doi:10.1016/j.femsle.2005.03.044 
Copyright © 2005 Federation of European Microbiological Societies Published by Elsevier B.V.
Marker-free chromosomal integration of the manganese superoxide dismutase gene (sodA) from Streptococcus thermophilus into Lactobacillus gasseri
José M. Bruno-Bárcenaa, M. Andrea Azcárate-Perilb, Todd R. Klaenhammera, b, c and Hosni M. Hassana, b, c, 
, 
aDepartment of Microbiology, North Carolina State University, P.O. Box 7615, Raleigh, NC 27695-7615, USA
bDepartment of Food Science, North Carolina State University, P.O. Box 7624, Raleigh, NC 27695-7624, USA
cDepartment of Southeast Dairy, Foods Research Center, North Carolina State University, P.O. Box 7624 Raleigh, NC 27695-7624, USA
Received 1 December 2004;
revised 1 February 2005;
accepted 26 March 2005.
Available online 7 April 2005.
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Abstract
A strategy for functional gene replacement in the chromosome of Lactobacillus gasseri is described. The phospho-β-galactosidase II gene (lacII) was functionally replaced by the manganese superoxide dismutase (MnSOD) gene (sodA) from Streptococcus thermophilus, by adapting the insertional inactivation method described for lactobacilli [Russell, W.M. and Klaenhammer, T.R. 2001 Efficient system for directed integration into the Lactobacillus acidophilus and Lactobacillus gasseri chromosomes via homologous recombination. Appl. Environ. Microbiol. 67, 4361–4364]. L. gasseri carrying the heterologous sodA gene grew on lactose as efficiently as the wild-type parent. An active MnSOD was expressed in the transgenic strain, and the enzyme migrated on PAGE-SOD activity gels to the same position as that of MnSOD from S. thermophilus. The expression of MnSOD from a single copy of sodA integrated in the chromosome of L. gasseri provided enhanced tolerance to hydrogen peroxide, and extended the viability of carbon/energy starved cultures stored at 25 °C. This is the first report showing the successful utilization of the pORI plasmids system to generate marker-free gene integration in L. gasseri strains.
Keywords: Functional gene replacement; Manganese superoxide dismutase; Oxidative stress; Lactobacillus gasseri; Lactic acid bacteria; Probiotics
Article Outline
- 1. Introduction
- 2. Materials and methods
- 2.1. Bacterial strains and growth conditions
- 2.2. Chemicals and enzymes
- 2.3. DNA isolation and manipulation
- 2.4. Oligonucleotides for PCR amplification
- 2.5. Preparation of cell-free extracts
- 2.6. Biochemical assays
- 2.7. Preparation of antibodies against S. thermophilus MnSOD and Western-blot hybridization
- 2.8. Quantitative real-time PCR
- 2.9. Growth kinetics
- 2.10. Survival of L. gasseri in the presence of hydrogen peroxide
- 3. Results
- 3.1. Construction of a site-specific integration plasmid for the insertional inactivation of lacII of L. gasseri NCK334
- 3.2. Functional chromosomal replacement of L. gasseri NCK334 lacII
- 3.3. Effect of the insertional inactivation of lacII on the growth of L. gasseri on lactose
- 3.4. Protection against hydrogen peroxide
- 3.5. Protection against carbon starvation
- 4. Discussion
- Acknowledgements
- References
Fig. 1. Construction of pJB34-S; an integrative plasmid containing the sodA gene from S. thermophilus AO54 & the two flanking sequences of lacII from L. gasseri NCK334. Symbols localize promoter (¬) and Rho independent terminator (
) regions in sodA.
Fig. 2. (a) Southern hybridization analysis of chromosomal DNA from L. gasseri strains. DNA was digested with HindIII (lane 1, plasmid pJB34-S; lane 2, NCK334; lanes 3–5, NC1502; lane 6, NC1503) and hybridized with a 1.43-kb internal fragment of lacII. (b) Schematic representation (i.e., not drawn to scale) of the relevant regions and HindIII fragments sizes of L. gasseri strains NCK334 (wild-type); NC1502 (single cross-over) and NC1503 (double cross-over). Symbols localize promoter (¬) and Rho independent terminator () regions.
Fig. 3. MnSOD-protein concentration (Western-blot) in cell-free extracts and transcription level of sodA (qRT-PCR) using RNA extracted from S. thermophilus AO54 and the different Lactobacillus strains harboring either pSodA or a single copy of sodA integrated into L. gasseri chromosome. Each lane was loaded with 10 μg of total protein that corresponded 300, 15.7, 2.25 and 2.64 mU of MnSOD, respectively. SD ± value for duplicated experiments was lower than 6%.
Fig. 4. Effects of hydrogen peroxide on the viability of wild-type L. gasseri and the different sodA bearing strains. Cells from exponential phase of growth were exposed to increasing concentrations of hydrogen peroxide for 90 min. The entire procedure was carried out in APT broth without sugar and under aerobic conditions.
Fig. 5. Effects of carbon starvation on the survival of the different strains of L. gasseri. Cells from the exponential growth phase were placed at 25 °C (a) or 4 °C (b). The entire procedure was carried out in APT broth without sugar under aerobic conditions.
Table 1.
Bacterial strains & plasmids used in this study
Ampr, ampicillin resistance; Cmr, chloramphenicol resistance; Emr, erythromycin resistance; Kmr, Kanamycin resistance.
NC & NCK culture collection at North Carolina State University, Raleigh, NC. ATCC, American type culture collection.
Table 2.
Primers utilized in this study
a Primers utilized for qRT-PCR experiments.
Table 3.
Maximum specific growth rates and final pH determined for the L. gasseri derivatives
Strains were grown aerobically at 37 °C in APT broth containing 0.5% (w/v) lactose.
No significant growth in the base APT medium without carbohydrate was observed. Results are based on three independent experiments, ±SD.
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