88D-3 |
Insertional inactivation of the S-layer protein gene slpA in Lactobacillus acidophilus NCFM |
E. ALTERMANN, W. M. Russell, and T. R. Klaenhammer. Department of Food Science, North Carolina State University, Box 7624, Raleigh, NC 27695 Lactic acid bacteria have been used for centuries for fermentation processes and recently gained increased attention due to their probiotic properties. We are investigating the genetic basis of probiotic properties of Lactobacillus acidophilus and applying genetic approaches in attempts to improve stability and functionality. S-layers found on the surface of lactobacilli interact with components of the epithelial cell layer of the mammalian gastrointestinal tract and have been proposed to effect adherence and cell survival properties. Using two primers designed within the slpA gene from the closely related Lb. acidophilus strain ATCC4356 (Boot, et al., 1993), we amplified an 826 bp fragment from Lb. acidophilus NCFM. Sequencing the amplicon revealed identity to the corresponding region of the Lb. acidophilus ATCC4356 gene, slpA. Insertional inactivation of slpA was carried out using a pORI-type integration system (Leenhouts, et al., 1998), modified for use in lactobacilli. Disruption of slpA was confirmed by Southern Blot analysis showing the corresponding junction fragments. Isolation of the surface-layer proteins from the slpA knockout strain showed that the S-layer protein band (47 kDa) was virtually absent in the knockout strain. A faint protein band with the same molecular weight was observed, possibly indicating expression of the slpB gene. The remaining part of the slpA gene was amplified via inverse PCR and the complete gene sequenced. A BlastN analysis confirmed the identity of the corresponding genes from Lb. acidophilus NCFM and ATCC4356. The slpA knockout strain was examined for its tolerance to various stresses (tolerance towards NaCl, bile salt and repeated freezing-thawing). Significant changes were found in comparison to the wildtype parent, Lb. acidophilus NCFM.
Session 88D, Food Microbiology: General
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