Development and Application of a upp -Based Counterselective Gene Replacement System for the Study of the S-Layer Protein SlpX of Lactobacillus acidophilus NCFM
- 15 May 2009
- journal article
- Published by American Society for Microbiology in Applied and Environmental Microbiology
- Vol. 75 (10), 3093-3105
- https://doi.org/10.1128/aem.02502-08
Abstract
In silico genome analysis of Lactobacillus acidophilus NCFM coupled with gene expression studies have identified putative genes and regulatory networks that are potentially important to this organism's survival, persistence, and activities in the gastrointestinal tract. Correlation of key genotypes to phenotypes requires an efficient gene replacement system. In this study, use of the upp -encoded uracil phosphoribosyltransferase (UPRTase) of L. acidophilus NCFM was explored as a counterselection marker to positively select for recombinants that have resolved from chromosomal integration of pORI-based plasmids. An isogenic mutant carrying a upp gene deletion was constructed and was resistant to 5-fluorouracil (5-FU), a toxic uracil analog that is also a substrate for UPRTase. A 3.0-kb pORI-based counterselectable integration vector bearing a upp expression cassette, pTRK935, was constructed and introduced into the Δ upp host harboring the pTRK669 helper plasmid. Extrachromosomal replication of pTRK935 complemented the mutated chromosomal upp allele and restored sensitivity to 5-FU. This host background provides a platform for a two-step plasmid integration and excision strategy that can select for plasmid-free recombinants with either the wild-type or mutated allele of the targeted gene in the presence of 5-FU. The efficacy of the system was demonstrated by in-frame deletion of the slpX gene (LBA0512) encoding a novel 51-kDa secreted protein associated with the S-layer complex of L. acidophilus . The resulting Δ slpX mutant exhibited lower growth rates, increased sensitivity to sodium dodecyl sulfate, and greater resistance to bile. Overall, this improved gene replacement system represents a valuable tool for investigating the mechanisms underlying the probiotic functionality of L. acidophilus .Keywords
This publication has 54 references indexed in Scilit:
- S layer protein A of Lactobacillus acidophilus NCFM regulates immature dendritic cell and T cell functionsProceedings of the National Academy of Sciences of the United States of America, 2008
- Genome Sequence of Lactobacillus helveticus , an Organism Distinguished by Selective Gene Loss and Insertion Sequence Element ExpansionJournal of Bacteriology, 2008
- Characterization of a Novel Bile-Inducible Operon Encoding a Two-Component Regulatory System inLactobacillus acidophilusJournal of Bacteriology, 2007
- Identification of a Putative Operon Involved in Fructooligosaccharide Utilization by Lactobacillus paracaseiApplied and Environmental Microbiology, 2006
- Uracil Salvage Pathway in Lactobacillus plantarum : Transcription and Genetic StudiesJournal of Bacteriology, 2006
- Genomic features of lactic acid bacteria effecting bioprocessing and healthFEMS Microbiology Reviews, 2005
- Improved Prediction of Signal Peptides: SignalP 3.0Journal of Molecular Biology, 2004
- Positive-negative KG cassettes for construction of multi-gene deletions using a single drug markerGene, 1996
- Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extensionGene, 1989
- A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistanceMolecular Genetics and Genomics, 1984