Enhancing oxidative stress resistance in Bifidobacterium thermophilum using a novel overexpression vector and transformation protocol

Accepted Manuscript Enhancing oxidative stress resistance in Bifidobacterium thermophilum using a novel overexpression vector and transformation protocol

Marc J.A. Stevens, Adriano Venturini, Christophe Lacroix, Leo Meile PII: DOI: Reference:

S0147-619X(17)30023-9 doi: 10.1016/j.plasmid.2017.06.002 YPLAS 2347

To appear in:

Plasmid

Received date: Revised date: Accepted date:

15 February 2017 9 June 2017 9 June 2017

Please cite this article as: Marc J.A. Stevens, Adriano Venturini, Christophe Lacroix, Leo Meile , Enhancing oxidative stress resistance in Bifidobacterium thermophilum using a novel overexpression vector and transformation protocol, Plasmid (2017), doi: 10.1016/ j.plasmid.2017.06.002

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ACCEPTED MANUSCRIPT Enhancing oxidative stress resistance in Bifidobacterium thermophilum using a novel overexpression vector and transformation protocol

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Marc J. A. Stevens*, Adriano Venturini, Christophe Lacroix and Leo Meile

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Schmelzbergstrasse 7, 8092 Zurich, Switzerland

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Laboratory of Food Biotechnology, Institute of Food, Nutrition and Health, ETH Zürich,

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*corresponding author:

Fax: +41 44 632 14 03

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E-Mail: [email protected]

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Telephone: +41 44 632 48 67

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ACCEPTED MANUSCRIPT Abstract Bifidobacterium thermophilum is encountered in the GI-tract of pigs and infants. Here we provide a transformation protocol for B. thermophilum and a novel expression vector for this species. The protocol resulted in transformation rates of 1x103 transformed cells per

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µg DNA. Transformation was shown to be dependent on the presence of fructo-

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oligosaccharides during growth, polyethylene glycol in the electroporation buffer, and on

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methylation of the vector.

The Escherichia coli - B. thermophilum shuttle vector pLFB1012 for heterologous gene

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expression was constructed harbouring the glyceraldehyde 3-phosphate dehydrogenase

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promoter from Bifidobacterium longum (Pgap). Activity of the β-glucoronidase gene gusA under control of Pgap could be detected at a 20-fold higher rate compared to the wild type,

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showing activity of the promoter in B. thermophilum. Thereafter, the B. longum gene

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bl_1404, previously proposed to be involved in oxidative stress resistance, was cloned under control of the Pgap. The wild type cell numbers of B. thermophilum RBL 67

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decreased at least 9 log after a 20-mM H2O2 treatment for 60 minutes whereas the mutant

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strain expressing bl_1404 showed an increased survival of 2 logs compared to the wild type strain. To our knowledge this is the first report on transformation of B.

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thermophilum. Further, it is shown that pLFB1002 is suitable for engineering B. thermophilum and that bl_1404 from B. longum is involved in peroxide resistance in bifidobacteria.

Keywords: Bifidobacterium thermophilum, transformation, overexpression, peroxide

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1. Introduction Bifidobacteria are strict anaerobic members of the Actinobacteria phylum that colonize the gastrointestinal tract of humans and many animals at an early stage of life (1). Bifidobacteria play an important role in the infant gut microbiota and their presence in

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the newborn gut seems at least partially regulated via mother-child transfer using breast

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milk as vector (2, 3). Functional genomic studies have revealed several bifidobacterial niche factors such as pili, exopolysaccharides, and ability to grown on human milk

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oligosaccharides (4-6). However, molecular tools and protocols for bifidobacteria are still limited and studies which confirm genetic traits are rare (7).

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Bifidobacteria are Gram-positive bacteria and possess a complex and thick cell wall,

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which is a strong barrier for uptake of external DNA and transformation (8). In addition,

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bifidobacteria are sensitive to prolonged oxygen exposure, resulting in stress during the transformation procedure and lower transformation rates (9). Bifidobacteria frequently

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possess protection mechanisms against foreign DNA, such as restriction-modification

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(RM) systems that degrade non-modified plasmid DNA. The barrier formed by such RM systems can be overcome via methylation of the DNA or by mutation of the restriction

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sites (10, 11). Transformation protocols are available for bifidobacteria with transformation rates ranging from 5.0 x 101 to 1.0 x 105 colony forming units (CFU)/µg DNA and protocols and efficiencies are species and even strain dependent (11-13). High transformation rates of 10exp7 CFU/ugDNA were obtained in B. breve UCC2003 and of 10exp6 CFU/ugDNA in B. longum NCIMB8809, allowing construction of integration mutants (4, 10, 14). In addition, integration mutants were made in Bifidobacterium longum NCC2705 by using a temperature-sensitive plasmid (15). 3

ACCEPTED MANUSCRIPT Bifidobacterium thermophilum is an animal-associated commensal species which is also found in some infants (16). Peptidoglycans of B. thermophilum improve cytotoxic activity of mice lymphocytes and protect mice and chicken against Escherichia coli infections (17-19). Furthermore, infant faeces isolate B. thermophilum strain RBL67 is

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active in vitro against Salmonella enterica subsp. enterica serovar Typhimurium, Listeria

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monocytogenes and rotavirus (20-24). In this study, we attempted to transform B.

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thermophilum by testing and optimizing protocols that result in successful transformation of other bifidobacteria. Furthermore, we constructed an overexpression plasmid for

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heterologous gene expression. The efficiency of the overexpression was tested with β-

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glucoronidase gene gusA, already applied in bifidobacteria previously (25, 26). To test suitability of the plasmid for strain engineering, the stability of the plasmid was

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determined and a gene putatively involved in peroxide resistance was overexpressed.

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2. Material and Methods 2.1 Bacterial strains, media, and growth conditions Bacterial strains used in this study are listed in Table 1. Bifidobacterium strains were grown anaerobically at 37° in sterile filtered MRS medium ((27), Biolife Italiana, Milan,

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Italy) supplemented with 0.05 % (w/v) L-cysteine (cMRS). E. coli strains were grown

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aerobically in TY medium (Difco Laboratories, Detroit, USA) with agitation at 37 °C. When appropriate, chloramphenicol was added to the medium at 3 µg/mL for

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Bifidobacterium strains and 10 µg/mL for E. coli strains, except when stated otherwise. Anaerobic incubations were performed using the AnaeroGen oxygen scavenger system

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(Oxoid, Basingstoke, United Kingdom). Medium supplements were obtained from

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Sigma-Aldrich Chemie GmbH (Buchs, Switzerland), except when stated otherwise.

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Bacterial growth was monitored by measuring the optical density at 600 nm (OD600) using a Biophotometer (Vaudaux-Eppendorf AG, Schönenbuch, Switzerland).

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2.2 DNA manipulation

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Plasmid-DNA isolations from E. coli were performed using a Maxiprep Kit (Qiagen, Basel, Switzerland). Restriction enzymes were obtained from New England Biolabs

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(Frankfurt am Main, Germany), except for Ecl136II (Fermentas GmbH, Le Mont-sur Lausanne, Switzerland). Proof-reading Phusion-polymerase and GpC methyltransferase were obtained from New England Biolabs and T4-ligase from Invitrogen (Basel, Switzerland). PCR using Taq polymerase was performed using 2x PCR Mastermix (Thermo Scientific, Waltham, USA). Primers were purchased from Microsynth (Balgach, Switzerland). Restriction, methylation and ligation reactions were performed according to the manufacturer’s instructions. 5

ACCEPTED MANUSCRIPT 2.3 Construction of plasmids The E. coli-Bifidobacterium shuttle vector pCSC5 contains the bifidobacterial repA gene from pLME201 and the rep genes from pUC19 (Fig. 1, (9)). The pCSC5 plasmid was restricted with PvuI and Ecl136II and the 3.2-kb fragment containing the

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chloramphenicol resistance gene cat and the repA genes was purified. Additionally, the

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pUC19 rep genes were amplified using the primers Rep_for and Rep_rev (Table 2). The

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generated 0.7-kb amplicon was ligated to the 3.2-kb fragment from pCSC5 resulting in the vector pLFB1011, a 4.0-kb E. coli-Bifidobacterium shuttle vector with a cat gene and

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the E. coli rep gene in the same orientation (Fig. 1). Construction and orientation of

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pLFB1011 was confirmed by PCR and restriction analyses.

For construction of the overexpression vector pLFB1012, a 531-bp fragment containing

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the glyceraldehyde-3-phosphate dehydrogenase promoter (Pgap) from B. longum DSM

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20088 was amplified using the primer pair Pgap-5/Pgap-3 (Table 2). The amplicons were restricted with PstI and SacII due to restriction sites introduced in primers and finally

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cloned into a similarly restricted pLFB1011, resulting in pLFB1012 (Fig. 1).

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A 1825-bp fragment containing gusA was obtained by PCR using pNZ276 (28) as template and the primer pair Gus_start/Gus_end (Table 2). The fragment was restricted

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with SphI and SacII (restriction sites introduced in primers) and ligated to the 3.7-kb fragment of a similarly restricted pLFB1012. This resulted in pLFB1013, a vector containing gusA under control of the B. longum Pgap. A similar strategy was applied for the construction of pLFB1404. The gene encoded by bl_1404 was amplified using the primers bl_1404_start/bl_1404_end (Table 2) and the resulting fragment was restricted with SphI and XhoI. The restricted fragment was ligated

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ACCEPTED MANUSCRIPT to a similarly restricted pLFB1012, resulting in pLFB1014, a vector containing bl_1404 under control of the B. longum Pgap. 2.4 Susceptibility tests The minimum inhibitory concentration (MIC) of chloramphenicol for B. thermophilum

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RBL67 was determined in an antibiotic susceptibility test according to CLSI guidelines

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2.5 Transformation of B. thermophilum RBL67

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as described previously (29). The tests were performed anaerobically in cMRS at 37°C.

A number of electroporation buffers were tested in this study to transform B. Growth

was

performed

in

cMRS

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thermophilum.

supplemented

with

16%

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fructooligosaccharides (FOS) according to Serafini et al (13), except when stated otherwise. All tested electroporation buffers were made in 1 mM citrate buffer at pH 8.0.

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An overnight pre-culture of B. thermophilum RBL67 in cMRS supplemented with 16 %

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(w/v) FOS (Cosucra, Warcoing, Belgium) was used to inoculate 8 mL of the same medium at 20%. The culture was incubated anaerobically at 37 °C for 2 – 5 h until an

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OD600 of 0.7 – 0.9 was reached in the late exponential phase. Thereafter, the cells were

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chilled on ice for 20 min and harvested by centrifugation (4’000 x g, 4 °C, 5 min). The pellet was washed twice in the appropriate tested electroporation solution (Table 3), then

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resuspended in 260 µL of the respective solution and used for electroporation. Cells (100 µL) and plasmid DNA were mixed in pre-cooled electroporation cuvettes with an interelectrode distance of 0.2 cm (EquiBio, Witec AG, Littau, Switzerland) and set on ice for 15 min. The cuvettes were placed in a Gene PulserTM (Bio-Rad, Richmond USA) and an electric pulse was delivered using a Pulse ControllerTM apparatus (Bio-Rad). The pulse controller was set at 200 Ω parallel resistance and an electrical capacity of 25 µF.

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ACCEPTED MANUSCRIPT Immediately after electroporation, 1 mL of cMRS with 16 % FOS was pipetted into the cuvettes followed by anaerobic incubation at 37 °C for 3 – 5 h to regenerate the cells. Thereafter, appropriate dilutions were plated on cMRS plates supplemented with 3 µg/mL chloramphenicol and incubated anaerobically at 37 °C for 5 to 7 days. All

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protocols were tested at least 2 times, except for those that did not result in successful

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transformation of cells.

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To measure the spontaneous chloramphenicol-resistance mutation rate, negative controls were performed by electroporation of competent bifidobacterial cells without plasmid

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DNA added. To determine the amount of viable cells at different steps during the

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transformation procedure, plate counts were performed using an agar spot method where 10 µL of appropriate dilutions were spotted on cMRS agar and incubated at 37 °C

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anaerobically for 24 h.

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2.6 GUS Assay

The β-glucuronidase (Gus)-activity was determined using a protocol adapted from

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Platteeuw et al. (28) and Axelsson et al. (30). E. coli pNZ276 (28), constitutively

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expressing gusA was used as positive control. Shortly, the appropriate strain was grown overnight and the next day diluted at an OD600 = 1.0, harvested, washed once and

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subsequently resupended in 1 mL GUS buffer (50 mM NaPi, 10 mM β-mercaptoethanol, 1 mM EDTA, 0.1% (w/v) Triton X-100, pH 7.0). Cells were disrupted by bead-beating them for two times 30 s at speed 4.0 using a Fastprep cell disrupter (QBiogene Inc., Carlsbad, Ca, USA), interspaced with cooling intervals on ice. The cells debris was centrifuged (10’000 x g, 5 min, 4° C) and 100 μl of the cytosolic proteins containing supernatant was mixed with 900 μl 1.25 mM 4-nitrophenyl-β-D-glucuronic acid solved in

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ACCEPTED MANUSCRIPT 50 mM NaPi buffer at pH 7.0. The reaction mixture was incubated at 37°C in a Macro photometer cuvette (Greiner Bio-One GmbH, Frickenhausen, Germany) and the absorption at 405 nm (A405) was measured every 5 min for 90 min. Four independent biological and two technical replicates were performed. The A405 was plotted in time and

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the linear regression calculated using the least squares method in Microsoft Excel. The β-

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glucuronidase activity was defined as the slope of the trend line normalized by the protein

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concentration of the sample. Protein concentrations were determined as described by Bradford (31). The experiment was performed in triplicate and significance of differential

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activity was analysed using the Student's test in Excel 2016 (Microsoft, Redmond, USA).

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2.8 Peroxide assay

B. thermophilum RBL67 and derivatives were grown overnight until an OD600 of ~ 1.5

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after which H2O2 was added to a final concentration of 20 mM. Samples were taken

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before H2O2 addition and after 30, and 60 min. Strains were grown in the exponential phase at an OD600 0.5 to test lower H2O2 levels of 10 mM. Samples were immediately

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diluted and appropriate dilution were plated on cMRS Agar incubated anaerobically at 37

duplicate.

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°C for 3 days, after which colonies were counted. The experiments were performed in

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2.9 Plasmid stability tests The stability of pLFB1011 in B. thermophilum in absence of selection pressure was determined by propagating the strains in 4 repeated cultures in cMRS without chloramphenicol for approximately 25 generations. The cultures were incubated anaerobically at 37 °C and appropriate dilutions were plated on cMRS plates with 3 µg/mL chloramphenicol daily. The experiment was performed in triplicate.

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3. Results 3.1 Chloramphenicol susceptibility of strains The plasmid pLFB1011 and its derivatives harbour a chloramphenicol acetyltransferase (cat) gene which allows selection of transformants on chloramphenicol. To find the

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lowest chloramphenicol concentration suitable for selection of transformed cells, the MIC

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of chloramphenicol was determined for B. thermophilum RBL67. The MIC was between 1 and 2 µg/mL and hence the selection of genetic variants was done on plates containing

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3 µg/mL chloramphenicol in all transformation experiments.

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Transformation of Bifidobacterium thermophilum

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To transform B. thermophilum RBL67, we first tested different growth stages and

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conditions for their effect on transformation efficiency. Growth in standard cMRS did not result in successful transformation, independent of growth stage or tested electroporation

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buffers (data not shown). Also addition of up to 0.5% glycine or of 0.5 M sucrose to the

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medium did not result in successful transformation (data not shown). Only growth and regeneration in cMRS supplemented with 16% FOS (13) and harvesting the cells in the

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late exponential phase resulted in detectable transformation of B. thermophilum. These growth conditions were used in all additional tests. Thereafter the survival rate of B. thermophilum RBL67 during transformation was determined. B. thermophilum RBL67 viable cell count remained constant during the transformation procedure and no difference in survival was observed between an electrical pulse of 10 or 12.5 kV/cm (data not shown). High pulses are generally thought

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ACCEPTED MANUSCRIPT to give better transformation results (32), and hence all further transformations were conducted with 12.5 kV/cm. The transformation was then optimized by testing different electroporation solutions (Table 3), all used previously to transform bifidobacteria or Lactobacillus plantarum (13,

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33, 34). The buffer 30% PEG 1450 resulted in 2.13 log transformed cells/µg DNA, which

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is significantly more compared to other tested electroporation solutions (Table 3).

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The restriction barrier protects bifidobacteria against foreign DNA, but is also a hurdle for their transformation (10). Therefore, the E. coli derived plasmid DNA was methylated

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to protect it against restriction enzymes. Methylation protected the DNA against SacII

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digestion (data not shown), demonstrating the methylation of the plasmid. Transformation with methylated plasmid resulted in an approximately one log increase in

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transformation efficiencies, independent of the electroporation buffer (Table 3). The

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buffer 30% PEG 1450 resulted in the highest efficiency of log 3.01 cells/µg methylated DNA.

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Taken together, growth in cMRS supplemented with16% FOS, harvesting cells in late

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exponential phase, using PEG-1450 in the electroporation buffer and methylating the

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plasmids results in the highest transformation rate for B. thermophilum RBL67.

Overexpression of genes in B. thermophilum RBL67 The pLFB1012 overexpression vector containing the Pgap was constructed to enable consecutive expression of genes in bifidobacteria. The suitability of the vector pLBF1012 was tested by cloning the β-glucuronidase gusA behind Pgap and measuring the βglucuronidase activity. B. thermophilum RBL67 transformed with the empty vector

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ACCEPTED MANUSCRIPT pLFB1012 showed a minimal enzymatic activity of 5.7 ± 2.85 10-5 min-1 mg-1prot (Fig. 2). B. thermophilum RBL67 harbouring its derivative pLFB1013 showed a 20 fold higher enzymatic activity of 1.0 ± 0.02 10-3 min-1 mg-1prot, showing that the overexpression vector is suitable for heterologous gene expression.

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To further test the suitability of the vector for engineering bifidobacteria, the protein

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encoded by ORF bl_1404 from B. longum NCC2705 was expressed in B. thermophilum

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RBL67. Bl_1404 encodes a putative membrane protein and is proposed to be involved in oxidative stress survival (35). The viable cell number of B. thermophilum RBL67

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harbouring the empty vector pLFB1012 reduced 4 log after exposure to 20 mM H2O2 for

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30 minutes and no viable cells were detected after 60 min (Fig 3a). The method has a detection limit 102 CFU/mL so the reduction after 60 minutes was of at least 7 logs. The

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viability of the strain expressing gusA was reduced by 5 log after of exposure to 20 mM

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H2O2 for 30 minutes and no viable cells could be detected after 60 min. The viable cell number of strain RBL67 expressing bl_1404 was reduced by 4 log after 30 minutes and 5

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log after 60 minutes. The expression of bl_1404 in RBL67 yielded an approximately 100-

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fold increased survival after exposure to 20 mM H2O2 rate compared to the wild type strains (Fig. 3a).

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Because no wild-type-like strains survived 20 mM H2O2 treatment after 60 minutes, we performed an addition analyses with 10 mM H2O2 using exponential growing cell and analysed survival after 15 and 30 minutes (Fig. 3b). The viable cell numbers of B. thermophilum RBL67 harbouring pLFB1012 or pLFB1013 reduced 3 logs after 15 minutes and another 3 logs after 30 min. The strain harbouring pLFB1404 reduced 1 log

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ACCEPTED MANUSCRIPT after 15 minutes and another 1.5 logs after 30 minutes. The test with 10 mM H2O2 confirmed the increased robustness to peroxide of the strain expressing bl_1404.

Stability of pLFB1011 and its derivatives in Bifidobacterium thermophilum

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The stability of the pLFB1011 without selection pressure was determined in 4 serial batch

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cultures and the number of cells harbouring pLFB1011 and of total cells determined in

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the early stationary phase. The total cell count was 3.42 ± 1.5 x 109 log CFU/mL and counts for cell harbouring pLFB1011 2.23 ± 1.1 x 109 log CFU/mL, revealing no

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significant difference in cell counts (p = 0.27 in Student's test). The plasmid is therefore

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stably replicating for at least 20 generations without selection.

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ACCEPTED MANUSCRIPT Discussion Here we present a protocol to transform Bifidobacterium thermophilum and the vector pLFB1012 to overexpress proteins in this species. The vector contains pUC18 origin for replication in E. coli and a repA gene from a plasmid isolated from Bifidobacterium

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longum (9). The plasmid was stable for at least 20 generations thus the activity of the

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repA gene seems sufficient for plasmid maintenance. This enables the use of the plasmid

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in prolonged experiments and application without selection pressure. However, plasmid stability in bifidobacteria decreases rapidly with increasing size (36) and stability of

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pLFB1012 derivatives with increased sizes should be tested separately.

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The transformation efficiency is affected by many factors, including voltage, permeabilization of the membrane and resistance of the plasmid DNA to degradation

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(32). The activity of many restriction enzymes is blocked by methylation, including that

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of bifidobacterial restriction enzymes (11). The enhanced transformation rate of B. thermophilum with methylated plasmids points strongly toward the presence of a

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restriction/modification system. However, in contrast to many other bifidobacteria the

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genome B. thermophilum RBL67 does not encode a restriction/modification system (37), and the reason why methylation of the plasmid enhances transformation in B.

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thermophilum remains unclear. In parallel, the role of FOS in the growth and regeneration media is not fully understood, but addition of 16% FOS might protect and osmotically stabilise the cells (13, 38). Polyethylene glycol 1450 (PEG) in the electroporation buffer enhanced transformation significantly (Table 3). PEG was already used to transform Streptomyces species (39), just as Bifidobacterium members of the Actinobacteria. PEG is known to enhance transformation efficiency for many microbes (40) but to our

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ACCEPTED MANUSCRIPT knowledge it was so far not used for bifidobacteria. Low molecular weight PEG 200-400 is cytotoxic in eukaryotes (41). B. thermophilum survived during the transformation procedure and the potential toxicity of PEG1450 is likely not affecting the transformation rate.

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The glucuronidase activity of B. thermophilum strains harbouring pLFB1013 containing

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gusA under control of Pgap indicated that the promoter is strong enough to alter the

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phenotype. The Pgap was used previously for expressing of gusA and was shown to be the strongest of all tested bifidobacterial promoter (42). The GUS activity in gusA harbouring

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cells was at 20 fold higher compared to wild type cells, similar to previous data reported

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for bifidobacteria (42). The functionality of the Pgap to drive heterologous gene expression was further tested by expression of the B. longum bl_1404. This gene is one of

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two genes constitutively higher expressed in a mutant strain with enhanced peroxide

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resistance (35). Here, we confirm that bl_1404 overexpression leads to enhanced peroxide resistance. Moreover, enhanced peroxide tolerance of strains harbouring

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pLFB1404 suggests that the gene encoded by bl_1404 is a target for engineering of

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peroxide resistance of bifidobacteria. Bifidobacteria are sensitive to oxygen, yet might encounter a severe oxidative stresses during production, storage and ingestion (43).

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Moreover, oxidative stress occurs in the GI-tract and some probiotic strains exhibit antioxidant properties that are presumed to be beneficial for the host (44). Engineering of oxidative stress resistance is therefore important to enhance viability of bacteria and functionality of bifidobacteria containing products (45). Our study provides a protocol and a tool to engineer oxidative stress resistance in B. thermophilum. We assume the tool can be used to engineer other stress resistances in B. thermophilum.

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ACCEPTED MANUSCRIPT Tables Table 1: Strains and plasmids used in this study and their relevant characteristics Relevant characteristics a

Sourceb

B. thermophilum RBL67

Baby faeces isolate

(46)

B. longum subsp. longum DSM20219

Type strain, adult intestine isolate

DSMZ

B. longum NCC2705 HPR2

Peroxide resistant mutant derivative of NCC2705

(35)

B. longum subsp. infantis DSM20088

Type strain, adult intestine isolate

DSMZ

E. coli MC1061

Cloning host

Material

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Strains

Plasmids

(47)

5.770 kb, shuttle vector, CmR, AmpR

pLFB1011

3.972 kb, pCSC5 derivative, CmR

This study

pLFB1012

3.809 kb, pLFB1011 derivative containing the promoter

This study

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pCSC5

(9)

PGAP, CmR

5.627 kb, pLFB1012 derivative containing the gusA gene

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pLFB1013

This study

under control of PGAP, CmR 4.514 kb, pLFB1012 derivative containing the gene encoded

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pLFB1404

a

by bl_1404 under control of P GAP, CmR 6.113 kb, vector containing the gusA gene, CmR

(28)

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pNZ276

This study

CmR, chloramphenicol resistance; AmpR, ampicillin resistance.

b

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DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany; NCC, Nestlé

culture collection, Lausanne, Switzerland;

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ACCEPTED MANUSCRIPT Table 2 Oligonucleotide primers used in this study 5’ – 3’ Sequence

Rep_for

ATG TGA GCA AAA GGC CAG CA

Rep_rev

ACC AAA ATC CCT TAA CGT GAG TT

Cat_for

AGG CCT AAT GAC TGG CTT TT

Pgap-5

GAT CCT GCA GTC CCA CTC CGA TGT TTC GCT

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Primer

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GAT CCC GCG GGG TAC CGC ATG CTA ATT CTC CCT Pgap-3a

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TGT AGG GTG

GAT CGC ATG CTA CGT CCT GTA GAA ACC

Gus_end

GAT CCC GCG GTC ATT GTT TGC CTC CCT GC

bl_1404_start

GAT CGC ATG CCC AAC CAC AAC ATC GCC

bl_1404_end

GAT CCT CGA GTC ACT GCA ACG TGA AGG TAT G

repA_pAV_for

ACT CTC ACG GCC AAA TTC AC

repA_pAV_rev

GAA CGT CGT CCC AAA ATT CA

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restriction sites introduced in primers are underlined.

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a

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Gus_start

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ACCEPTED MANUSCRIPT Table 3: Transformation rates of B. longum RBL67 with different plasmids and electroporation conditions Electroporation solution

Log of Efficiency per µg DNA*

pLFB1011

30% (w/v) PEG-1450

2.13 ± 0.55c

pLFB1011

0.5 M sucrose

1.81 ± 0.68b

pLFB1011

10% (w/v) glycerol

1.27 ± 0.07a

Plasmid

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pLFB1011

30% (w/v) PEG-1450

pLFB1011

16% (w/v) FOS

pLFB1011

0.5 M sucrose

pLFB1011

10% (w/v) glycerol

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methylated

3.01 ± 0.06e

2.37 ± 0.15 c,d 2.43 ± 0.16d

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2.16 ± 0.17 c

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different characters indicate significantly difference (p < 0.05) in a student's t-test

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*

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non-methylated

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ACCEPTED MANUSCRIPT Figures PvuI

PvuI

pLFB1011

pCSC5

3972 bp SacII

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5770 bp

PstI

pLFB1012 3809 bp

pLFB1013 5621 bp

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AflII/SalI XhoI

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Ecl136II

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SphI/KpnI/SacI

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Figure 1: Construction of the overexpression plasmid pLFB1013 to be used in

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bifidobacteria. Genes originating from E. coli pUC19 are depicted in grey, other genes in black and the Pgap in white. Restriction sites used in this study or suitable for cloning

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are indicated. CmR = chloramphenicol resistance, AmpR = ampicillin resistance, repA = bifidobacterial replication gene repA, pBR322 rep = replication gene for pBR322, LacZ’ = truncated lacZ, gusA = glucuronidase gusA, Pgap = gap promoter.

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0.00012

0.00008

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0.00006

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0.00004

0.00002

0 1

2

RBL67 pAV-GAP-gusA

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ΔA405 mgprot-1 min-1

0.0001

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RBL67

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Figure 2: GUS activity in B. thermophilum strains. RBL67 is the wild type strain,

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RBL67 pAV-GAP-gusA harbors gusA under the control of the promotor Pgap. Average

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results of three independent experiments are shown

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ACCEPTED MANUSCRIPT A 1.00E+10

1.00E+08

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1.00E+04

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CFU/ml

1.00E+06

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1.00E+02

1.00E+00

30 t/min

60

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0

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B

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1.00E+08

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1.00E+10

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ACCEPTED MANUSCRIPT Figure 3: Plasmid-dependent survival of B. thermophilum strains at upon exposure to hydrogen peroxide. A) exposure to 20 mM hydrogen peroxide, B) exposure to 10 mM hydrogen peroxide. White = RBL67 harbouring empty vector pLFB1012, Grey = RBL67 harbouring pLFB1404, Black = RBL67 harbouring pLFB1013. The dashed line

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indicates the detection limit. Average results of two independent experiments are shown.

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Highlights

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Bifidobacterium thermophilum was successfully transformed A vector for heterologous gene expression was constructed Increased peroxide resistance was achieved via genetic engineering of Bifidobacterium thermophilum

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