Effects of small heat shock proteins from thermotolerant bacteria on the stress resistance of Escherichia coli to temperature, pH, and hyperosmolarity

Small heat shock proteins (HSPs), such as HSP20, represent cellular thermal resistance mechanisms, to avoid protein aggregation at elevated temperatures. Recombinantly expressed HSP20s serve as a molecular tool for improving the tolerance of living cells to various physical and chemical stressors. Here, we aimed to heterologously express 18 HSP20s from 12 thermotolerant bacteria in Escherichia coli and evaluate their effects on various physical and chemical cellular stresses. Seventeen HSP20s were successfully expressed as soluble proteins. Recombinant E. coli cells were subjected to heat, cold, acidic, alkaline, and hyperosmolar stress to evaluate the effects of HSP20 proteins on stress resistance. Notably, the overexpression of 15 HSP20s enhanced the stress resistance of E. coli compared to that of the control strain. In particular, HSPs from Tepidimonas sediminis and Oceanithermus profundus improved the stress tolerance of E. coli under all tested conditions. In addition, E. coli harboring HSP20 from T. sediminis retained cell viability even after heat treatment at 52 °C for 5 days. To our knowledge, this is the first report of E. coli tolerance to prolonged (> 100 h) high-temperature stress. These findings indicate the potential of thermotolerant HSPs as molecular tools for improving stress tolerance in E. coli. Supplementary Information The online version contains supplementary material available at 10.1007/s00792-023-01326-y.


Introduction
Living cells are equipped with various molecular machineries to adapt to physical and chemical stresses, such as cold, heat, acid, alkali, and salinity.Heat shock proteins (HSPs) are one such machinery which are found ubiquitously.On exposure to environmental stress, cells increase the expression of HSPs that function as molecular chaperones to prevent protein aggregation (Whitley et al. 1999).HSPs are classified into the following five major families: HSP100, HSP90, HSP70, HSP60, and small HSP.Among them, small HSPs are defined as those with molecular masses ranging from 12 to 43 kDa (Basha et al. 2012), with majority of the HSPs between 14 and 27 kDa (Narberhaus 2002).
Small HSPs are ATP-independent molecular chaperones which prevent protein aggregation in living cells (Bepperling et al. 2012).In particular, HSP20, a type of small HSP, has been well studied in eukaryotes, archaea, and bacteria, and plays crucial roles against many physical and chemical stresses (Tanguay and Hightower 2015).The transcription of small HSPs is induced by multiple stresses, such as heat, cold, starvation, pH changes, chemicals, biomodulators, and posttranslational modifications (e.g., Guo et al. 2020;Cong et al. 2020), to adopt these stresses.For instance, endogenous small HSP20s from Escherichia coli (ibpA and ibpB) contribute to heat and hydrogen peroxide resistance (Kitagawa et al. 2000).In addition, HSP20s encoded by a genomic island improved the cell viability of E. coli at 60 °C (Li and Gänzle 2016).Besides heat stress, the co-expression of HSP20, glutaredoxin-3, iron-binding protein, and 2Fe-2S ferredoxin in Deinococcus radiodurans enhances its resistance to hydrogen peroxide (Singh et al. 2014).HSP20 is also essential for desiccation tolerance in Azotobacter vinelandii (Cocotl-Yañez et al. 2014).HSP20s are also found in several types of bacteriophages (Maaroufi and Tanguay 2013), and are presumably involved in the maturation of capsid proteins and/or stress resistance in their hosts (Sullivan et al. 2010;Chen et al. 2020).Furthermore, heterologous overexpression of HSP20 derived from multiple organisms can enhance cellular tolerance to diverse stresses in E. coli cells (Table 1).Ezemaduka et al. (2014) demonstrated that the heterologous expression of small HSPs derived from the nematode Caenorhabditis elegans allowed E. coli to grow at 50 °C, which is 3 °C higher than its maximum growth temperature.This finding is notable, particularly because C. elegans is a mesophilic organism (growth temperature range 15-25 °C), which is incapable of growing at such high temperatures  (Gupta et al. 2007).This finding encouraged us to investigate the effect of introducing HSPs from organisms with high thermal resistance.Thermophilic and thermotolerant archaea also harbor small HSPs involved in cell maintenance at high temperatures (Laksanalamai et al. 2004;Lemmens et al. 2018;Roy et al. 2022).In addition, proteins from thermophiles and hyperthermophiles exhibit excellent tolerance not only to high temperatures, but also to other stresses, such as those caused by organic solvents and detergents (Owusu and Cowan 1989;Atomi 2005).In this study, we aimed to introduce 18 small HSP20s from 12 thermophilic and thermotolerant bacteria into E. coli (Table 2) and evaluate their effects on various physical and chemical cellular stresses.

Strains and culture condition
The strains and plasmids used in this study are summarized in Table 3. E. coli strain One Shot TOP10 (Invitrogen, Carlsbad, CA, USA) was used for gene cloning, and Rosetta 2 (DE3) pLysS (Novagen, Merck, Darmstadt, Germany) or BW25113 was used for the expression of genes encoding each HSP20.E. coli strains or their transformants were cultivated in Luria Bertani (LB) medium at 37 °C at 180 rpm.

Construction of expression plasmids for small HSPs
Two types of plasmids were constructed for the expression analyses of HSP20s using the pET28a and pBAD30 vectors as the backbone.Primers used in this study are listed (Online Resource 1).Genomic DNA was extracted from cultured bacteria using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA).Genes encoding HSP20s were amplified using PrimeSTAR GXL DNA polymerase (Takara Bio, Osaka, Japan).For constructing pET28abased plasmids, the amplicons and pET28a were digested with NcoI-HF/SacI-HF or NcoI-HF/HindIII-HF.The GGA codon for glycine was added behind the start codon to avoid a frameshift in NcoI-HF site.The digested products were purified using the Wizard SV Gel and PCR Clean-up System (Promega) and ligated using T4 DNA ligase (Nippon Gene, Tokyo, Japan), following the manufacturer's instructions.For constructing pBAD30-based plasmids, the amplified fragments were assembled using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, MA, USA).The constructed plasmids were introduced into E. coli TOP10 cells through a brief heat shock (42 °C, 45 s), and the transformants were cultivated for 14-18 h at 37 °C with appropriate antibiotics (50 µg mL -1 of kanamycin for pET28a-based plasmids; 100 µg mL -1 of ampicillin for pBAD30-based plasmids).Plasmid extraction from the transformants was performed using the Wizard Plus SV Minipreps DNA Purification System (Promega).The nucleotide sequences of the constructed plasmids were confirmed using Sanger sequencing.

Verification of HSP20 expression in E. coli
The expression of HSP20s was confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).The pET28a-based plasmids were introduced into E. coli Rosetta2 (DE3) pLysS competent cells.The transformants were pre-cultivated overnight in LB broth including 50 µg mL -1 kanamycin and 17 µg mL -1 chloramphenicol.The culture was inoculated in fresh medium with the same antibiotics at 37 °C at 180 rpm.Isopropylβ-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.2 mM in the early logarithmic phase (OD 600 = 0.2-0.4) to induce the expression of each HSP20.
After induction for 5 h, cells were washed twice with 50 mM Tris-HCl (pH 7.5) and resuspended in the same buffer (200 mg wet cells ml −1 ).Each suspension was subjected to cell disruption via sonication using an ultrasonic disruptor (UD-201; Tomy Seiko, Tokyo, Japan).The sonication conditions were 10 flashes (output 3, duty 75) and cooling for 10 cycles (30 s on ice).The crude lysate was centrifuged at 15,000 × g at 4 °C for 15 min to separate the soluble and insoluble fractions.The soluble fraction was heat treated at 70 or 80 °C for 30 min to evaluate the heat resistance of small HSPs briefly.The 5 µL of each fraction was subjected to SDS-PAGE, and proteins were visualized by staining the gels with Coomassie Brilliant Blue R250.

Survival assay under abiotic stresses
The viability of E. coli cells was evaluated under extreme conditions (heat, cold, acidic, alkaline, and osmophilic).
The transformants of E. coli strain Rosetta2 (DE3) pLysS were cultivated to the logarithmic stage (OD 600 = ca.0.7) at 37 °C in LB medium with 50 µg mL -1 kanamycin and 17 µg mL -1 chloramphenicol.The preculture (50 µL) was transferred into 5 mL of fresh medium supplemented with 0.2 mM IPTG, 50 µg mL -1 kanamycin and 17 µg mL -1 chloramphenicol.After induction for 15 h, 1 mL of the culture was centrifuged at 10,000 × g for 3 min.Cell pellets were washed twice with an equal volume of 0.8% sodium chloride (NaCl) and subjected to various stress conditions.For heat and cold treatments, the washed pellets were resuspended in 1 mL LB medium (room temperature, pH 7, and 1% (w/v) of NaCl).The cell resuspension was transferred to 1.5 mL tubes, and the tubes were incubated at 52 °C (heat stress) for 30 min in a water bath or -25 °C (cold stress) for 6 h in a freezer.Heat-or cold-treated samples were collected and serially diluted in 0.8% NaCl solution.To calculate accurate viability with or without the expression of hsp20 genes, each diluted sample was spotted onto a solid LB medium without antibiotics or inducers.The colony-forming units (CFU) in each sample were counted in quintuplicate as biological replicates.Cell viability was calculated by comparing the CFU before and after treatment.As a negative control, E. coli cells transformed with empty pET28a (+) vector (Novagen) or pET28a-ivy coding Ivy family C-type lysozyme inhibitor in E. coli (158 aa), a protein with similar molecular weight of HSP20, were used.
As a positive control, E. coli cells harboring pET28a with genes coding small HSPs from E. coli (ibpA or ibpB), containing functional domains similar to those of the HSP20s from thermotolerant bacteria, or small HSP from C. elegans (CE) were used (Table 2).For acidic, alkaline, and osmophilic conditions, the washed pellets were resuspended in the following three different types of modified LB media: medium adjusted to pH 3 with HCl, medium adjusted to pH 11 with NaOH, or medium containing 10% (w/v) NaCl.Cell viability was evaluated in the same manner as that for heat and cold treatments.
Statistical analyses were conducted using the oneway ANOVA test to compare the control strain harboring pET28a-ivy with the HSP20-expressed strains, and p-values below 0.05 and 0.01 were set as significance thresholds for statistical significance in this study.

Effect of long-term heat stress on cell viability
Viability of the recombinant E. coli expressing thermophilic HSP20 was tested after long-term heat treatment.Two hsp20 genes (O2 and TS in Table 2) that enhance the thermotolerance of E. coli were evaluated.To avoid cell toxicity due to excess HSP20 production by the IPTG-induced systems, plasmids were reconstructed using pBAD30 containing an arabinose-induced promoter.A plasmid harboring the HSP17 gene from C. elegans and an empty vector (pBAD30) was used as positive and negative controls, respectively.Each plasmid was introduced into E. coli strain BW25113.The transformants were cultivated in LB medium containing 0.02% arabinose and 100 µg mL −1 of ampicillin at 37 °C overnight.The culture was exposed to high temperatures (52 °C) in a water bath for 5 days.Sampling was performed at 0, 0.5, 8, 24, 48, and 120 h to check cell viability using CFU on LB agar plates.To determine the proliferation ability of long-term heat-treated cells, the culture of each strain after 120 h was inoculated and cultured in LB medium at 37 °C and 180 rpm.

Phylogenetic analysis of small HSPs
Phylogenetic analyses were performed using the amino acid sequences of small HSPs used in this and previous studies.In this study, 18 small HSP20s were selected from 13 thermophilic bacteria belonging to various taxonomic groups.The amino acid sequences were obtained from the Kyoto Encyclopedia of Genes and Genomes.Multiple sequence alignments were performed using Clustal Omega (https:// www.ebi.ac.uk/ Tools/ msa/ clust alo/).A phylogenetic tree was constructed by the neighbor-joining method using Genome Workbench version 3.5.0and was visualized by iTOL version 6 (https:// itol.embl.de/).

Overexpression of HSPs
Eighteen HSP20s derived from 12 genera of thermotolerant bacteria were tested to evaluate their effects on the viability of E. coli under harsh conditions (  For expression of hsp20 from Geobacillus kaustophilus pET28a-G2 For expression of hsp20 from Geobacillus kaustophilus pET28a-G3 For expression of hsp20 from Geobacillus kaustophilus pET28a-R1 For expression of hsp20 from Rhodothermus marinus pET28a-R2 For expression of hsp20 from Rhodothermus marinus pET28a-R3 For expression of hsp20 from Rhodothermus marinus pET28a-O1 For expression of hsp20 from Oceanithermus profundus pET28a-O2 For expression of hsp20 from Oceanithermus profundus pET28a-O3 For expression of hsp20 from Oceanithermus profundus pET28a-TK For expression of hsp20 from Thermoanaerobacter kivui pET28a-KO For expression of hsp20 from Kosmotoga olearia pET28a-TA For expression of hsp20 from Thermovibrio ammonificans pET28a-TE For expression of hsp20 from Thermosynechococcus elongatus pET28a-TS For expression of hsp20 from Tepidimonas sediminis pET28a-TM For expression of hsp20 from Thermotoga maritima pET28a-PH For expression of hsp20 from Pseudidiomarina halophila pET28a-HT For expression of hsp20 from Hydrogenobacter thermophilus pET28a-DT For expression of hsp20 from Desulfurobacterium thermolithotrophum pET28a-CE For expression of hsp20 from Caenorhabditis elegans (nematode) pET28a-EA For expression of hsp20 from Escherichia coli pET28a-EB For expression of hsp20 from Escherichia coli pBAD30 Empty vector (resistance, ampicillin; inducer, arabinose) For long-term heat assay pBAD30-O2 For expression of hsp20 from Oceanithermus profundus pBAD30-TS For expression of hsp20 from Tepidimonas sediminis pBAD30-PH For expression of hsp20 from Pseudidiomarina halophila pBAD30-CE For expression of hsp20 from Caenorhabditis elegans (nematode) SDS-PAGE confirmed the presence of 17 HSP20s, except for O3 (OP2086 from Oceanithermus profundus), in the soluble fraction, suggesting their successful soluble expression (Fig. 1).This result is consistent with those of previous studies where various small HSPs from eukaryotes and prokaryotes were successfully expressed in E. coli (Table 1).In addition, the expression of each HSP20 did not seem to defect the growth of the host strains under the non-stress condition at 37 °C in comparison with that of control strains expressing a non-HSP20 protein with similar molecular weight (ivy; see Supplementary Method and Online Resource 3).HSP20s derived from the thermophilic bacteria used in this study remained in soluble forms even after heat treatment (80 °C, 30 min), indicating their thermostable structure.In contrast, the HSP20 from mesophilic bacterium Pseudidiomarina halophila was found in the supernatant after exposure to 70 °C but not to 80 °C for 30 min.P. halophila is mesophilic, and its optimum growth temperature was the lowest among the bacteria tested in this study (Table 2).Therefore, the structures of the protein chaperones from thermophiles were more stable than those from mesophilic P. halophila, as expected.

Temperature resistance of HSP20-expressed strains
To reveal the effect of each HSP20 on elevated and cold temperatures, we determined cell viability after exposure to elevated temperature (52 °C, 30 min) and cold temperature (-25 °C, 6 h).Most HSP20s from thermotolerant bacteria used in this study improved the resistance of E. coli to high and low temperatures as well as host's small HSPs (EA and EB) (Fig. 2).Majority of the transformants demonstrated higher viability after heat treatment than that of the control strains harboring pET28a or pET28a-ivy.In particular, the expression of O2, TE, or TS increased the cell viability equal to or greater than that of EA, EB, and CE from mesophilic organisms, which allowed the growth of E. coli at temperatures higher than its maximum growth temperature (Ezemaduka et al. 2014).On the other hand, eight HSP20s improved the cell viability after cold treatment.The cell viability of the control strain with empty vector (NC) was significantly low, possibly due to the gradual freezing process from room temperature to -25 °C.The control strain (ivy) overexpresses the small non-HSP protein also showed improved viability, suggesting that an excess of low-molecular-weight proteins in the cells may reduce cell stress like a compatible solute.These results are consistent with those of previous studies suggesting that HSP20s confer resistance to host cells at cold and high temperatures (Table 1).
However, TA and DT did not significantly improve stress resistance.Their amino acid sequences were 86.7% identical, which was higher than that (45.1% or less) between TA or DT and the other HSP20s used in this study (Online Resource 4).Alignment analyses suggested the amino acid residues, which are possibly involved in the chaperone activity of the proteins, in the α-crystallin domain of TA and DT (Online Resource 5).In addition, the molecular weights of TA and DT (approximately 20 kDa) were considerably higher than those of the other HSP20s (15-18 kDa).Both HSP20s were derived from thermophilic bacteria belonging to a similar taxonomic group (Aquificae).Therefore, these HSPs may be functionally different from other HSP20s.The abbreviation of HSP20 is corresponding to that in Table 2. "NC" and "ivy" represent specific strains of E. coli Rosetta 2 (DE3) pLysS carrying different plasmids (pET28a and pET28a-ivy, respectively).The x-marks and filled circles represent the actual data points and the average values, respectively.Symbol mark indicates statistical differences with the control strain harboring pET28a-ivy by the one-way ANOVA method (*p-value < 0.05; **p-value < 0.01)

Multiple resistance of HSP20-expressed strains: acidic, alkalic, and osmophilic
To reveal the effect of each HSP20 on the other stresses except for temperature, we investigated the cell viability after exposure to acidic (pH 3, 1 h), alkalic (pH 11, 1 h), and hyperosmotic (10% NaCl, 6 h) conditions (Fig. 3).Compared to the control strains harboring pET28a (NC) or pET28a-ivy (ivy), HSP20 also improved the viability of E. coli to multiple stresses other than extreme temperatures.For seven HSP20s (R1, O1, O2, TK, TE, TS, and PH), cell viability under acidic conditions significantly increased than that of the control strain with pET28a-ivy and were more than 100-fold higher compared to that of the control strains harboring empty vector (NC), suggesting that most HSP20s including small HSPs from E. coli enhanced the acid tolerance of E. coli (Fig. 3a).Several HSP20s also improved the cell viability under alkaline conditions (Fig. 3b).Especially, O2 and TS improved cell viability with statistical significance by more than 100-fold in comparison with the ive-expressing strain.In addition, some HSP20s, including O2 and TS, enhanced the viability of E. coli after exposure to high osmotic pressure (10% [w/v] NaCl) (Fig. 3c).Two types of HSP20s, O2 and TS, successfully improved tolerance to a variety of stresses in E. coli.On the other hand, TA and DT did not improve the viability under most stress conditions in comparison to the other HSP20s.Although we have identified the two amino acid residues (alanine in positions 149 and 150) conserved specifically in TS and O2 and five residues (Positions 103,106,122,128,and 156) found only in TA and DT (Online Resource 5), the impact of these residues on stress tolerance remains unclear.2. "NC" and "ivy" represent specific strains of E. coli Rosetta 2 (DE3) pLysS carrying different plasmids (pET28a and pET28a-ivy, respectively).The faction marks and filled circles represent the points of each measurement and the average values, respectively.Symbol mark indicates statistical differences with the control strain harboring pET28a-ivy by the one-way ANOVA method (*p-value < 0.05; **p-value < 0.01)

Cell viability under long-term heat stress conditions
We verified whether the maximum growth temperature of E. coli could be increased by HSP20s by O2 or TS expression.Although two sets of expression systems, pET28a/Rosetta 2 (DE3) pLysS and pBAD30/BW25113, were tested, HSP20 expression did not affect the maximum growth temperature (47 °C) of E. coli (Online Resource 6).The maximum growth temperature was consistent with the value in the previous study (Schink et al. 2022).In contrast, for strain BW25113 harboring pBAD30-TS, some viable cells were identified after long-term heat treatment (52 °C, 5 days) using the colony-forming assay (Fig. 4a).In addition, the strain could proliferate at 37 °C after the treatment for 5 days (Fig. 4b), although the other strains, including negative (with empty vector) and positive (expressed CE) control strains, did not proliferate.Therefore, HSP20 from Tepidimonas affords E. coli to survive after prolonged (> 100 h) hightemperature stress.
We further investigated how HSP20 (TS) contributes to the homeostasis of E. coli under severe conditions.Compared with the other thermophiles used in this study, TS (beta-proteobacteria) is phylogenetically similar to E. coli (gammaproteobacteria).Therefore, the effective protection of E. coli cellular proteins by HSP20 (TS) may be due to their phylogenetic proximity and compatibility with structurally similar proteins.We intend to elucidate the detailed mechanisms of this phenomenon in future studies.
In conclusion, we demonstrated the improvement in E. coli stress tolerance by the heterologous expression of HSP20s from thermotolerant microorganisms.Expression of several HSP20s enhanced stress tolerance in E. coli as much as or more than those of ibpA and ibpB from E. coli.In particular, E. coli with thermotolerant HSPs, such as O2 and TS, exhibited remarkable stress tolerance, comparable to that of C. elegans HSP20.These findings indicate the potential of thermotolerant HSPs as molecular tools for improving stress tolerance in E. coli.

Fig. 1
Fig. 1 Sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis of 18 heat shock protein (HSP)20s from insoluble and soluble fractions expressed in Escherichia coli.Lane M, molecular mass marker (Precision Plus Protein Dual Color Standards, BIORAD); Lane I, insoluble fraction; Lane S, soluble fraction; Lane H, soluble fraction after heat treatment at 80 °C for 30 min.Only for Pseudidiomarina halophila, lane H indicates soluble fraction after heat treatment at 70 or 80 °C for 30 min, respectively

Fig. 2
Fig.2Viability of each mutant after temperature variation: a viability of each mutant after heat treatment (52 °C for 30 min).b Viability of each mutant after freeze-thaw treatment (− 25 °C for 6 h).The abbreviation of HSP20 is corresponding to that in Table2."NC" and "ivy" represent specific strains of E. coli Rosetta 2 (DE3) pLysS carrying different plasmids (pET28a and pET28a-ivy, respectively).The x-marks and filled circles represent the actual data points and the average values, respectively.Symbol mark indicates statistical differences with the control strain harboring pET28a-ivy by the one-way ANOVA method (*p-value < 0.05; **p-value < 0.01)

Fig. 3
Fig. 3 Viability of each mutant under multiple stress conditions: a viability of each mutant exposed to acidic condition (pH 3 for 1 h); b viability of each mutant exposed to alkaline condition (pH 11 for 1 h); c viability of each mutant exposed to high osmotic condition [10%(w/v) of NaCl for 6 h].The abbreviation of each HSP20 is corresponding to that in Table2."NC" and "ivy" represent specific strains of E. coli Rosetta 2 (DE3) pLysS carrying different plasmids (pET28a and pET28a-ivy, respectively).The faction marks and filled circles represent the points of each measurement and the average values, respectively.Symbol mark indicates statistical differences with the control strain harboring pET28a-ivy by the one-way ANOVA method (*p-value < 0.05; **p-value < 0.01)

Fig. 4
Fig. 4 Cell viability after long-term heat treatment.a The time course of cell viability of strain BW25113 harboring pBAD30-TS after longterm heat treatment (52 °C).The faction marks and filled circles represent the points of each measurement and the average values, respectively.Most probable number (MPN) shows the estimated number of viable cells in long-term heat-treated samples.b The cultivation results at 37 °C for 3 days using strains exposed to 52 °C for 5 days.N/A indicates the time point of no measurement

Table 1
Previous studies for small HSPs on stress resistance of Escherichia coli

Table 2
HSP20s used in this study

Table 3
Strains and plasmids used in this study