1 Unité de Recherches Laitières et de Génétique Appliquée, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy-en-Josas cedex, France
2 Institute of Biological Sciences, Federal University of Minas Gerais (UFMG-ICB), Belo Horizonte, MG, Brazil
Correspondence
P. Langella
philippe.langella{at}jouy.inra.fr
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ABSTRACT |
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Present address: Unité d'Ecologie et Physiologie du Système Digestif, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy-en-Josas cedex, France.
Present address: Unité Mixte de Recherche Génétique et Horticulture, 42 rue Georges Morel, BP 60057 49071, Beaucouzé cedex, France.
These authors contributed equally to this work.
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INTRODUCTION |
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Bacterial species are more or less sensitive to oxygen according to the enzymic equipment they possess to prevent or repair ROS damage. Some lactic acid bacteria, such as Lactococcus lactis, widely used in the production of fermented food products, produce a superoxide dismutase, which degrades to generate H2O2 (Sanders et al., 1995
). However, they lack catalases, antioxidant metalloenzymes that catalyse the reaction in which toxic H2O2 is reduced to two H2O molecules and O2. In contrast, catalase activity has been reported in several Lactobacillus species during the last decade (Igarashi et al., 1996
; Knauf et al., 1992
). In the absence of catalase, H2O2 produced by the cell or present in the environment accumulates, and may lead, especially in presence of iron, to the production of the more toxic OH· (Fridovich, 1998
; Imlay, 2003
).
Bacterial catalases are widespread in aerobes (facultative or not) such as Escherichia coli and Bacillus subtilis. Two classes of catalases have been distinguished, according to their active-site composition: one is haem-dependent, and the other, also named pseudocatalase, is manganese-dependent for the reduction of H2O2. Catalases of two lactobacilli have been successfully transferred and phenotypically expressed in heterologous hosts deficient in catalase activity (Abriouel et al., 2004; Knauf et al., 1992
; Noonpakdee et al., 2004
). Strains of lactic acid bacteria expressing high levels of catalases could be useful in both traditional food applications and new therapeutic uses. A probiotic antioxidant strain able to eliminate ROS in the digestive tract of animals and humans could have applications for treatment of inflammatory diseases or post-cancer drug treatments.
In this work, we tested the effect of the production of the B. subtilis haem-catalase KatE (Engelmann et al., 1995) on the oxidative stress resistance of L. lactis.
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METHODS |
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Cloning of B. subtilis katE.
The katE gene was amplified by PCR from the chromosome of B. subtilis 168 using the primers 5'-AATGTGCTCTAGAATGTTTCGTTTTAAA and 3'-TTTTCCTCGAGTTCGTAAGCTTCAGTGAGC, designed using the sequenced genome of B. subtilis (Kunst et al., 1997). The restriction sites used for subsequent cloning are underlined: XbaI and XhoI for the 5' and 3' primers, respectively. The PCR product was then inserted into pBS : KS+ (Stratagene; hereafter called pBS) cut by XbaI and XhoI in E. coli TG1, resulting in pBS : katE. The promoter P23 fragment (van der Vossen et al., 1987
) was obtained after digestion of plasmid pUC1318 : P23 (kindly provided by Y. Le Loir) by EcoRI and XbaI. It was then cloned in pBS : katE cut by EcoRI and XbaI, resulting in pBS : P23 : katE. The expression cassette P23 : katE, obtained after double digestion of pBS : P23 : katE by EcoRI and XhoI, was inserted into EcoRI/XhoI-cut high-copy-number pILN12, resulting in pILN12 : P23 : katE obtained in L. lactis. The plasmid pSEC : Nuc (Table 1
; Bermudez-Humaran et al., 2003
) was used in this work: (i) to clone the katE gene under transcriptional control of the lactococcal nisin-inducible promoter PnisA (de Ruyter et al., 1996a
) and (ii) to secrete the KatE catalase into the extracellular medium. This plasmid contains the ribosome-binding site (RBSUsp45) and the sequence encoding the signal peptide (SPUsp45) of the usp45 gene (van Asseldonk et al., 1990
) and the mature part of the staphylococcal nuclease (Nuc; Le Loir et al., 1998
). An NsiI site was introduced at the 3' end of RBSUsp45 to allow replacement of the nuc coding sequence by a DNA fragment encoding the B. subtilis katE. Thus, the 2172 bp katE gene was PCR-amplified from the plasmid pBS : P23 : katE (Table 1
). Primers, containing one artificial restriction site at each end, were designed according to the genomic DNA sequence of katE (GenBank accession no. D83026). Two primers were used: (i) SCkatE5' for the coding strand [5'-GGATGCATCAAGTGATGACCAAAAC-3', where the NsiI site is underlined and CA (in bold) was added to adapt the reading frame of SPUsp45]; and (ii) SCkatE3' for the complementary strand [5'-GGCTCGAGTCAAATTCGTCTATCCC-3', where the XhoI site is underlined]. The resulting amplified product was then cloned into the pCRII-TOPO vector (Table 1
), generating the intermediate construction pTPS : katE (Table 1
) in E. coli TOP10 (Table 1
). This construction was then digested with NsiI and XhoI, allowing purification of fragments containing the katE gene. This fragment was cloned into NsiI/XhoI-cut pSEC : Nuc vector, resulting in plasmid pSEC : KatE (Table 1
), established in E. coli TG1 (Table 1
). The sequence of the insert was checked.
Construction of L. lactis strains to analyse the impact of KatE production.
To analyse the effects of katE expression on bacterial physiology, two strains were constructed: L. lactis MG1363 containing pILN12 : KatE, hereafter called MG(pILN12 : KatE), and L. lactis NZ9000 (carrying the nisRK genes necessary for PnisA induction; Table 1, Kuipers et al., 1998
) containing pSEC : KatE, hereafter called NZ(pSEC : KatE). The production and the activity of KatE and its potential impact on the survival rate of these KatE+ strains after H2O2 exposure were successively analysed. Finally, we evaluated the influence of katE expression (using the construction pSEC : KatE) on long-term survival in aerated cultures. As the wild-type strain of L. lactis is not appropriate for these experiments, we constructed a derivative of L. lactis NZ9000 unable to respire (inactivated in the cytochrome oxidase gene cydA; NZ9000 cydA, or NZ cydA for short) by conjugation as previously described (Langella et al., 1993
). The donor strain was the erythromycin-resistant (EmR) MG1363 cydA (Duwat et al., 2001
) and the recipient was a spontaneously streptomycin- and rifampicin-resistant (SmR RifR) mutant of NZ9000 (Bermudez-Humaran et al., 2002
). Transconjugants were first isolated on GM17 containing Em, Sm and Rif. Then, one transconjugant that contained nisR/K genes was selected; pSEC : Nuc was introduced into this transconjugant and a TBD-agar test was performed in the presence of 1 ng nisin ml1 to check nuc induction by detection of Nuc activity as previously described (Le Loir et al., 1994
). OD600 and pH measurements of aerated cultures, supplemented with haemin, of the selected transconjugant showed that it was still unable to respire, like the parental cydA strain (OD600 <2·5 and pH <6). As the negative control we used the plasmid pVE3655 (Le Loir et al., 2001
), which was obtained after XhoI digestion of pNZ8010 (de Ruyter et al., 1996b
) to delete the gus gene and maintain only PnisA. The two plasmids pVE3655 and pSEC : KatE were introduced into this transconjugant, giving strains hereafter called NZ cydA(pVE3655) and NZ cydA(pSEC : KatE), respectively. In this context, where addition of haemin has no influence on O2 utilization (Duwat et al., 2001
), the impact of KatE on L. lactis survival could be analysed in aerated stress conditions. Finally, the strain NZ9000 cydA/recA (NZ cydA/recA for short), unable to respire (with aeration and in the presence of haemin) and unable to repair DNA damage (because of recA inactivation) was constructed by the same procedure: a mating was performed between MG1363 recA (Duwat et al., 1995
) and NZ cydA. The two plasmids pVE3655 and pSEC : KatE were introduced into a selected transconjugant, giving strains hereafter called respectively NZ cydA/recA(pVE3655) and NZ cydA/recA(pSEC : KatE), and the influence of KatE on DNA integrity in cultures exposed to long-term aeration was analysed. Noninduced cultures and L. lactis NZ strains harbouring the control plasmid pVE3655 (Table 1
) were used as negative controls.
Detection of catalase activity in L. lactis strains.
Two millilitres of exponentially growing cultures of each L. lactis strains, induced or not for 1 hour by addition of 1 ng nisin ml1, were harvested and resuspended in 30 µl TES buffer (50 mM Tris/HCl pH 8·0, 1 mM EDTA, 25 % Sucrose). Samples (20 µl) of TES-resuspended cells were mixed with 10 µl H2O2 (8 M). To detect catalase activity in the culture medium, H2O2 was directly mixed with 20 µl harvested supernatant. The presence of catalase activity leads to bubble formation resulting from the transformation of H2O2 to H2O and gaseous O2. Quantitative assay of catalase activity was performed on cell suspensions by the method of Sinha (1972). Briefly, exponentially growing cells (induced for NZ derivative strains) were centrifuged, resuspended in phosphate buffer (0·1 M, pH 7) at 109 c.f.u. ml1, and mixed with 0·8 mmol H2O2 in phosphate buffer. H2O2 concentration was determined each minute by mixing, at a ratio of 1 : 3, an aliquot with a solution of dichromate in acetic acid (1/3 dipotassium chromate 50 g l1; 2/3 glacial acetic acid). The samples were then boiled and centrifuged to remove cells; the absorbance was measured at 570 nm. Catalase activity was expressed as µmol H2O2 degraded per min per 109 c.f.u. The results presented correspond to the mean of three different assays.
Preparation of cellular and supernatant protein fractions of L. lactis for SDS-PAGE.
For fractionation between cell and supernatant fractions, 2 ml samples of nisin-induced L. lactis cultures were centrifuged for 5 min at 6000 g at 4 °C. Protein extracts were then prepared as previously described (Le Loir et al., 1998).
Amino-terminal sequencing.
To determine the nature of the catalase form detected in the cell fraction of NZ(pSEC : KatE), the corresponding band was cut from the SDS-PAGE gel and submitted to N-terminal microsequencing (performed by J. C. Huet at UBSP, INRA Jouy-en-Josas) on a gas-phase sequencer (model 477A/HPLC 120A; Perkin Elmer).
Survival after H2O2 exposure.
Cultures in stationary phase were diluted 1/50 in GM17, supplemented with haemin, grown to OD600 0·5 and divided into two samples, nisin-induced or not, for the construction containing PnisA. To measure the survival of the oxygen-sensitive strain in the presence of KatE, induced cultures of NZ(pSEC : KatE) or NZ(pVE3655) were mixed (at 1 : 1) with an exponential-phase culture of MG(pIL253). Before mixing, the cultures were centrifuged and resuspended in GM17 devoid of Cm or Em. For oxidative challenge, the cultures were incubated with different concentrations of H2O2 (0, 2 and 4 mM) for 1 h. H2O2 was then removed by addition of bovine catalase (10 U ml1, Sigma), and enumerations were performed by plating appropriate dilutions on GM17 using a spiral plater. Mixed cultures were plated and counted on GM17 containing either Cm or Em. Survival rates were compared between control and catalase-producing strains and between induced and noninduced strains. All results presented correspond to the mean of three assays.
Long-term survival of aerated cultures.
Saturated overnight cultures of control and catalase-producing strains were diluted 1000-fold in GM17 supplemented with haemin, Em and Cm, and with or without nisin for induced or noninduced cultures. Aeration was performed by stirring to 240 r.p.m. throughout the incubation. Survival was determined by plate counts on GM17 agar at days 1, 2 and 3 after cultures reached the stationary phase (when cell division stops, viable cells counts indicate the survival). All results presented correspond to the mean of three assays.
Agarose gel electrophoresis of cell lysates from aerated cultures.
Long-term aerated cultures of strains NZ cydA/recA(pVE3655) and NZ cydA/recA(pSEC : KatE) were performed. GM17 was supplemented with haemin, Em, Tc and Cm, with or without nisin. Each 24 h for 5 days, enumerations and whole-cell lysates were performed. Equal cell amounts of each culture were harvested based on their OD600, supernatant was removed and cells were resuspended in 100 µl buffer I (TES/lysozyme 1 mg ml1/RNase 1 mg ml1) and incubated for 2 h at 37 °C. Then 150 µl buffer II (TES/SDS 5 %/Proteinase K 50 µg ml1/bromophenol blue) were added, followed by incubation for 2 h at 56 °C. Samples (15 µl) of whole-cell lysates were run on agarose gel (0·7 %) overnight at 30 mV for a clean separation. The results presented are representative of three assays.
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RESULTS |
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DISCUSSION |
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We initially introduced the katE gene into L. lactis under control of a constitutive lactococcal promoter. The resulting recombinant KatE+ L. lactis strain possessed catalase activity but no increased H2O2 resistance was measured. Combination of an inducible promoter and fusion with a signal peptide led to production of an active KatE able to confer resistance to oxidative stress generated either by the presence of high levels of H2O2 or by aerated conditions. The quantity of KatE expressed under control of the constitutive promoter is probably too low to confer a resistance phenotype at 2 mM H2O2, and this was confirmed by the catalase activity assay. In the case of KatE expression under PnisA, the major form detected in the cellular protein fraction corresponds to the precursor form SPUsp45 : KatE (Fig. 2). No mature form was observed in the supernatant fraction by Coomassie staining of SDS-PAGE gels. This was confirmed by the absence of detectable catalase activity in culture supernatants. Interestingly, precursors of naturally secreted proteins are in a partially unfolded conformation which is translocation-competent, and they are generally considered as inactive (Simonen & Palva, 1993
). At present, we cannot determine whether the observed KatE activity is attributable to SPUsp45 : KatE, which might be properly conformed into the cytoplasm, or to weak processing of the precursor that would lead to release of active mature forms in the supernatant. The lack of anti-KatE antibodies and our inability to visualize catalase activity on non-denaturing gels (data not shown) hamper the confirmation of this hypothesis via determination of the cellular distribution of KatE.
The significant increase in the survival rate of the KatE-producing L. lactis strain in aerated conditions supports the use of the present strategy for improving antioxidant properties by cloning heterologous catalases. We observed that the presence of KatE protects cells and DNA from oxidative damage, not only upon exposure to H2O2, but also under conditions of aerated growth. The effects of KatE were observed in a DNA repair-defective strain. Prevention of DNA damage could be one of the main reasons for the better survival rate of the KatE-producing L. lactis strain.
This KatE-producing strain might also eliminate H2O2 from the environment and thus could be used as new antioxidant strain to deliver this antioxidant enzyme in vivo at the mucosal level. Mucosal tissue damage and dysfunction in chronic inflammatory bowel diseases or in radio-induced inflammation are partly caused by an exposure to excessive amounts of ROS, which can destroy biomolecules (Kruidenier et al., 2003a, b
, c
). In healthy individuals, the harmful effects of ROS are counteracted in the intestinal mucosa by an extensive system of antioxidants. A previous study established that the production of an active superoxide dismutase by two strains of lactobacilli protected them efficiently against oxidative stress by removing
ions (Bruno-Barcena et al., 2004
). The supply of antioxidant enzymes like catalase, alone or combined with superoxide dismutase, into the intestinal mucosa via ingestion of lactic acid bacteria could eliminate ROS and offer a promising new therapeutic strategy.
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ACKNOWLEDGEMENTS |
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Received 30 December 2004;
revised 29 May 2005;
accepted 6 June 2005.
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