ArticleApplied and environmental microbiology2009
Development and application of a upp-based counterselective gene replacement system for the study of the S-layer protein SlpX of Lactobacillus acidophilus NCFM.
Article in Applied and environmental microbiology, 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 84 papers.
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Who cites it
84 citing papers in PubMed, 155 citations in OpenAlex.
- The SlpX protein of Lactobacillus acidophilus CICC6074 has anti-inflammatory properties and protects Caco-2 intestinal epithelial cells from TNF-α-induced barrier damage.Journal of the science of food and agriculture · 2026Article
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- Ecology and engineering to modify the bile acid output of a defined microbial community.bioRxiv : the preprint server for biology · 2026Article
- Antiviral Role of Surface Layer Protein A (SlpA) ofPathogens (Basel, Switzerland) · 2026Article
- Development of a markerless tool for targeted chromosome modification in the thermophilic and methylotrophic bacterium Bacillus methanolicus.Microbial cell factories · 2025Article
- Being a better version of yourself: genetically engineered probiotic bacteria as host defense enhancers in the control of intestinal pathogens.Gut microbes · 2025Review
- The curated Lactobacillus acidophilus NCFM genome provides insights into strain specificity and microevolution.BMC genomics · 2025Article
- Scarless gene disruption enabled by a dual-plasmid knockout platform in a clinical infant-derivedFrontiers in microbiology · 2025Article
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- Identification of NpdA as the protein forming the surface layer inAccess microbiology · 2023Article
- Elimination of Deoxynivalenol, Aflatoxin B1, and Zearalenone by Gram-Positive Microbes (Toxins · 2022Article
- Development of a Markerless Deletion Mutagenesis System in Nitrate-Reducing Bacterium Rhodanobacter denitrificans.Applied and environmental microbiology · 2022Article
- AI-2E Family Transporter Protein inFrontiers in microbiology · 2022Article
24 more citing papers are in PubMed but not listed here.
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Authors and funding
8 authors at 2 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
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 Deltaupp 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 DeltaslpX 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.
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