Curr Microbiol DOI 10.1007/s00284-015-0811-0
Overexpression of Small Heat Shock Protein Enhances Heat- and Salt-Stress Tolerance of Bifidobacterium longum NCC2705 Gul Bahar Khaskheli1 • FangLei Zuo1,2 • Rui Yu1,2 • ShangWu Chen1,2
Received: 15 October 2014 / Accepted: 23 February 2015 Springer Science+Business Media New York 2015
Abstract Bifidobacteria are probiotics that are incorporated live into various dairy products. They confer healthpromotive effects via gastrointestinal tract colonization. However, to provide their health-beneficial properties, they must battle the various abiotic stresses in that environment, such as bile salts, acids, oxygen, and heat. In this study, Bifidobacterium longum salt- and heat-stress tolerance was enhanced by homologous overexpression of a small heat shock protein (sHsp). A positive contribution of overproduced sHsp to abiotic stress tolerance was observed when the bacterium was exposed to heat and salt stresses. Significantly higher survival of B. longum NCC2705 overexpressing sHsp was observed at 30 and 60 min into heat (55 C) and salt (5 M NaCl) treatment, respectively. Thermotolerance analysis at 47 C with sampling every 2 h also revealed the great potential tolerance of the engineered strain. Cell density and acid production rate increased for the sHsp-overexpressing strain after 8 and 10 h of both heat and salt stresses. In addition, tolerance to bile salts, low pH (3.5) and low temperature (4 C) was also increased by homologous overexpression of the sHsp hsp20 in B. longum. Results revealed that hsp20 overexpression in Blongum NCC2705 plays a positive cross-protective role in upregulating abiotic responses, ensuring the organism’s & ShangWu Chen [email protected] 1
Key Laboratory of Functional Dairy Science of Chinese Ministry of Education and Municipal Government of Beijing, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, People0 s Republic of China
2
Beijing Engineering Research Center of Function Agricultural Microbiology, Beijing 100083, People0 s Republic of China
tolerance to various stress conditions; therefore, sHspoverexpressing B. longum is advised for fermented dairy foods and other probiotic product applications.
Introduction Bifidobacteria are considered to be probiotic due to their contribution to the maintenance of gastrointestinal health [21]. Health benefits offered by bifidobacteria to its host, as supported by clinical evidence, have led to their wide application as probiotic components of health-promoting foods, especially in fermented dairy products [20,21,24]. Today, several probiotic species of the genus Bifidobacterium are applied in dairy products and infant foods [8], as well as in high numbers as live bacteria in numerous food products with various health claims [16]. When bifidobacteria are utilized for probiotic applications, high temperature, low water activity, osmotic shock, and oxidative stresses are frequently encountered. Genes belonging to the different heat shock protein (Hsp) families, for example groEL, groES, dnaK, dnaJ, grpE, clpB, clpC, and clpP, are induced to respond upon exposure of bifidobacterial cultures to these stressful conditions [28]. NonHsps such as heme-dependent catalase (Kat) and Mn2?dependent superoxide dismutase (Sod) have been investigated for synergistic protection of Bifidobacteriumlongum via the oxidative stress response [33]. However, the small heat shock proteins (sHsps) have recently garnered much interest in bacteria, including bifidobacteria, yeast, plants and animal cells for their role in stress tolerance [9,11,12,17,29]. The sHsps are ubiquitous ATP-independent molecular chaperones that prevent protein aggregation upon stressinduced unfolding [1]. These proteins contain a conserved
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a-crystallin domain and commonly form large dynamic oligomers, often composed of up to 24 subunits [22]. Overexpression of sHsps in some bacteria has been shown to increase tolerance to a variety of stresses [11]. Expression analysis of an analog of the sHsp gene hsp20 in Bifidobacteriumbreve showed significant induction of its expression during heat and osmotic stresses [29,31]; hsp20 (BL0576) is the most rapidly and strongly induced component in B. longum NCC2705 under heat shock, oxidative stress, and starvation conditions [16]. Bioinformatic analysis revealed that bifidobacteria hsp20 analogs may have been obtained via horizontal gene transfer from Firmicutes [18]. Although various aspects of the response of sHsps in bifidobacteria have already been studied, the high level of their constitutive expression and their role in heat- and saltstress responses are not well documented for B. longum. To investigate and confirm the function of the sHsps in bifidobacteria, hsp20 gene was cloned and homologously overexpressed in B. longum NCC2705. A positive contribution of the overproduced sHsp to the bacterial abiotic stress response, when exposed to a variety of environmental stresses, such as heat and salt stress and a multicross-protective mechanism was observed.
Materials and Methods Bacterial Strains, Plasmids, and Culture Conditions The bacterial strains and plasmids used in this study are listed in Table 1. Bifidobacteria strains were generally grown at 37 C in MRS medium supplemented with
0.05 % (wt/vol) L-cysteine HCl (MRSc) under 80 % N2– 10 % H2–10 % CO2 unless otherwise noted. Escherichia coli strains were grown at 37 C in Luria–Bertani (LB) broth with vigorous shaking. Antibiotics (Sigma) were added to the appropriate media at the following concentrations: 100 l ml-1 ampicillin and 100 l ml-1 spectinomycin for E. coli and 75 l ml-1 spectinomycin for bifidobacteria. Cloning of hsp20 (BL0576) from Bifidobacterium longum Strain NCC2705 Genomic DNA from B. longum NCC2705 was isolated following the method of Zuo et al. [32]. The open reading frame (ORF) of hsp20 (BL0576) encoding the sHsp was amplified by polymerase chain reaction (PCR) using genomic DNA as the template and the primer pair sHsp-F (50 ACCATGGCAATGTTTCCGGCTTT-30 ) and sHsp-R (50 GGTACCTCAGCCCTCAATCGCGAT-30 ). The underlined bases represent NcoI and KpnI recognition sites, respectively. The PCR was carried out in a 25-ll reaction mixture for 30 cycles at 94 C for 1 min, 65 C for 0.5 min, and 72 C for 1 min, with a final extension at 72 C for 10 min. The amplified product was analyzed by electrophoresis and purified using the Gel Band Purification kit (Biomed, Beijing, China). Construction of Expression Vector and Transformation The purified hsp20 PCR fragment was ligated to the pMD19-T simple vector and transformed into E. coli strain
Table 1 List of plasmids and strains used in the current study Plasmid or strain
Feature or sequence
Source or reference
pMD19-T simple
2.7-kb E. coli cloning vector, Ampr
Takara, Dalian, China
pMD19PmcsT
3.1-kb pMD19-PIT derivate, a linker containing a multiple cloning site was substituted for Bifidobacterium breve Sec2 protein signal peptide-coding region and human IL-10-encoding gene
[33]
pDP870
4.3-kb E. coli-B. longum shuttle cloning vector, Spr
[16]
pMD19PsHsp
pMD19-T simple derivate, containing Pgap–sHsp–Thup expression cassette
This work
pDP401sHsp
5.6-kb; pDP870 derivate, contain Pgap–sHsp–Thup expression cassette
This work
NCC2705
Wild-type B. longum NCC2705, isolated from adult human feces (GenBank Accession no. AE014295)
[25]
NCC2705pDP401
B. longum NCC2705 harboring plasmid pDP401
[34]
NCC2705sHsp
Hsp20-overexpressing B. longum NCC2705
This work
Plasmids
Strains
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DH5a to form pMD19-sHsp. pMD19-sHsp was cut with NcoI and KpnI, and the hsp20-containing fragment was ligated to the NcoI- and KpnI-digested backbone fragment of pMD19-PmcsT [33] and transformed into E. coli DH5a to form pMD19-PsHsp. Then pMD19-PsHsp was digested with NdeI and XhoI, and the Pgap–sHsp–Thup expression cassette fragment was ligated to the NdeI- and XhoI-digested backbone fragment of pDP870 and transformed into E. coli DH5a to form pDP401-PsHsp. The recombinant plasmid pDP401-PsHsp was sequenced in an ABI 3730XL DNA Analyzer (Applied Biosystems). Recombinant plasmid pDP401-PsHsp and the corresponding control plasmid pDP401 were introduced into B. longum NCC2705 electrocompetent cells by electroporation following the method of Argnani et al. [3]. Transformants were confirmed by colony PCR and plasmid DNA extraction. Recombinant B. longum strains harboring plasmid pDP401-PsHsp (designated NCC2705-sHsp) or pDP401 (control, designated NCC2705-pDP401) were confirmed by plasmid DNA extraction and PCR detection or 15 % polyacrylamide gel electrophoresis, respectively. Determination of the Stress Tolerance of Hsp20Overproducing B. longum NCC2705 Two cultures of B. longum NCC2705-control and NCC2705-sHsp transformants were grown in MRSc broth supplemented with spectinomycin to an OD600 of 1.0. Thermotolerance of B. longum NCC2705 was investigated by monitoring survival in MRSc broth at 55 C for 60 min. Aliquots were removed at 0, 30 and 60 min, and serial dilutions were spread on MRSc agar and then anaerobically incubated at 37 C for 48 h. Salt tolerance was determined by adding 5 M NaCl and monitoring culture viability for 1 h with aliquots taken at 0, 30, and 60 min. To monitor the growth of B. longum NCC2705 under long-term stress conditions, overnight growth culture was inoculated (2 %) in 50 ml MRSc broth supplemented with spectinomycin. For long-term heat stress, the cultures were grown at 47 C and absorbance was measured at 2-h intervals. For the long-term salt-stress assay, MRSc broth was supplemented with 200 mM NaCl, the culture was grown at 37 C, and absorbance was measured at 2-h intervals. To determine the tolerance of sHsp-overproducing B. longum NCC2705 to multiple stresses, the exponentially growing B. longum cells were inoculated in MRSc broth (pH 3.5 or bile salt 0.1 %) for pH or bile salt stress. Aliquots were taken at 0, 30, and 60 min, and serial dilutions were spread on MRSc agar and then anaerobically incubated at 37 C for 48 h. For low-temperature (4 C) stress, overnight culture was incubated at 4 C and aliquots were removed at 0, 6, 12 and 24 h; serial dilutions were spread
on MRSc agar and then anaerobically incubated at 37 C for 48 h. Quantitative Reverse Transcription PCR (qRTPCR) Total RNA from exponentially growing (OD600 of 0.6) B. longum strains was isolated using TRIzol Reagent (Invitrogen) and then treated with RNase-free DNase I (Takara). RNA concentrations were determined by spectrophotometry at 260 nm. The absence of residual DNA in the DNase I-digested total RNA was confirmed by PCR. Reverse transcription was carried out using M-MLV Reverse Transcriptase (Promega) in a 20-ll reaction volume containing 150 ng of random primers and 1 mM dNTP mix (Tiangen, China). Primer pairs (sHsp-F1: 50 -CCGTAATGCCCGC CAACA-30 and sHsp-B1: 50 -GCAGCCACTTGCCC GAAT-30 ) were designed with Primer Premier 5 software (Palo Alto, CA) to amplify 234-bp regions of hsp20 for qRTPCR. Real-Time PCR was performed using a SYBR Green assay kit (Takara) and optimized primer concentrations in a 7500 Fast Real-Time PCR system (Applied Biosystems). Gene expressions were normalized by the DDCT method [27] and using 16S ribosomal DNA as the reference gene for the calculations [30]. The experiment was performed in triplicate and the average results are reported. Statistical Analysis All of the data from three independent experiments were analyzed by two-tailed Student’s t test. All analyses were performed using Microsoft Office Excel, version 2007. Values of P \ 0.05 were considered significant.
Results Overexpression of sHsp (hsp20 BL0576) in B. longum NCC2705 A ca. 513-bp PCR product, consistent with the theoretical length of sHsp (501 bp), was digested and used to construct the expression vector pDP401-sHsp, which was then verified by DNA sequencing. The 501-bp ORF encodes 167 amino acids with a calculated molecular mass of 18.8 kDa. B. longum transformation with plasmid pDP401 as a control (NCC2705-pDP401) or pDP401-sHsp (NCC2705sHsp) was confirmed by colony PCR and plasmid isolation (data not shown). In the exponential phase of NCC2705sHsp growth, transcript level of sHsp was 7.1-fold higher than that in the control NCC2705 detected by qRT-PCR (Fig. 1), suggesting that the sHsp was successfully overexpressed in NCC2705-sHsp.
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Fig. 1 Relative expression fold change in hsp20 gene expression in B. longum NCC2705 transformants detected by reverse transcription PCR. The fold change was calculated as the ratio between signals observed in hsp20-overexpressing strain (sHsp) and strain harboring pDP401 (Control). They were normalized using housekeeping 16S rRNA as an internal control. The mean values from three independent determinations ±SD are shown
Heat-Stress Tolerance of sHsp-Overproducing B. longum NCC2705 Heat tolerance of B. longum overexpressing sHsp (NCC2705-sHsp) was evaluated (Fig. 2a). Variation was observed in both sHsp-overexpressing and control B. longum strains. The average survival (%) of NCC2705sHsp at 55 C was 0.056 and 0.078, compared to 0.016 and 0.00 for NCC2705-pDP401, at 30 and 60 min, respectively (Fig. 2a). The overall survival rate of NCC2705-sHsp was 3.44-fold that of the control strain after 30 min at 55 C (P \ 0.05). It was also noted that the NCC2705-sHsp strain continued to grow while the NCC2707-pDP401 (control) strain stopped growing completely from 30 to 60 min at 55 C (Fig. 2a). The experimental analysis of thermotolerance was extended to a study of long-term growth performed at 47 C with monitoring at 2-h intervals. Samples were analyzed for optical density (OD600) and pH and the results are summarized in Fig. 2b. A slight numerical difference in both OD600 and pH was revealed with time. NCC2705-sHsp increased, whereas NCC2705-pDP401 grew more slowly after 6 h. The pH values of both NCC2705-sHsp and the control strain (NCC2705-pDP401) decreased with time, but the pH values of NCC2705-sHsp were lower than those of NCC2705-pDP401 at all time points (Fig. 2b). Salt-Stress Tolerance of sHsp-Overproducing B. longum NCC2705 The results obtained for salt tolerance (to 5 M NaCl) of B. longum (NCC2705-sHsp and NCC2705-pDP401) are
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Fig. 2 Effects of hsp20 overexpression on heat tolerance of B. longum NCC2705 a Survival of B. longum strains under heat stress (55 C). Light bars and dark bars represent B. longum control (NCC2705-pDP401) and hsp20-overexpressing (NCC2705-sHsp) strains, respectively, after 30 and 60 min of heat treatment. b Longterm heat tolerance to heat stress (47 C): cell density, measured as OD600, shows changes in growth rate in MRSc medium for NCC2705-sHsp (filled square) and NCC2705-pDP401 (unfilled square), and changes in acid production (pH) for NCC2705-sHsp (filled circle) and NCC2705-pDP401 (unfilled circle)
presented in Fig. 3a. The mean survival (%) of strain NCC2705-sHsp was 0.035 and 0.026, and that of NCC2705-pDP401 was 0.030 and 0.002 under salt stress for 30 and 60 min, respectively (Fig. 3a). Overall survival of NCC2705-sHsp was higher than the control strain, being 1.17- and 9.21-fold that of the control strain under 5 M NaCl at 30 and 60 min (P \ 0.05), respectively. As with the heat stress, strain NCC2705-sHsp continued to grow while strain NCC2705-pDP401 showed reduced growth under osmotic stress for 60 min (Fig. 3a). To monitor the continued salt tolerance, long-term growth analysis was carried out by inoculating the experimental strains in MRSc medium containing 200 mM NaCl at 37 C and taking aliquots at 2-h intervals for OD600 and pH determinations. The results are
G. B. Khaskheli et al.: Overexpression of Small Heat Shock Protein…
Fig. 3 Effects of hsp20 overexpression on tolerance to salt stress in B. longum NCC2705 a Survival of B. longum strains under salt stress (5 M NaCl). Light bars and dark bars indicate B. longum NCC2705pDP401 and NCC2705-sHsp, respectively, after 30 and 60 min of salt treatment. b Long-term tolerance to salt stress (200 mM NaCl): cell density, measured as OD600, shows changes in growth rate in MRSc medium for NCC2705-sHsp (filled square), and NCC2705-pDP401 (filled circle), and changes in acid production (pH) for NCC2705sHsp (unfilled square) and NCC2705-pDP401 (unfilled circle)
summarized in Fig. 3b. A marked difference was observed for OD600 and pH values of both B. longum overexpressing sHsp and control strains with time. Both strains grew well up to 8 h incubation; thereafter, NCC2705-sHsp grew more than NCC2705-pDP401 (Fig. 3b). Similar to the heat tolerance results, the pH values of NCC2705-sHsp and NCC2705-pDP401 decreased with time, and the pH values of NCC2705-sHsp were lower than those of the control strain (NCC2705pDP401) at all time points (Fig. 3b). Multiple Stress Tolerance of Hsp20-Overexpressing B. longum NCC2705 In addition to abiotic stresses (temperature and salt), experiments were carried out with bile salts (0.1 %), low pH (3.5), and low temperature (4 C) as selective criteria for stress tolerance of hsp20-overexpressing strains. The results are presented in Fig. 4. All
treatments revealed the prominent stress tolerance of strain NCC2705-sHsp compared to the control (NCC2705-pDP401). The mean survival (%) of NCC2705-sHsp was 0.027 and 0.114, compared to 0.024 and 0.002 for NCC2705pDP401 under pH stress (3.5) at 30 and 60 min, respectively (Fig. 4a). Furthermore, a statistically significant difference in survival (%) of B. longum NCC2705 overexpressing sHsp was observed compared to NCC2705pDP401, and overall survival rate of NCC2705-sHsp increased to 4.8- and 13.19-fold that of the control strain after 30 and 60 min, respectively. The average survival (%) of strain NCC2705-sHsp was higher (0.045 and 0.015) than that of NCC2705-pDP401 (0.025 and 0.002) under bile salt stress for 30 and 60 min, respectively (Fig. 4b). Moreover, NCC2705-sHsp survival increased 1.80- and 5.3-fold that of the control strain at 30 and 60 min, respectively. Mean survival (%) of sHsp-overexpressing B. longum (NCC2705-sHsp) was 0.056, 0.065, and 0.008 versus 0.052, 0.028, and 0.003 for control B. longum (NCC2705-pDP401) under low-temperature (4 C) stress at 6, 12, and 24 h, respectively. Similar to pH and bile salt stress, B. longum overexpressing sHsp (NCC2705-sHsp) seemed to adapt to the low temperature, growing 1.08-, 2.34- and 2.58-fold more than control B. longum (NCC2705-pDP401) at 6, 12, and 24 h, respectively.
Discussion Bifidobacteria are utilized for probiotic applications in the gastrointestinal tract (GIT) of mammals. As autochtonous bacteria in the GIT, they generally encounter a variety of abiotic stresses. However, proteins of different Hsp families are active in the bifidobacteria’s response to stressful conditions [28], with sHsps being the most attractive subject for studies of stress tolerance potential [9,11,12] because their overexpression in bacteria results in increased tolerance to a variety of stresses [11]. Expression analysis of the hsp20 analog in B. breve showed significant induction during heat and osmotic stresses [16,29,31]. B. longum NCC2705 contains different sHsps, including hsp20 which is believed to be a common stress response factor, being the most rapidly and highly induced gene under heat shock, oxidative stress, and starvation conditions [16]. We confirmed homologous overexpression of this sHsp in B. longum NCC2705, accompanied by improved tolerance to heat and salt stress. In addition to the newly characterized response to abiotic heat and osmotic stresses, the effects of abiotic and/or biotic stresses—i.e., low pH, bile salts
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Characteristics of Heat- and Salt-Stress Tolerance of hsp20-Overproducing B. longum NCC2705
Fig. 4 Effect of homologous overexpression of hsp20 on survival rates of B. longum strains. a Survival of B. longum strains under low pH (3.5), showing the B. longum NCC2705-pDP401 and NCC2705sHsp cells after 30 and 60 min of low pH treatment, respectively. b Survival of B. longum strains under bile salt (0.1 %), indicating B. longum NCC2705-pDP401 and NCC2705-sHsp cells at 30 and 60 min of bile salt treatment, respectively. c Survival of B.longum strains under low temperature (4 C); viable cells of B. longum NCC2705-pDP401 and NCC2705-sHsp were assayed after 6, 12, and 24 h incubation at 4 C. Light and dark bars indicate B. longum NCC2705-pDP401 and NCC2705-sHsp, respectively. Asterisks indicate a statistically significant difference (*P \ 0.05, **P \ 0.01) between B. longum strains NCC2705-pDP401 and NCC2705-sHsp
and low temperature—on the Hsp20-overexpressing B. longum strains were demonstrated, as discussed further on.
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Variation in growth was observed between engineered and control strains. A significantly higher number of colonies (P \ 0.005) were found for NCC2705-sHsp vs. controls at 55 C for 60 min. In addition, the overall survival rate of strain NCC2705-sHsp was higher than that of NCC2705pDP401 under these conditions. NCC2705-sHsp continued to grow at 55 C for 60 min while NCC2707-pDP401 did not. These findings show the great potential for thermotolerance of NCC2705-sHsp in comparison to NCC2707pDP401. Further, thermotolerance analysis at 47 C with monitoring at 2-h intervals also revealed the great tolerance potential of the engineered strain in the current study. However, differences were observed for both strains in optical density (OD600) and pH values with time: the OD600 for NCC2705-sHsp was higher than that of the controls after 8- and 10-h intervals. The pH values of the heattreated NCC2705-sHsp were lower than those of NCC2705-pDP401. From a technological point of view, bifidobacteria are not robust microorganisms. In general, their optimal growth temperature range is 36–38 and 41–43 C for strains of human and animal origin, respectively, although B. thermacidophilum can grow at 49 C and B. psychraerophilum at 4 C. The optimal pH range for growth is between 6.5 and 7.0, but B. animalis and B. thermacidophilum survive well at pH 3.5–4.0. These observations highlight the narrow choice of oxygen, temperature, and acidic conditions that are suitable for bifidobacterial survival, although some bifidobacteria are able to survive under more extreme conditions (e.g. B. animalis subsp. lactis strains), whereas others display a low tolerance to stress (e.g. some B. bifidum and B. longum strains); nevertheless, the physiology underlying their adaptation is far from understood. The mechanisms most extensively studied in response to technological stress of bifidobacteria are those related to heat, oxygen, and acid, as reviewed by Ruiz et al. [23]. There is some disparity in the results of studies on stress tolerance in bifidobacteria and in the stress-resistant mutants that sometimes arise spontaneously [6]. Regarding technological stresses, it is worth noting that heat-tolerant mutants of B. longum strains are obtained by successive sublethal heat-shock treatments, and the genetic basis for this adaptation has been recently documented [5, 21, 23]. Another study states that sHsps cooperate with other Hsps by binding to non-native proteins to refold them. Our results support those of Kitagawa et al. [15], who claimed that overexpression of sHsp results in enhanced heat and oxidative tolerance potential. In the current study, strong induction of sHsp was found in NCC2705-sHsp compared to NCC2705-pDP401 in response to stress. Similarly, Klijn et al. [16] observed that
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hsp20 (BL0576) expression is a common stress response in B. longum NCC2705, being the most rapidly and strongly induced gene in a variety of stresses (heat shock, oxidative stress, and starvation). Similar trends for OD and pH were noted with respect to salt tolerance, with a significantly higher number of colonies found for NCC2705-sHsp compared to NCC2705pDP401 after 30 min exposure to 5 M NaCl. In the current study, the engineered strain expressed great potential for salt tolerance, surviving longer than the control strain. Kim et al. [14], in an experiment with L. acidophilus cells, observed that 2 and 18 % (w/v) NaCl cause sublethal and lethal osmotic stress, respectively. Although B. longum NCC2705 endured the stress of 5 M NaCl, the duration of the stress could also be an important factor in strain survival. Furthermore, NCC2705-sHsp showed better salt tolerance (0.035 and 0.026) than the control strain (0.030 and 0.002) at 30 and 60 min, respectively. In previous experiments, the adaptive response to NaCl stress revealed cross-protection against heat stress [14], but not vice versa, resulting in the conclusion that Hsps might be induced by salt stress, but elevated temperature may not necessarily induce the salt stress response. Our results support the hypothesis that a sHsp-overexpressing strain can endure both salt and heat tolerance. It is, therefore, suggested that further studies be carried out on the mechanism underlying the sHsp-overexpressing strain’s tolerance to abiotic stresses (elevated temperature and salt), as well as exposure to bile salt, low temperature, and acidic conditions. Bile salts and acids are pumped into the GIT at concentrations typically ranging from 10 mM in the small intestine to 1 mM in the cecum [13]. Bile salts in the intestinal tract are a particularly difficult environmental challenge for probiotic bacteria [4]. Flahaut et al. [10] and Schmidt and Zink [26] confirmed that DnaK and GroEL are induced by bile salts. Another study also reported that cells pre-exposed to bile salts show greater resistance to elevated temperature; on the other hand, pre-exposure to heat stress did not increase resistance to lethal bile stress. Kim et al. [14] reported that bile at 0.05 % (w/v) is sublethal (cells grew slowly) and bile at 0.5 % (w/v) is lethal (most of the cells were killed at this level) to L. acidophilus. In the current study, we found a prominent difference in the growth of the sHsp-overexpressing transformant vs. the control strain. Lorca et al. [19] revealed that Hsp family factors DnaK, DnaJ, GrpE, GroES and GroEL are induced by acid stress. We also investigated the sHsp-overexpressing strain’s response to acid stress (pH 3.5): it grew markedly better than the non-overexpressing strain (Fig. 4a). Interestingly, not only did we find remarkable growth of NCC2705-sHsp under acidic conditions, but we also detected a similar pattern in the response to low temperature (4 C) and bile salt treatments.
Resistance to bile salts in bifidobacteria may be governed by a different mechanism, such as metabolic changes [2]. The hsp20 gene has been predicted to be present in the genomes of B. breve, B. longum, and B. adolescentis. Transcription of this sHsp in B. breve is upregulated under heat stress (50 C) and osmotic stress (0.7 M NaCl) [29]. Coucheney et al. [7] revealed that the sHsp gene Lo18 encodes a membrane-associated protein in Oenococcus oeni that maintains the integrity of the cell membrane during heat, ethanol, and benzyl alcohol stress. Our research findings reinforce the notion that sHsp overexpression in bifidobacteria plays a prominent role in cell viability and increased tolerance to multiple stresses.
Conclusions We conclude that overexpression of hsp20 in B. longum NCC2705 ensures the organism’s tolerance of heat and salt stresses, and provides cross-protection against low pH levels, bile salts, and low temperature (4 C) conditions which might exist in the surrounding environment. These functional properties of sHsps in bifidobacteria, an organism that is frequently added to fermented dairy and therapeutic products, may enhance protection against the various stresses imposed during the commercial process, from manufacturing to consumption. Acknowledgments This work was funded by the National Natural Science Foundation of China (No. 31071507), the National High Technology Research and Development Program (‘‘863’’ Program, No. 2008AA10Z310), and the National Science and Technology Support Program, Ministry of Science and Technology of China (2011BAD09B03).
References 1. Alexander B, Ferdinand A, Thomas K, Nathalie B, Sevil W, Michael G, Martin H, Johannes B (2012) Alternative bacterial two-component small heat shock protein systems. Proc Natl Acad Sci USA 109(50):20407–20412 2. Alp G, Aslim B (2010) Relationship between the resistance to bile salts and low pH with exopolysaccharide (EPS) production of Bifidobacterium spp. isolated from infants feces and breast milk. Anaerobe 16:101–105 3. Argnani A, Leer RJ, van Luijk N, Pouwels PH (1996) A convenient and reproducible method to genetically transform bacteria of the genus Bifidobacterium. Microbiology 142:109–114 4. Begley M, Gahan CGM, Hill C (2005) The interaction between bacteria and bile. FEMS Microbiol Rev 29:625–651 5. Berger B, Moine D, Mansourian R, Arigoni F (2010) HspR mutations are naturally selected in Bifidobacterium longum when successive heat shock treatments are applied. J Bacteriol 192:256–263 6. Collado MC, Sanz Y (2006) Method for direct selection of potentially probiotic Bifidobacterium strains from human feces based on their acid-adaptation ability. J Microbiol Method 66:560–563
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G. B. Khaskheli et al.: Overexpression of Small Heat Shock Protein… 7. Coucheney F, Gal L, Beney L, Lherminier J, Gervais P, Guzzo J (2005) A small HSP Lo18, interacts with the cell membrane and modulates lipid physical state under heat shock conditions in a lactic acid bacterium. Biochim Biophys Acta Biomembr 1720:92–98 8. De Dea Lindne J, Canchaya C, Zhang ZD, Neviani E, Fitzgerald GF, van Sinderen D, Ventura M (2007) Exploiting Bifidobacterium genomes: The molecular basis of stress response. Int J Food Microbiol 120:13–24 9. Eylesa SJ, Gierasch LM (2010) Nature’s molecular sponges: Small heat shock proteins grow into their chaperone roles. Proc Natl Acad Sci USA 107(7):2727–2728 10. Flahaut S, Frere J, Boutibonnes P, Auffray Y (1996) Comparison of the bile salts and sodium dodecyl sulfate stress responses in Enterococcus faecalis. Appl Environ Microbiol 62:2416–2420 11. Guzzo J (2012) Biotechnical applications of small heat shock proteins from bacteria. Int J Biochem Cell Biol 44:1698–1705 12. Han MJ, Yun H, Lee SY (2008) Microbial small heat shock proteins and their use in biotechnology. Biotechnol Adv 26:591–609 13. Hofmann AF (1999) The continuing importance of bile acids in liver and intestinal disease. Arch Intern Med 159:2647–2658 14. Kim WS, Perl L, Park JH, Tandianus JE, Dunn NW (2001) Assessment of stress response of the probiotic Lactobacillus acidophilus. Curr Microbiol 43:346–350 15. Kitagawa M, Matsumura Y, Tsuchido T (2000) Small heat shock proteins, IbpA and IbpB are involved in resistances to heat and superoxide stresses in Escherichia coli. FEMS Microbiol Lett 184(2):165–171 16. Klijn A, Mercenier A, Arigoni F (2005) Lessons from the genomes of bifidobacteria. FEMS Microbiol Rev 29:491–509 17. Landry J, Chre´tien P, Lambert H, Hickey E, Weber LA (1989) Heat shock resistance conferred by expression of the human HSP27 gene in rodent cells. J Cell Biol 101(1):7–15 18. Lee JH, O’Sullivan DJ (2010) Genomic insights into bifidobacteria. Microbiol Mol Biol Rev 74(3):378–416 19. Lorca GL, Font de Valdez G, Ljungh A (2002) Characterization of the protein synthesis dependent adaptive acid tolerance response in Lactobacillus acidophilus. J Mol Microbiol Biotechnol 4:525–532 20. Michelle C, Marco V, Gerald FF, van Douwe S (2011) Progress in genomics, metabolism and biotechnology of bifidobacteria. Int J Food Microbiol 149(1):4–18 21. Ouwehand AC, Salminen S, Isolauri E (2002) Probiotics: an overview of beneficial effects. Antonie Leeuwenhoek 82:279–289 22. Richter K, Haslbeck M, Buchner J (2010) The heat shock response: life on the verge of death molecular. Cell 40:253–266
123
23. Ruiz L, Patricia RM, Miguel G, Clara GDLR, Abelardo M, Sanchez Borja (2011) How do bifidobacteria counteract environmental challenges? Mechanisms involved and physiological consequences. Genes Nutr 6:307–318 24. Savijoki K, Suokko A, Palva A, Valmu L, Kalkkinen N, Varmanen P (2005) Effect of heat-shock and bile salts on protein synthesis of Bifidobacterium longum revealed by (35S) methionine labelling and two dimensional gel electrophoresis. FEMS Microbiol Lett 248:207–215 25. Schell MA, Karmirantzou M, Snel B, Vilanova D, Berger B, Pessi G, Zwahlen MC, Desiere F, Bork P, Delley M, Pridmore RD, Arigoni F (2002) The genome sequence of Bifidobacterium longum reflects its adaptation to the human gastrointestinal tract. Proc Natl Acad Sci USA 99(22):14422–14427 26. Schmidt G, Zink R (2000) Basic features of the stress response in three species of bifidobacteria: B. longum, B. adolescentis, and B. breve. Int J Food Microbiol 55:41–45 27. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101–1108 28. Ventura M, Canchaya C, Zhang Z, Bernini V, Fitzgerald GF, van Sinderen D (2006) How high GC gram-positive bacteria and in particular bifidobacteria cope with heat stress: protein players and regulators. FEMS Microbiol Rev 30:734–759 29. Ventura M, Canchaya C, Zhang Z, Fitzgerald GF, van Sinderen D (2007) Molecular characterization of hsp20, encoding a small heat shock protein of Bifidobacterium breve UCC2003. Appl Environ Microbiol 73:4695–4703 30. Xiao M, Xu P, Zhao J, Wang Z, Zuo F, Zhang J, Ren F, Li P, Chen S, Ma H (2011) Oxidative stress-related responses of Bifidobacterium longum subsp. longum BBMN68 at the proteomic level after exposure to oxygen. Microbiology 157:1573–1588 31. Zomer A, Fernandez M, Kearney B, Fitzgerald GF, Ventura M, van Sinderen D (2009) An interactive regulatory network controls stress response in Bifidobacterium breve UCC2003. J Bacteriol 191:7039–7049 32. Zuo FL, Feng XJ, Sun XF, Du C, Chen SW (2013) Characterization of plasmid pML21 of Enterococcus faecalis ML21 from koumiss. Curr Microbiol 66(2):103–105 33. Zuo FL, Yu R, Feng XJ, Khaskheli GB, Chen LL, Ma HQ, Chen SW (2014) Combination of heterogeneous catalase and superoxide dismutase protects Bifidobacterium longum strain NCC2705 from oxidative stress. Appl Microbiol Biotechnol 98:7523–7534 34. Zuo FL, Yu R, Khaskheli GB, Ma HQ, Chen LL, Zeng Z, Mao AJ, Chen SW (2014) Homologous overexpression of alkyl hydroperoxide reductase subunit C (ahpC) protects Bifidobacterium longum strain NCC2705 from oxidative stress. Res Microbiol 165:581–589
Overexpression of Small Heat Shock Protein Enhances Heat- and Salt-Stress Tolerance of Bifidobacterium longum NCC2705 Gul Bahar Khaskheli1 • FangLei Zuo1,2 • Rui Yu1,2 • ShangWu Chen1,2
Received: 15 October 2014 / Accepted: 23 February 2015 Springer Science+Business Media New York 2015
Abstract Bifidobacteria are probiotics that are incorporated live into various dairy products. They confer healthpromotive effects via gastrointestinal tract colonization. However, to provide their health-beneficial properties, they must battle the various abiotic stresses in that environment, such as bile salts, acids, oxygen, and heat. In this study, Bifidobacterium longum salt- and heat-stress tolerance was enhanced by homologous overexpression of a small heat shock protein (sHsp). A positive contribution of overproduced sHsp to abiotic stress tolerance was observed when the bacterium was exposed to heat and salt stresses. Significantly higher survival of B. longum NCC2705 overexpressing sHsp was observed at 30 and 60 min into heat (55 C) and salt (5 M NaCl) treatment, respectively. Thermotolerance analysis at 47 C with sampling every 2 h also revealed the great potential tolerance of the engineered strain. Cell density and acid production rate increased for the sHsp-overexpressing strain after 8 and 10 h of both heat and salt stresses. In addition, tolerance to bile salts, low pH (3.5) and low temperature (4 C) was also increased by homologous overexpression of the sHsp hsp20 in B. longum. Results revealed that hsp20 overexpression in Blongum NCC2705 plays a positive cross-protective role in upregulating abiotic responses, ensuring the organism’s & ShangWu Chen [email protected] 1
Key Laboratory of Functional Dairy Science of Chinese Ministry of Education and Municipal Government of Beijing, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, People0 s Republic of China
2
Beijing Engineering Research Center of Function Agricultural Microbiology, Beijing 100083, People0 s Republic of China
tolerance to various stress conditions; therefore, sHspoverexpressing B. longum is advised for fermented dairy foods and other probiotic product applications.
Introduction Bifidobacteria are considered to be probiotic due to their contribution to the maintenance of gastrointestinal health [21]. Health benefits offered by bifidobacteria to its host, as supported by clinical evidence, have led to their wide application as probiotic components of health-promoting foods, especially in fermented dairy products [20,21,24]. Today, several probiotic species of the genus Bifidobacterium are applied in dairy products and infant foods [8], as well as in high numbers as live bacteria in numerous food products with various health claims [16]. When bifidobacteria are utilized for probiotic applications, high temperature, low water activity, osmotic shock, and oxidative stresses are frequently encountered. Genes belonging to the different heat shock protein (Hsp) families, for example groEL, groES, dnaK, dnaJ, grpE, clpB, clpC, and clpP, are induced to respond upon exposure of bifidobacterial cultures to these stressful conditions [28]. NonHsps such as heme-dependent catalase (Kat) and Mn2?dependent superoxide dismutase (Sod) have been investigated for synergistic protection of Bifidobacteriumlongum via the oxidative stress response [33]. However, the small heat shock proteins (sHsps) have recently garnered much interest in bacteria, including bifidobacteria, yeast, plants and animal cells for their role in stress tolerance [9,11,12,17,29]. The sHsps are ubiquitous ATP-independent molecular chaperones that prevent protein aggregation upon stressinduced unfolding [1]. These proteins contain a conserved
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a-crystallin domain and commonly form large dynamic oligomers, often composed of up to 24 subunits [22]. Overexpression of sHsps in some bacteria has been shown to increase tolerance to a variety of stresses [11]. Expression analysis of an analog of the sHsp gene hsp20 in Bifidobacteriumbreve showed significant induction of its expression during heat and osmotic stresses [29,31]; hsp20 (BL0576) is the most rapidly and strongly induced component in B. longum NCC2705 under heat shock, oxidative stress, and starvation conditions [16]. Bioinformatic analysis revealed that bifidobacteria hsp20 analogs may have been obtained via horizontal gene transfer from Firmicutes [18]. Although various aspects of the response of sHsps in bifidobacteria have already been studied, the high level of their constitutive expression and their role in heat- and saltstress responses are not well documented for B. longum. To investigate and confirm the function of the sHsps in bifidobacteria, hsp20 gene was cloned and homologously overexpressed in B. longum NCC2705. A positive contribution of the overproduced sHsp to the bacterial abiotic stress response, when exposed to a variety of environmental stresses, such as heat and salt stress and a multicross-protective mechanism was observed.
Materials and Methods Bacterial Strains, Plasmids, and Culture Conditions The bacterial strains and plasmids used in this study are listed in Table 1. Bifidobacteria strains were generally grown at 37 C in MRS medium supplemented with
0.05 % (wt/vol) L-cysteine HCl (MRSc) under 80 % N2– 10 % H2–10 % CO2 unless otherwise noted. Escherichia coli strains were grown at 37 C in Luria–Bertani (LB) broth with vigorous shaking. Antibiotics (Sigma) were added to the appropriate media at the following concentrations: 100 l ml-1 ampicillin and 100 l ml-1 spectinomycin for E. coli and 75 l ml-1 spectinomycin for bifidobacteria. Cloning of hsp20 (BL0576) from Bifidobacterium longum Strain NCC2705 Genomic DNA from B. longum NCC2705 was isolated following the method of Zuo et al. [32]. The open reading frame (ORF) of hsp20 (BL0576) encoding the sHsp was amplified by polymerase chain reaction (PCR) using genomic DNA as the template and the primer pair sHsp-F (50 ACCATGGCAATGTTTCCGGCTTT-30 ) and sHsp-R (50 GGTACCTCAGCCCTCAATCGCGAT-30 ). The underlined bases represent NcoI and KpnI recognition sites, respectively. The PCR was carried out in a 25-ll reaction mixture for 30 cycles at 94 C for 1 min, 65 C for 0.5 min, and 72 C for 1 min, with a final extension at 72 C for 10 min. The amplified product was analyzed by electrophoresis and purified using the Gel Band Purification kit (Biomed, Beijing, China). Construction of Expression Vector and Transformation The purified hsp20 PCR fragment was ligated to the pMD19-T simple vector and transformed into E. coli strain
Table 1 List of plasmids and strains used in the current study Plasmid or strain
Feature or sequence
Source or reference
pMD19-T simple
2.7-kb E. coli cloning vector, Ampr
Takara, Dalian, China
pMD19PmcsT
3.1-kb pMD19-PIT derivate, a linker containing a multiple cloning site was substituted for Bifidobacterium breve Sec2 protein signal peptide-coding region and human IL-10-encoding gene
[33]
pDP870
4.3-kb E. coli-B. longum shuttle cloning vector, Spr
[16]
pMD19PsHsp
pMD19-T simple derivate, containing Pgap–sHsp–Thup expression cassette
This work
pDP401sHsp
5.6-kb; pDP870 derivate, contain Pgap–sHsp–Thup expression cassette
This work
NCC2705
Wild-type B. longum NCC2705, isolated from adult human feces (GenBank Accession no. AE014295)
[25]
NCC2705pDP401
B. longum NCC2705 harboring plasmid pDP401
[34]
NCC2705sHsp
Hsp20-overexpressing B. longum NCC2705
This work
Plasmids
Strains
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DH5a to form pMD19-sHsp. pMD19-sHsp was cut with NcoI and KpnI, and the hsp20-containing fragment was ligated to the NcoI- and KpnI-digested backbone fragment of pMD19-PmcsT [33] and transformed into E. coli DH5a to form pMD19-PsHsp. Then pMD19-PsHsp was digested with NdeI and XhoI, and the Pgap–sHsp–Thup expression cassette fragment was ligated to the NdeI- and XhoI-digested backbone fragment of pDP870 and transformed into E. coli DH5a to form pDP401-PsHsp. The recombinant plasmid pDP401-PsHsp was sequenced in an ABI 3730XL DNA Analyzer (Applied Biosystems). Recombinant plasmid pDP401-PsHsp and the corresponding control plasmid pDP401 were introduced into B. longum NCC2705 electrocompetent cells by electroporation following the method of Argnani et al. [3]. Transformants were confirmed by colony PCR and plasmid DNA extraction. Recombinant B. longum strains harboring plasmid pDP401-PsHsp (designated NCC2705-sHsp) or pDP401 (control, designated NCC2705-pDP401) were confirmed by plasmid DNA extraction and PCR detection or 15 % polyacrylamide gel electrophoresis, respectively. Determination of the Stress Tolerance of Hsp20Overproducing B. longum NCC2705 Two cultures of B. longum NCC2705-control and NCC2705-sHsp transformants were grown in MRSc broth supplemented with spectinomycin to an OD600 of 1.0. Thermotolerance of B. longum NCC2705 was investigated by monitoring survival in MRSc broth at 55 C for 60 min. Aliquots were removed at 0, 30 and 60 min, and serial dilutions were spread on MRSc agar and then anaerobically incubated at 37 C for 48 h. Salt tolerance was determined by adding 5 M NaCl and monitoring culture viability for 1 h with aliquots taken at 0, 30, and 60 min. To monitor the growth of B. longum NCC2705 under long-term stress conditions, overnight growth culture was inoculated (2 %) in 50 ml MRSc broth supplemented with spectinomycin. For long-term heat stress, the cultures were grown at 47 C and absorbance was measured at 2-h intervals. For the long-term salt-stress assay, MRSc broth was supplemented with 200 mM NaCl, the culture was grown at 37 C, and absorbance was measured at 2-h intervals. To determine the tolerance of sHsp-overproducing B. longum NCC2705 to multiple stresses, the exponentially growing B. longum cells were inoculated in MRSc broth (pH 3.5 or bile salt 0.1 %) for pH or bile salt stress. Aliquots were taken at 0, 30, and 60 min, and serial dilutions were spread on MRSc agar and then anaerobically incubated at 37 C for 48 h. For low-temperature (4 C) stress, overnight culture was incubated at 4 C and aliquots were removed at 0, 6, 12 and 24 h; serial dilutions were spread
on MRSc agar and then anaerobically incubated at 37 C for 48 h. Quantitative Reverse Transcription PCR (qRTPCR) Total RNA from exponentially growing (OD600 of 0.6) B. longum strains was isolated using TRIzol Reagent (Invitrogen) and then treated with RNase-free DNase I (Takara). RNA concentrations were determined by spectrophotometry at 260 nm. The absence of residual DNA in the DNase I-digested total RNA was confirmed by PCR. Reverse transcription was carried out using M-MLV Reverse Transcriptase (Promega) in a 20-ll reaction volume containing 150 ng of random primers and 1 mM dNTP mix (Tiangen, China). Primer pairs (sHsp-F1: 50 -CCGTAATGCCCGC CAACA-30 and sHsp-B1: 50 -GCAGCCACTTGCCC GAAT-30 ) were designed with Primer Premier 5 software (Palo Alto, CA) to amplify 234-bp regions of hsp20 for qRTPCR. Real-Time PCR was performed using a SYBR Green assay kit (Takara) and optimized primer concentrations in a 7500 Fast Real-Time PCR system (Applied Biosystems). Gene expressions were normalized by the DDCT method [27] and using 16S ribosomal DNA as the reference gene for the calculations [30]. The experiment was performed in triplicate and the average results are reported. Statistical Analysis All of the data from three independent experiments were analyzed by two-tailed Student’s t test. All analyses were performed using Microsoft Office Excel, version 2007. Values of P \ 0.05 were considered significant.
Results Overexpression of sHsp (hsp20 BL0576) in B. longum NCC2705 A ca. 513-bp PCR product, consistent with the theoretical length of sHsp (501 bp), was digested and used to construct the expression vector pDP401-sHsp, which was then verified by DNA sequencing. The 501-bp ORF encodes 167 amino acids with a calculated molecular mass of 18.8 kDa. B. longum transformation with plasmid pDP401 as a control (NCC2705-pDP401) or pDP401-sHsp (NCC2705sHsp) was confirmed by colony PCR and plasmid isolation (data not shown). In the exponential phase of NCC2705sHsp growth, transcript level of sHsp was 7.1-fold higher than that in the control NCC2705 detected by qRT-PCR (Fig. 1), suggesting that the sHsp was successfully overexpressed in NCC2705-sHsp.
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Fig. 1 Relative expression fold change in hsp20 gene expression in B. longum NCC2705 transformants detected by reverse transcription PCR. The fold change was calculated as the ratio between signals observed in hsp20-overexpressing strain (sHsp) and strain harboring pDP401 (Control). They were normalized using housekeeping 16S rRNA as an internal control. The mean values from three independent determinations ±SD are shown
Heat-Stress Tolerance of sHsp-Overproducing B. longum NCC2705 Heat tolerance of B. longum overexpressing sHsp (NCC2705-sHsp) was evaluated (Fig. 2a). Variation was observed in both sHsp-overexpressing and control B. longum strains. The average survival (%) of NCC2705sHsp at 55 C was 0.056 and 0.078, compared to 0.016 and 0.00 for NCC2705-pDP401, at 30 and 60 min, respectively (Fig. 2a). The overall survival rate of NCC2705-sHsp was 3.44-fold that of the control strain after 30 min at 55 C (P \ 0.05). It was also noted that the NCC2705-sHsp strain continued to grow while the NCC2707-pDP401 (control) strain stopped growing completely from 30 to 60 min at 55 C (Fig. 2a). The experimental analysis of thermotolerance was extended to a study of long-term growth performed at 47 C with monitoring at 2-h intervals. Samples were analyzed for optical density (OD600) and pH and the results are summarized in Fig. 2b. A slight numerical difference in both OD600 and pH was revealed with time. NCC2705-sHsp increased, whereas NCC2705-pDP401 grew more slowly after 6 h. The pH values of both NCC2705-sHsp and the control strain (NCC2705-pDP401) decreased with time, but the pH values of NCC2705-sHsp were lower than those of NCC2705-pDP401 at all time points (Fig. 2b). Salt-Stress Tolerance of sHsp-Overproducing B. longum NCC2705 The results obtained for salt tolerance (to 5 M NaCl) of B. longum (NCC2705-sHsp and NCC2705-pDP401) are
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Fig. 2 Effects of hsp20 overexpression on heat tolerance of B. longum NCC2705 a Survival of B. longum strains under heat stress (55 C). Light bars and dark bars represent B. longum control (NCC2705-pDP401) and hsp20-overexpressing (NCC2705-sHsp) strains, respectively, after 30 and 60 min of heat treatment. b Longterm heat tolerance to heat stress (47 C): cell density, measured as OD600, shows changes in growth rate in MRSc medium for NCC2705-sHsp (filled square) and NCC2705-pDP401 (unfilled square), and changes in acid production (pH) for NCC2705-sHsp (filled circle) and NCC2705-pDP401 (unfilled circle)
presented in Fig. 3a. The mean survival (%) of strain NCC2705-sHsp was 0.035 and 0.026, and that of NCC2705-pDP401 was 0.030 and 0.002 under salt stress for 30 and 60 min, respectively (Fig. 3a). Overall survival of NCC2705-sHsp was higher than the control strain, being 1.17- and 9.21-fold that of the control strain under 5 M NaCl at 30 and 60 min (P \ 0.05), respectively. As with the heat stress, strain NCC2705-sHsp continued to grow while strain NCC2705-pDP401 showed reduced growth under osmotic stress for 60 min (Fig. 3a). To monitor the continued salt tolerance, long-term growth analysis was carried out by inoculating the experimental strains in MRSc medium containing 200 mM NaCl at 37 C and taking aliquots at 2-h intervals for OD600 and pH determinations. The results are
G. B. Khaskheli et al.: Overexpression of Small Heat Shock Protein…
Fig. 3 Effects of hsp20 overexpression on tolerance to salt stress in B. longum NCC2705 a Survival of B. longum strains under salt stress (5 M NaCl). Light bars and dark bars indicate B. longum NCC2705pDP401 and NCC2705-sHsp, respectively, after 30 and 60 min of salt treatment. b Long-term tolerance to salt stress (200 mM NaCl): cell density, measured as OD600, shows changes in growth rate in MRSc medium for NCC2705-sHsp (filled square), and NCC2705-pDP401 (filled circle), and changes in acid production (pH) for NCC2705sHsp (unfilled square) and NCC2705-pDP401 (unfilled circle)
summarized in Fig. 3b. A marked difference was observed for OD600 and pH values of both B. longum overexpressing sHsp and control strains with time. Both strains grew well up to 8 h incubation; thereafter, NCC2705-sHsp grew more than NCC2705-pDP401 (Fig. 3b). Similar to the heat tolerance results, the pH values of NCC2705-sHsp and NCC2705-pDP401 decreased with time, and the pH values of NCC2705-sHsp were lower than those of the control strain (NCC2705pDP401) at all time points (Fig. 3b). Multiple Stress Tolerance of Hsp20-Overexpressing B. longum NCC2705 In addition to abiotic stresses (temperature and salt), experiments were carried out with bile salts (0.1 %), low pH (3.5), and low temperature (4 C) as selective criteria for stress tolerance of hsp20-overexpressing strains. The results are presented in Fig. 4. All
treatments revealed the prominent stress tolerance of strain NCC2705-sHsp compared to the control (NCC2705-pDP401). The mean survival (%) of NCC2705-sHsp was 0.027 and 0.114, compared to 0.024 and 0.002 for NCC2705pDP401 under pH stress (3.5) at 30 and 60 min, respectively (Fig. 4a). Furthermore, a statistically significant difference in survival (%) of B. longum NCC2705 overexpressing sHsp was observed compared to NCC2705pDP401, and overall survival rate of NCC2705-sHsp increased to 4.8- and 13.19-fold that of the control strain after 30 and 60 min, respectively. The average survival (%) of strain NCC2705-sHsp was higher (0.045 and 0.015) than that of NCC2705-pDP401 (0.025 and 0.002) under bile salt stress for 30 and 60 min, respectively (Fig. 4b). Moreover, NCC2705-sHsp survival increased 1.80- and 5.3-fold that of the control strain at 30 and 60 min, respectively. Mean survival (%) of sHsp-overexpressing B. longum (NCC2705-sHsp) was 0.056, 0.065, and 0.008 versus 0.052, 0.028, and 0.003 for control B. longum (NCC2705-pDP401) under low-temperature (4 C) stress at 6, 12, and 24 h, respectively. Similar to pH and bile salt stress, B. longum overexpressing sHsp (NCC2705-sHsp) seemed to adapt to the low temperature, growing 1.08-, 2.34- and 2.58-fold more than control B. longum (NCC2705-pDP401) at 6, 12, and 24 h, respectively.
Discussion Bifidobacteria are utilized for probiotic applications in the gastrointestinal tract (GIT) of mammals. As autochtonous bacteria in the GIT, they generally encounter a variety of abiotic stresses. However, proteins of different Hsp families are active in the bifidobacteria’s response to stressful conditions [28], with sHsps being the most attractive subject for studies of stress tolerance potential [9,11,12] because their overexpression in bacteria results in increased tolerance to a variety of stresses [11]. Expression analysis of the hsp20 analog in B. breve showed significant induction during heat and osmotic stresses [16,29,31]. B. longum NCC2705 contains different sHsps, including hsp20 which is believed to be a common stress response factor, being the most rapidly and highly induced gene under heat shock, oxidative stress, and starvation conditions [16]. We confirmed homologous overexpression of this sHsp in B. longum NCC2705, accompanied by improved tolerance to heat and salt stress. In addition to the newly characterized response to abiotic heat and osmotic stresses, the effects of abiotic and/or biotic stresses—i.e., low pH, bile salts
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Characteristics of Heat- and Salt-Stress Tolerance of hsp20-Overproducing B. longum NCC2705
Fig. 4 Effect of homologous overexpression of hsp20 on survival rates of B. longum strains. a Survival of B. longum strains under low pH (3.5), showing the B. longum NCC2705-pDP401 and NCC2705sHsp cells after 30 and 60 min of low pH treatment, respectively. b Survival of B. longum strains under bile salt (0.1 %), indicating B. longum NCC2705-pDP401 and NCC2705-sHsp cells at 30 and 60 min of bile salt treatment, respectively. c Survival of B.longum strains under low temperature (4 C); viable cells of B. longum NCC2705-pDP401 and NCC2705-sHsp were assayed after 6, 12, and 24 h incubation at 4 C. Light and dark bars indicate B. longum NCC2705-pDP401 and NCC2705-sHsp, respectively. Asterisks indicate a statistically significant difference (*P \ 0.05, **P \ 0.01) between B. longum strains NCC2705-pDP401 and NCC2705-sHsp
and low temperature—on the Hsp20-overexpressing B. longum strains were demonstrated, as discussed further on.
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Variation in growth was observed between engineered and control strains. A significantly higher number of colonies (P \ 0.005) were found for NCC2705-sHsp vs. controls at 55 C for 60 min. In addition, the overall survival rate of strain NCC2705-sHsp was higher than that of NCC2705pDP401 under these conditions. NCC2705-sHsp continued to grow at 55 C for 60 min while NCC2707-pDP401 did not. These findings show the great potential for thermotolerance of NCC2705-sHsp in comparison to NCC2707pDP401. Further, thermotolerance analysis at 47 C with monitoring at 2-h intervals also revealed the great tolerance potential of the engineered strain in the current study. However, differences were observed for both strains in optical density (OD600) and pH values with time: the OD600 for NCC2705-sHsp was higher than that of the controls after 8- and 10-h intervals. The pH values of the heattreated NCC2705-sHsp were lower than those of NCC2705-pDP401. From a technological point of view, bifidobacteria are not robust microorganisms. In general, their optimal growth temperature range is 36–38 and 41–43 C for strains of human and animal origin, respectively, although B. thermacidophilum can grow at 49 C and B. psychraerophilum at 4 C. The optimal pH range for growth is between 6.5 and 7.0, but B. animalis and B. thermacidophilum survive well at pH 3.5–4.0. These observations highlight the narrow choice of oxygen, temperature, and acidic conditions that are suitable for bifidobacterial survival, although some bifidobacteria are able to survive under more extreme conditions (e.g. B. animalis subsp. lactis strains), whereas others display a low tolerance to stress (e.g. some B. bifidum and B. longum strains); nevertheless, the physiology underlying their adaptation is far from understood. The mechanisms most extensively studied in response to technological stress of bifidobacteria are those related to heat, oxygen, and acid, as reviewed by Ruiz et al. [23]. There is some disparity in the results of studies on stress tolerance in bifidobacteria and in the stress-resistant mutants that sometimes arise spontaneously [6]. Regarding technological stresses, it is worth noting that heat-tolerant mutants of B. longum strains are obtained by successive sublethal heat-shock treatments, and the genetic basis for this adaptation has been recently documented [5, 21, 23]. Another study states that sHsps cooperate with other Hsps by binding to non-native proteins to refold them. Our results support those of Kitagawa et al. [15], who claimed that overexpression of sHsp results in enhanced heat and oxidative tolerance potential. In the current study, strong induction of sHsp was found in NCC2705-sHsp compared to NCC2705-pDP401 in response to stress. Similarly, Klijn et al. [16] observed that
G. B. Khaskheli et al.: Overexpression of Small Heat Shock Protein…
hsp20 (BL0576) expression is a common stress response in B. longum NCC2705, being the most rapidly and strongly induced gene in a variety of stresses (heat shock, oxidative stress, and starvation). Similar trends for OD and pH were noted with respect to salt tolerance, with a significantly higher number of colonies found for NCC2705-sHsp compared to NCC2705pDP401 after 30 min exposure to 5 M NaCl. In the current study, the engineered strain expressed great potential for salt tolerance, surviving longer than the control strain. Kim et al. [14], in an experiment with L. acidophilus cells, observed that 2 and 18 % (w/v) NaCl cause sublethal and lethal osmotic stress, respectively. Although B. longum NCC2705 endured the stress of 5 M NaCl, the duration of the stress could also be an important factor in strain survival. Furthermore, NCC2705-sHsp showed better salt tolerance (0.035 and 0.026) than the control strain (0.030 and 0.002) at 30 and 60 min, respectively. In previous experiments, the adaptive response to NaCl stress revealed cross-protection against heat stress [14], but not vice versa, resulting in the conclusion that Hsps might be induced by salt stress, but elevated temperature may not necessarily induce the salt stress response. Our results support the hypothesis that a sHsp-overexpressing strain can endure both salt and heat tolerance. It is, therefore, suggested that further studies be carried out on the mechanism underlying the sHsp-overexpressing strain’s tolerance to abiotic stresses (elevated temperature and salt), as well as exposure to bile salt, low temperature, and acidic conditions. Bile salts and acids are pumped into the GIT at concentrations typically ranging from 10 mM in the small intestine to 1 mM in the cecum [13]. Bile salts in the intestinal tract are a particularly difficult environmental challenge for probiotic bacteria [4]. Flahaut et al. [10] and Schmidt and Zink [26] confirmed that DnaK and GroEL are induced by bile salts. Another study also reported that cells pre-exposed to bile salts show greater resistance to elevated temperature; on the other hand, pre-exposure to heat stress did not increase resistance to lethal bile stress. Kim et al. [14] reported that bile at 0.05 % (w/v) is sublethal (cells grew slowly) and bile at 0.5 % (w/v) is lethal (most of the cells were killed at this level) to L. acidophilus. In the current study, we found a prominent difference in the growth of the sHsp-overexpressing transformant vs. the control strain. Lorca et al. [19] revealed that Hsp family factors DnaK, DnaJ, GrpE, GroES and GroEL are induced by acid stress. We also investigated the sHsp-overexpressing strain’s response to acid stress (pH 3.5): it grew markedly better than the non-overexpressing strain (Fig. 4a). Interestingly, not only did we find remarkable growth of NCC2705-sHsp under acidic conditions, but we also detected a similar pattern in the response to low temperature (4 C) and bile salt treatments.
Resistance to bile salts in bifidobacteria may be governed by a different mechanism, such as metabolic changes [2]. The hsp20 gene has been predicted to be present in the genomes of B. breve, B. longum, and B. adolescentis. Transcription of this sHsp in B. breve is upregulated under heat stress (50 C) and osmotic stress (0.7 M NaCl) [29]. Coucheney et al. [7] revealed that the sHsp gene Lo18 encodes a membrane-associated protein in Oenococcus oeni that maintains the integrity of the cell membrane during heat, ethanol, and benzyl alcohol stress. Our research findings reinforce the notion that sHsp overexpression in bifidobacteria plays a prominent role in cell viability and increased tolerance to multiple stresses.
Conclusions We conclude that overexpression of hsp20 in B. longum NCC2705 ensures the organism’s tolerance of heat and salt stresses, and provides cross-protection against low pH levels, bile salts, and low temperature (4 C) conditions which might exist in the surrounding environment. These functional properties of sHsps in bifidobacteria, an organism that is frequently added to fermented dairy and therapeutic products, may enhance protection against the various stresses imposed during the commercial process, from manufacturing to consumption. Acknowledgments This work was funded by the National Natural Science Foundation of China (No. 31071507), the National High Technology Research and Development Program (‘‘863’’ Program, No. 2008AA10Z310), and the National Science and Technology Support Program, Ministry of Science and Technology of China (2011BAD09B03).
References 1. Alexander B, Ferdinand A, Thomas K, Nathalie B, Sevil W, Michael G, Martin H, Johannes B (2012) Alternative bacterial two-component small heat shock protein systems. Proc Natl Acad Sci USA 109(50):20407–20412 2. Alp G, Aslim B (2010) Relationship between the resistance to bile salts and low pH with exopolysaccharide (EPS) production of Bifidobacterium spp. isolated from infants feces and breast milk. Anaerobe 16:101–105 3. Argnani A, Leer RJ, van Luijk N, Pouwels PH (1996) A convenient and reproducible method to genetically transform bacteria of the genus Bifidobacterium. Microbiology 142:109–114 4. Begley M, Gahan CGM, Hill C (2005) The interaction between bacteria and bile. FEMS Microbiol Rev 29:625–651 5. Berger B, Moine D, Mansourian R, Arigoni F (2010) HspR mutations are naturally selected in Bifidobacterium longum when successive heat shock treatments are applied. J Bacteriol 192:256–263 6. Collado MC, Sanz Y (2006) Method for direct selection of potentially probiotic Bifidobacterium strains from human feces based on their acid-adaptation ability. J Microbiol Method 66:560–563
123
G. B. Khaskheli et al.: Overexpression of Small Heat Shock Protein… 7. Coucheney F, Gal L, Beney L, Lherminier J, Gervais P, Guzzo J (2005) A small HSP Lo18, interacts with the cell membrane and modulates lipid physical state under heat shock conditions in a lactic acid bacterium. Biochim Biophys Acta Biomembr 1720:92–98 8. De Dea Lindne J, Canchaya C, Zhang ZD, Neviani E, Fitzgerald GF, van Sinderen D, Ventura M (2007) Exploiting Bifidobacterium genomes: The molecular basis of stress response. Int J Food Microbiol 120:13–24 9. Eylesa SJ, Gierasch LM (2010) Nature’s molecular sponges: Small heat shock proteins grow into their chaperone roles. Proc Natl Acad Sci USA 107(7):2727–2728 10. Flahaut S, Frere J, Boutibonnes P, Auffray Y (1996) Comparison of the bile salts and sodium dodecyl sulfate stress responses in Enterococcus faecalis. Appl Environ Microbiol 62:2416–2420 11. Guzzo J (2012) Biotechnical applications of small heat shock proteins from bacteria. Int J Biochem Cell Biol 44:1698–1705 12. Han MJ, Yun H, Lee SY (2008) Microbial small heat shock proteins and their use in biotechnology. Biotechnol Adv 26:591–609 13. Hofmann AF (1999) The continuing importance of bile acids in liver and intestinal disease. Arch Intern Med 159:2647–2658 14. Kim WS, Perl L, Park JH, Tandianus JE, Dunn NW (2001) Assessment of stress response of the probiotic Lactobacillus acidophilus. Curr Microbiol 43:346–350 15. Kitagawa M, Matsumura Y, Tsuchido T (2000) Small heat shock proteins, IbpA and IbpB are involved in resistances to heat and superoxide stresses in Escherichia coli. FEMS Microbiol Lett 184(2):165–171 16. Klijn A, Mercenier A, Arigoni F (2005) Lessons from the genomes of bifidobacteria. FEMS Microbiol Rev 29:491–509 17. Landry J, Chre´tien P, Lambert H, Hickey E, Weber LA (1989) Heat shock resistance conferred by expression of the human HSP27 gene in rodent cells. J Cell Biol 101(1):7–15 18. Lee JH, O’Sullivan DJ (2010) Genomic insights into bifidobacteria. Microbiol Mol Biol Rev 74(3):378–416 19. Lorca GL, Font de Valdez G, Ljungh A (2002) Characterization of the protein synthesis dependent adaptive acid tolerance response in Lactobacillus acidophilus. J Mol Microbiol Biotechnol 4:525–532 20. Michelle C, Marco V, Gerald FF, van Douwe S (2011) Progress in genomics, metabolism and biotechnology of bifidobacteria. Int J Food Microbiol 149(1):4–18 21. Ouwehand AC, Salminen S, Isolauri E (2002) Probiotics: an overview of beneficial effects. Antonie Leeuwenhoek 82:279–289 22. Richter K, Haslbeck M, Buchner J (2010) The heat shock response: life on the verge of death molecular. Cell 40:253–266
123
23. Ruiz L, Patricia RM, Miguel G, Clara GDLR, Abelardo M, Sanchez Borja (2011) How do bifidobacteria counteract environmental challenges? Mechanisms involved and physiological consequences. Genes Nutr 6:307–318 24. Savijoki K, Suokko A, Palva A, Valmu L, Kalkkinen N, Varmanen P (2005) Effect of heat-shock and bile salts on protein synthesis of Bifidobacterium longum revealed by (35S) methionine labelling and two dimensional gel electrophoresis. FEMS Microbiol Lett 248:207–215 25. Schell MA, Karmirantzou M, Snel B, Vilanova D, Berger B, Pessi G, Zwahlen MC, Desiere F, Bork P, Delley M, Pridmore RD, Arigoni F (2002) The genome sequence of Bifidobacterium longum reflects its adaptation to the human gastrointestinal tract. Proc Natl Acad Sci USA 99(22):14422–14427 26. Schmidt G, Zink R (2000) Basic features of the stress response in three species of bifidobacteria: B. longum, B. adolescentis, and B. breve. Int J Food Microbiol 55:41–45 27. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101–1108 28. Ventura M, Canchaya C, Zhang Z, Bernini V, Fitzgerald GF, van Sinderen D (2006) How high GC gram-positive bacteria and in particular bifidobacteria cope with heat stress: protein players and regulators. FEMS Microbiol Rev 30:734–759 29. Ventura M, Canchaya C, Zhang Z, Fitzgerald GF, van Sinderen D (2007) Molecular characterization of hsp20, encoding a small heat shock protein of Bifidobacterium breve UCC2003. Appl Environ Microbiol 73:4695–4703 30. Xiao M, Xu P, Zhao J, Wang Z, Zuo F, Zhang J, Ren F, Li P, Chen S, Ma H (2011) Oxidative stress-related responses of Bifidobacterium longum subsp. longum BBMN68 at the proteomic level after exposure to oxygen. Microbiology 157:1573–1588 31. Zomer A, Fernandez M, Kearney B, Fitzgerald GF, Ventura M, van Sinderen D (2009) An interactive regulatory network controls stress response in Bifidobacterium breve UCC2003. J Bacteriol 191:7039–7049 32. Zuo FL, Feng XJ, Sun XF, Du C, Chen SW (2013) Characterization of plasmid pML21 of Enterococcus faecalis ML21 from koumiss. Curr Microbiol 66(2):103–105 33. Zuo FL, Yu R, Feng XJ, Khaskheli GB, Chen LL, Ma HQ, Chen SW (2014) Combination of heterogeneous catalase and superoxide dismutase protects Bifidobacterium longum strain NCC2705 from oxidative stress. Appl Microbiol Biotechnol 98:7523–7534 34. Zuo FL, Yu R, Khaskheli GB, Ma HQ, Chen LL, Zeng Z, Mao AJ, Chen SW (2014) Homologous overexpression of alkyl hydroperoxide reductase subunit C (ahpC) protects Bifidobacterium longum strain NCC2705 from oxidative stress. Res Microbiol 165:581–589
