAPE pathogens or E. coli (as a positive control) and induced with arabinose, E. faecium, S. aureus, and P. aeruginosa GroES/GroEL chaperone systems could not rescue GroES/ GroEL-deficient LG6 cells ( Fig. 2A). This was also the case for untransformed LG6 and LG6 transformed with pBAD-driven empty vector ( Fig. 2A). We reasoned this could be due to inappropriate protein levels and thus probed the system with lower levels of ESKAPE and E. coli GroES/GroEL. Plasmid antibiotic selection alone (without inducing agent) demonstrated that there was significant transcriptional leakage from the pBAD promoter of the groESL plasmids, and the same ESKAPE groESL plasmids rescued LG6 similarly as when induced with arabinose (compare Fig. 2A and B). Addition of dextrose to the agar plate, to suppress transcriptional leakage, reduced the number of viable colonies of K. pneumoniae, A. baumannii, and Enterobacter cloacae, but failed to produce any viable LG6 colonies in E. faecium, S. aureus, and P. aeruginosa groESL-transformed cells (Fig. 2C). Surprised by these results, we induced the chromosomal E. coli groESL operon to demonstrate that LG6 could still be rescued by GroESL Coli . Despite adequate levels of GroESL Coli , we found that in the presence of the ESKAPE groESL plasmids, E. faecium, S. aureus, and P. aeruginosa GroES/GroEL-containing cells were unable to produce viable organisms. However, empty vector, K. pneumoniae, A. baumannii, E. cloacae, and E. coli groESL plasmid-containing LG6 cells were viable in the presence of wild-type, chromosomal GroES/GroEL stimulated by IPTG induction (Fig. 2D, E, and F).
All pBAD groESL plasmids from Gram-negative KAPE pathogens rescued transformed AI90 cells after the sacB pACYC E. coli groESL plasmid was counterselected. After ruling out gene dosage (Fig. 2) and codon bias (Table S3) as factors that prevented E. faecium, S. aureus, and P. aeruginosa groESL from rescuing LG6 cells, we sought to determine the cause of these observed dominant-negative phenotypes. To rule out ring mixing between GroEL Coli and GroEL ESKAPE as the cause of the dominantnegative effect, we expressed ESKAPE GroES/GroEL in AI90 E. coli cells, in which groES is present, but groEL is absent from the chromosome. AI90 is maintained by an E. coli groESL plasmid capable of negative selection due to the presence of sacB within this pACYC construct (7). AI90 cells were transformed with ESKAPE groESL plasmids in the presence of sucrose (negative pACYC E. coli groESL sacB selection) and kanamycin (positive ESKAPE groESL selection). This selection shuffled out the E. coli groESL plasmid, forcing reliance on the ESKAPE groESL plasmids for survival. This platform eliminated the possibility of forming mixed-GroEL tetradecamers (active or inactive) and more conclusively tested the compatibility of ESKAPE GroES/GroEL in E. coli (Fig. 3A). We found that all ESKAPE pathogen GroELs that could complement LG6 also rescued E. coli groEL-null AI90 (Fig. 3B). Because sacB is subject to mutations that render its gene product unable to kill cells that retain this plasmid (26), postselection colonies were amplified and plasmid DNA purified to confirm the absence of the pACYC groESL plasmids and the presence of an ESKAPE groESL plasmid (Fig. 3C). P. aeruginosa GroES/ GroEL was able to complement AI90, but not LG6, supporting our previous observation that P. aeruginosa GroEL formed inactive/underactive mixed-GroEL rings in the presence of E. coli GroEL (Fig. 2) and is responsible for the dominant-negative effect seen in LG6. This observation could not explain the lack of AI90 E. coli rescue in the presence of E. faecium or S. aureus GroEL, although E. faecium GroEL may be incompatible with E. coli GroES in this system.
Viable ESKAPE groESL knock-ins were generated by k-red recombineering in MG1655. Because AI90 contains groES on the chromosome, we sought to eliminate the possibility of generating inactive GroESL Coli -GroESL ESKAPE mixed complexes within the E. coli chaperonin complex. To accomplish this, E. coli groESL was replaced by ESKAPE groESL using l-red recombination (27,28) (Fig. 4A). Because of the high base pair identity between the Gram-negative ESKAPE pathogen and E. coli groESL, complete knock-ins for these organisms could not initially be generated. Lower base pair homology between E. faecium and E. coli was sufficient for complete knock-in of E. faecium groESL into the E. coli groESL operon. The E. faecium groESL strain was then used as a template to knock in groESL from the remaining ESKAPE pathogens, and each FIG 3 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.
Differences between E. coli and ESKAPE GroES/GroEL ® successful knock-in cell line was confirmed by sequencing. In the absence of background E. coli GroES or GroEL, we discovered that all but one of the ESKAPE pathogen GroES/GroEL chaperone systems could replace E. coli groESL in MG1655 (Fig. 4B). The ability of E. faecium to rescue in this context, but not in AI90, indicates that GroES ring mixing between E. faecium and E. coli may produce a dominate-negative phenotype. It should not be discounted that GroES Coli and GroEL E. faecium may not interact or may form a trapped complex incapable of refolding clients. Complete S. aureus groESL knock-in was not possible using this system, but we have observed that S. aureus GroES/GroEL forms inclusion bodies when expressed in BL21 cells with host GroES/ GroEL. The underlying biochemical reason for this remains unknown and is under investigation.
Coexpression of GroEL ESKAPE and E. coli GroEL D473C/532D forms nonfunctionaltetradecameric GroEL hetero-oligomers. The genetic data from the AI90 rescue and l-red recombineering experiments argued for the formation of mixed GroEL complexes, but we wanted to demonstrate mixed complex formation and to determine if these had compromised biochemical function. It has been previously shown that coexpression of GroEL Coli and GroEL Coli-mutant monomers form mixed GroEL tetradecamers with monomer integration directly correlated with the level of expression of each GroEL monomer type (29,30). To investigate the formation of GroEL hetero-oligomers in E. coli, pBAD-driven GroEL ESKAPE (expressed alone in its respective knock-in strain  allowed to air oxidize. It is expected that the GroEL cysteine mutant (D473C/532D) will form a covalent bond with the TPS resin and elute after reduction with dithiothreitol (DTT), whereas GroEL ESKAPE will be found in the flowthrough and not in the DTT-eluted fractions because it lacks the reactive cysteine. Furthermore, to differentiate between GroEL ESKAPE and GroEL D473C/532D , active truncated E. coli GroEL (532D mutant) was used to determine GroEL identity by SDS-PAGE (Fig. 5D). Coexpressed GroEL D473C/532D and GroEL P. aeruginosa protein eluted with DTT from TPS resin was found to be a tetradecamer Malachite green ATPase assay using 50 nM GroEL and 100 mM ATP measured at 660 nm over time. Black, GroEL D473C/532D ; red, GroEL P. aeruginosa ; blue, GroEL E. faecium ; pink, GroEL P. aeruginosa/D473C/532D ; green, GroEL E. faecium/D473C/532D ; gold, ATP only (spontaneous ATP hydrolysis).
Differences between E. coli and ESKAPE GroES/GroEL ® by native PAGE (Fig. 5E). The DTT-eluted fraction species was present as a double band by PAGE under denaturing conditions, indicating the formation of hetero-oligomeric GroEL in vivo. (Fig. 5E). This experimental design was replicated using GroEL D473C/532D and GroEL E. faecium which yielded similar results (Fig. 5F). Importantly, these GroEL hetero-oligomers showed severely impaired ATPase activity (Fig. 5G) compared to purified homo-oligomers generated using strains from Fig. 4 or GroEL D473C/532D expressed in BL21 alone.
ESKAPE GroEL domain replacement by E. coli GroEL domains produces functional chimeras capable of rescuing GroES/GroEL-deficient E. coli. Based upon the E. faecium and P. aeruginosa GroES/GroEL dominant-negative phenotype in LG6 (Fig. 2) and the formation of GroEL ESKAPE and GroEL D473C/532D hetero-oligomers (Fig. 5), we investigated domain ( Fig. 6A and B) incompatibilities that could be responsible for the lack of GroEL hetero-oligomer activity in vivo. GroEL chimeras consisting of E. coli/P. aeruginosa or E. coli/E. faecium domain swaps were generated and tested for their ability to rescue LG6 (Fig. 6). Chimeric groEL was QuickStep cloned (31) into pBAD-promoted plasmids with upstream E. coli groES present. Expression of P. aeruginosa GroEL with the equatorial domain replaced by E. coli rescued LG6, but not E. coli GroEL with the equatorial domain replaced by P. aeruginosa (Fig. 6C). E. faecium GroEL with E. coli equatorial and apical domains, but not E. coli equatorial domain replacement alone, was required for functional rescue of LG6 (Fig. 6C). These results suggest P. aeruginosa and E. faecium GroEL equatorial domains, in a mixed oligomer with E. coli GroEL, may disrupt positive and/or negative ring allostery and loss of chaperonin function. Furthermore, incompatibility of the E. faecium apical domain in the presence of E. coli GroES may contribute to the dominant-negative phenotype seen in Fig. 2.  ) 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 display similar growth kinetics and GroES/GroEL induction at various temperatures compared to the parent strain, but present with elongated phenotypes. The MG1655 wild-type strain and ESKAPE groESL knockin strains were grown to mid-log phase at 24, 30, 37, or 42°C (for clarity, we only show data for 24 and 42°C) and imaged by bright-field microscopy at a total magnification of 400Â. MG1655 and A. baumannii groESL knock-in strains did not display phenotypic abnormalities (Fig. 7). However, E. faecium, K. pneumoniae, and E. cloacae groESL knockin strains displayed elongated phenotypes at 24°C, but not 42°C (Fig. 7), which is indicative of compromised GroEL function since GroEL is required for FtsZ function (7). P. aeruginosa groESL knock-ins displayed a normal phenotype at 24°C, but mild elongation compared to other strains at 42°C (Fig. 7). Despite these morphological changes at various temperatures, the growth rates of the ESKAPE groESL knock-in strains compared to the wild-type strain over 24 h appear unaffected from 24 to 42°C (Fig. 8A to D). To test the groESL operon response to heat stress, wild-type and ESKAPE knock-in strain GroES/GroEL induction at 24°C was compared to that in cells grown at 24°C and shifted to 42°C for 5 min. All strains were found to have heat-inducible GroES/GroEL as measured by SDS-PAGE, indicating preservation of functional groE operons (Fig. 8E). GroES/GroEL levels at 24 or 42°C were not found to be correlated with growth rate or phenotypic changes noted in Fig. 7

DISCUSSION
Previous studies that replaced E. coli GroES/GroEL with homologs such as Cpn10/ Cpn60 from Rhizobium leguminosarum or human mitochondrial Hsp10/Hsp60 have generated viable E. coli (32,33). This type of complementation lends to the idea that chaperonin amino acid conservation between species parallels with similar client scopes. Therefore, it is not surprising that the intrinsic refolding actions among these systems are sufficient to sustain other organisms in some cases. Although complete replacement of E. coli GroES/GroEL with the chaperonins from other organisms is possible (33,34), experiments where exogenous chaperonins were used to rescue GroES/ GroEL-deficient E. coli, LG6 (18), have produced unexpected results (35)(36)(37)(38). The most intriguing example came from the Mande group, where they studied the ability of Mycobacterium tuberculosis GroEL2 and E. coli/M. tuberculosis GroEL chimeras to rescue GroES/GroEL-deficient E. coli (39). Although M. tuberculosis GroEL2 is essential for M. tuberculosis survival, this chaperonin could not rescue GroES/GroEL-deficient E. coli Differences between E. coli and ESKAPE GroES/GroEL ® despite significant amino acid identity with that of E. coli GroEL. Several chimeras that were generated in these studies existed as tetradecamers and could prevent aggregation of citrate synthase, but they could not rescue GroES/GroEL-deficient E. coli. This suggests that the GroEL chimeras were assembled as nonfunctional tetradecamers capable of trapping denatured protein, but not able to refold clients in vivo. We  (Fig. 9). Because the refolding cycle of GroEL is dependent on the highly coordinated movement of multiple domains synchronized between subunits, positive allostery within the ring and/or negative allostery between the rings (40) could be altered by incorporation of dissimilar GroEL subunits. Some of these mixed tetradecamers may be hypofunctional or nonfunctional, perhaps capable of trapping misfolded protein, but unable to refold misfolded proteins in vivo. Organisms with multiple HSP60 isoforms seem to have evolved a mechanism to prevent mixture of endogenous HSP60s. Several groups have probed this and found that mixed endogenous oligomers were present at undetectable or very low levels (9,41,42); however, exceptions do exist (43,44). Despite this, coexpression of GroEL Coli and GroEL Coli-mutant subunits can produce mixed GroEL tetradecamers and have been used to study E. coli GroEL function (29,30).
E. coli strains were generated in which E. coli groESL was replaced by ESKAPE pathogen groESL to compare the extent to which these conserved chaperonin systems could function in E. coli. We predicted that the modest differences in amino acid similarity, isoelectric point, and total residue number between ESKAPE and E. coli GroES/GroEL (Table S2) were unlikely to cause divergence in chaperonin function or client recognition. Therefore, it was reasonable to predict that this set of chaperonin systems could complement a GroES/GroEL-deficient E. coli cell line. We discovered that the expression of GroES/GroEL from E. faecium, S. aureus, and P. aeruginosa in GroES/GroEL-deficient E. coli LG6 produced a dominant-negative phenotype that was not the result of codon bias or inappropriate gene dosage ( Fig. 2; Table S3). Conversely, three other Gram-negative pathogens were able to rescue this GroES/GroEL-deficient cell line, including A. baumannii, whose GroEL sequence is least like E. coli GroEL compared to the other Gram-negative pathogen GroEL. ESKAPE pathogen GroES/GroEL that were unable to rescue LG6 undermined cellular viability in ways other than lack of expression; this was evident when GroESL Coli was expressed from the chromosome of LG6 in the presence Differences between E. coli and ESKAPE GroES/GroEL of ESKAPE pathogen groESL plasmid (Fig. 2F). Expression of GroES/GroEL from the LG6 chromosome produced cellular rescue in the presence of E. coli, K. pneumoniae, A. baumanii, and E. cloacae groESL plasmids or empty vector. However, when E. faecium, S. aureus, or P. aeruginosa groESL plasmids were present, these still failed to rescue despite the expression of GroES/GroEL from the chromosome of LG6. Together, these observations indicate that GroESL Coli and GroESL ESKAPE were being translated within LG6. However, in the presence of E. faecium, S. aureus, or P. aeruginosa chaperonin systems, viable LG6 colonies were not observed.
We next set out to test if the formation of mixed-nonfunctional GroEL complexes was responsible for the observed dominant-negative effect in E. coli by removing GroEL Coli from the background of E. coli strain AI90, which retains a chromosomal copy of groES, but not groEL (Fig. 3). Here, AI90 is maintained by a plasmid copy of groESL, which can be selected against in the presence of sucrose. The formation of mixed-GroEL complexes is not possible in this system due to negative selection of the E. coli groESL and positive selection for ESKAPE groESL plasmid. GroEL ring mixing at the level of translation would be eliminated due to the absence of the E. coli groEL. This hypothesis was strengthened by the fact that P. aeruginosa, which previously could not rescue E. coli, was now able to rescue. We attribute this change to the loss of GroEL ring mixing, which was likely responsible for the dominant-negative phenotype seen with the P. aeruginosa GroES/GroEL chaperone system when expressed in the presence of GroEL Coli in LG6. This observation does not explain the lack of rescue for Gram-positive chaperonin systems from E. faecium and S. aureus. It is possible that the presence of E. coli GroES and E. faecium or S. aureus GroES may disrupt the efficient refolding of clients due to a perturbed GroES-GroEL interaction and/or GroES function.
Next, we completely removed the possibility of GroESL Coli and GroESL ESKAPE subunit mixing for both GroES and GroEL by replacing E. coli groESL with ESKAPE groESL, while maintaining the upstream and downstream components of the E. coli groE operon (Fig. 4). Along with the Gram-negative ESKAPE pathogen chaperonin systems, which were found to rescue in earlier experiments, Gram-positive E. faecium groESL knock-in was now able to rescue in the absence of GroESL Coli .
Previous work in E. coli has shown that coexpression of GroEL Coli and GroEL Coli-mutant produced mixed tetradecamers (29,30). This observation, along with work from the Lund and Mande groups, inspired the hypothesis that the coexpression of GroESL Coli and GroESL ESKAPE could produce the same phenomenon. This mixture of subunits could operate with enough functionality to maintain viable E. coli within some chaperone systems, but not others. Formation of mixed-GroEL complexes between E. coli chaperonin and E. faecium, S. aureus, and P. aeruginosa chaperonins, respectively, may perturb positive allostery within the GroEL ring and/or negative allostery between GroEL rings such that efficient refolding of essential gene products is compromised. This scenario would ultimately lead to loss of cell viability (8). Coexpression of GroEL ESKAPE and GroEL D473C/532D demonstrated that tetradecameric GroEL hetero-oligomers were formed in vivo (Fig. 5). Furthermore, P aeruginosa-E. coli and E. faecium-E. coli heterooligomers were found to be essentially devoid of ATPase activity (Fig. 5G), supporting our hypothesis regarding the dominant-negative phenotypes seen in the LG6 rescue experiment (Fig. 2).
Incompatibilities between GroEL ESKAPE and GroEL Coli domains were determined by generating GroEL chimeras (with the plasmid containing E. coli groES upstream of chimeric groEL) and screening for the functional rescue of LG6. For P. aeruginosa GroEL, rescue was possible by replacing the equatorial domain with the E. coli equatorial domain. For GroEL E. faecium to rescue LG6, it was required that both the equatorial and apical domains be replaced by the E. coli equatorial and apical domains (Fig. 6C). Cochaperonin specificity has been documented (45); therefore, it is possible that chaperoning ability is compromised if E. coli GroES cannot efficiently interact with the E. faecium apical domain. It is recognized that the lack of a traditional GGM repeat in the Cterminal tail of GroEL may contribute to premature client release and decreased rate of refolding (46)(47)(48); however, replacement of the GroEL E. faecium equatorial domain with that of GroEL Coli (which contains the C-terminal GGM repeat) did not aid in the rescue of AI90 chimeric GroEL E. faecium . Furthermore, a traditional C-terminal GGM repeat was not required for GroESL E. faecium rescue of E. coli (Fig. 4).
Each of the groESL knock-in strains displayed phenotypic changes at various temperatures, except for A. baumannii (Fig. 7). However, basal levels of GroES/GroEL or heat stress induction of knock-in ESKAPE groESL did not appear to affect growth rate compared to wild-type (Fig. 8). Inefficient FtsZ refolding by the GroES/GroEL chaperonin system (7, 49) may be responsible for the phenotypic changes seen with some of the ESKAPE groESL knock-in strains. It remains to be determined if these GroESL ESKAPE chaperonins have divergent client scopes that are specific to their respective hosts, and this opens the possibility that their structure, allostery, and/or refolding cycle rates may differ to accommodate their own proteome.
Conclusion. We herein report a stepwise approach to study the ability of GroESL ESKAPE chaperonin systems to rescue chaperonin-deficient E. coli. We found that the coexpression of GroESL Coli and GroESL ESKAPE generates mixed-subunit oligomers, some of which are nonfunctional and affect organism survival. These results build upon previous attempts to study exogenous GroES/GroEL within E. coli where background GroESL Coli was present. This work highlights the need to eliminate background GroES/GroEL from the host strain as a requisite for recombinant expression to further study exogenous chaperonin systems. Future efforts will involve characterization of ESKAPE GroES/GroEL using the ESKAPE groESL knock-in strains we have generated. We wish to determine if, despite high conservation between ESKAPE and E. coli GroES/GroEL, these chaperone systems have evolved to refold different scopes of clients. Furthermore, these strains can be used to express ESKAPE GroES/GroEL without interference from host strain GroES/GroEL. Additionally, elucidation of ESKAPE GroEL allostery, GroES-GroEL interactions, and GroES/GroEL structures will be pursued.

MATERIALS AND METHODS
Plasmids and strains. pBAD-promoted ESKAPE and E. coli groESL plasmids were generated by polymerase incomplete primer extension (PIPE) cloning using pSpeedET as the vector component and genomic DNA from Enterococcus faecium ATCC 51559, Staphylococcus aureus ATCC 25923, Klebsiella pneumonia ATCC 700603, Acinetobacter baumannii ATCC 19606, Pseudomonas aeruginosa ATCC 47085, Enterobacter cloacae ATCC 13047, and MG1655 K-12 groESL as insert components. pBAD-promoted chimera plasmids included E. coli groES upstream of chimeric groEL and were generated by QuickStep cloning (31). lacIq-Ptac GroEL D473C/532D mutagenesis was performed using the Naismith method (50). Plasmid transformation into LG6 (from Horwich lab) was done by incubation of cells with 100 ng of plasmid for 20 min on ice, followed by 45 s of heat shock at 42°C. Cells were immediately returned to wet ice and diluted with 1 ml SOB (super optimal broth) medium after 2 min of incubation. Transformants were shaken for 1 h at 37°C, with or without induction/suppression agents (arabinose/dextrose), and plated at multiple dilutions on separate agar-antibiotic 6 0.2% arabinose or 0.5% dextrose. This same procedure was used for AI90, with exception of addition of 5 to 10% sucrose to agar plates.
Gene knock-in. ESKAPE groESL was knocked-in to the MG1655 K-12 groE operon using modified Datsenko-Wanner protocol (27,28). MG1655 K-12 cells transformed with l-red pKD46 plasmid were grown to an optical density at 600 nm (OD 600 ) of 0.2 prior to induction with 0.2% arabinose. Cells were made electrocompetent after growth to an OD 600 of 0.35 to 0.4 using several washes with ice-cold water and 10% glycerol. ESKAPE groESL genes were individually cloned into pKIKOarsB using traditional methods. Insertion cassette PCR products (including 50-bp overhangs covering upstream and downstream of the MG1655 K-12 groE operon) were transformed into MG1655 K-12 l-red cells by electroporation using Bio-Rad Gene Pulser Xcell with 0.2-cm Gene Pulser electroporation cuvettes (C= 25 mF; PC = 200 X; V = 2.5 kV). Cells were shaken at 37°C in SOB medium for 3 h and plated on agar-antibiotic. Colonies that arose were picked and grown in LB medium/antibiotic, lysed by boiling at 100°C for 10 min, and then used as the DNA template in a PCR mixture containing primers that flank the groE operon. PCR products of potential knock-in colonies were sent for sequence confirmation. The chromosomal antibiotic resistance marker was removed using FLP-recombinase.
Microscopy. Knock-in strains or MG1655 K-12 cells were grown to an OD 600 of 0.6 in LB medium and prepared in triplicate to be imaged after growth at 24 or 42°C. Live cells were diluted and added to Fisherbrand microscope slides prior to imaging on a Nikon Eclipse 50i microscope at a total magnification of 400Â. Image colors were modified in Microsoft PowerPoint.
Bacterial growth rate. Knock-in strains or MG1655 K-12 cells were grown overnight in LB medium and prepared in triplicate after being diluted to an OD 600 of 0.050. Diluted samples were grown at 24, 30, 37, or 42°C, with OD 600 measurements taken at various time points, after which a growth curve was generated using GraphPad Prism.
Differences between E. coli and ESKAPE GroES/GroEL ® Chromosomal GroESL expression. ESKAPE knock-in strains or MG1655 K-12 cells were grown to an OD 600 of 0.6 at 24°C and then subjected to 5 min of heat shock at 42°C or continued growth at 24°C. Cells were pelleted, and supernatant was obtained by lysis with radioimmunoprecipitation assay (RIPA) buffer containing Halt protease cocktail (Thermo) and 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), followed by centrifugation at 22,500 Â g for 30 min at 4°C. Supernatant was diluted with Laemmli buffer and heated to 100°C for 10 min, then loaded onto a 15% polyacrylamide denaturing gel and resolved by electrophoresis. GroES and GroEL protein bands were visualized after staining with Coomassie blue.
ATPase activity. The malachite green assay (51) was used to detect the presence of inorganic phosphate post-ATP hydrolysis by GroEL. GroEL (50 nM) and ATP (100 mM) were incubated at room temperature in reaction buffer (50 mM Tris [pH 7.4], 50 mM KCl, 10 mM MgCl 2 , 1 mM DTT), with 50-ml aliquots removed from reaction mixture and added to 100 ml of malachite green in a 96-well clear plate (Greiner 655101) read at various time points using a SpectraMax ID5 plate reader at 660 nm.
GroEL purification. pBAD GroEL ESKAPE and lacIq-Ptac GroEL D473C/532D were transformed into BL21 in a stepwise fashion and induced for 45 min at 37°C using 0.2% arabinose and 0.5 mM IPTG in LB medium. Mixed complexes were first purified by Q Sepharose FF chromatography and then loaded onto thiopropyl Sepharose 4B resin and eluted with increasing amounts of DTT. Expression of GroEL ESKAPE was done by transformation of pBAD GroEL ESKAPE into the respective knock-in strain (Fig. 4) and induced with 0.2% arabinose for 4 h at 37°C after the culture reached an OD 600 of 0.6. GroEL D473C/532D expression was done by transformation of lacIq-Ptac GroEL D473C/532D into BL21 and induced with 0.5 mM IPTG for 4 h at 37°C after the culture reached an OD 600 of 0.6. Purification was carried out by Q Sepharose FF and TPS chromatography.
Data availability. Supporting information associated with this article can be found in the online version, which includes E. coli and ESKAPE pathogen amino acid conservation as well as codon usage for ESKAPE groESL.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.