Development and Application of a upp -Based Counterselective Gene Replacement System for the Study of the S-Layer Protein SlpX of Lactobacillus acidophilus NCFM

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 ⌬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 ⌬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.Lactobacillus acidophilus NCFM is a commercially established probiotic bacterium that is widely used in dietary supplements and in milk and fermented dairy products (47). Originally a human intestinal isolate from the 1970s (8), this strain has since been examined extensively for various desirable traits. Due to the importance of understanding the molecular mechanisms involved in probiotic functions, the complete genome sequence of L. acidophilus NCFM was determined (2). The NCFM genome sequence serves as a blueprint for in silico identification of candidate gene loci and gene regulatory networks that may play essential roles in the survival and host interactions of this microorganism in the gastrointestinal (GI) tract, including genes involved in acid tolerance (5), bile tolerance (38, 42), adherence factors (20), environmental sensing and response (7), prebiotic sugar utilization (9), polysaccharide biosynthesis, oxalate degradation (6), and bacteriocin production (22). In addition, ongoing microarray gene expression studies have revealed specific gene sets of interest that are being investigated further. The fundamental approach for establishing functional roles for important gene features involves inactivation of the genes ...

Sortase-deficient lactobacilli: effect on immunomodulation and gut retention

“…The DNA manipulation and cloning methodology used to construct DsrtA mutants followed those previously described (Goh et al, 2009). All primers were synthesized by Integrated DNA Technologies (Table 2).…”

“…Survival was measured over the course of 1 h in SGJ and 4 h in SIJ as determined by plating on MRS agar (Goh & Klaenhammer, 2010 Mucin and Caco-2 adherence assays. Adherence to porcine mucin (Sigma) and Caco-2 adenocarcinoma cells (ATCC HTB-37) was performed as described previously (Goh et al, 2009). Briefly, type III porcine gastric mucin (Sigma) was immobilized in 96-well flat bottom microtitre plates for 18 h at 4 uC.…”

“…Selected integrants with Em R and Cm S phenotypes were subcultured three times in MRS broth without antibiotic at 24 h intervals at 37 uC. Following the third passage, cultures were diluted and plated on glucose semi-defined medium (Kimmel & Roberts, 1998) supplemented with 100 mg 5-FU ml 21 (Goh et al, 2009) to select for 5-FU R colonies in which plasmid excision had occurred, resulting in reversion to either the WT or DsrtA genotype. Colonies with the DsrtA genotype were screened by PCR using primers flanking the deletion region (Table 2).…”

“…An in-frame deletion of the SrtA-encoding gene, srtA, in L. acidophilus NCFM (LBA1244) or L. gasseri ATCC 33323 (LGAS_0825) was constructed using a uppcounterselective gene replacement system (Goh et al, 2009;Selle et al, 2014). Artemis software (Rutherford et al, 2000) was used to visualize the specific chromosomal region associated with each of the srtA genes, and primers (Table 2) were designed to amplify homologous regions of approximately 600 bp upstream and downstream of the deletion target.…”

“…Sortase deletion mutants of L. acidophilus NCFM and L. gasseri ATCC 33323 were generated using upp-based counterselection gene replacement systems (Goh et al, 2009;Selle et al, 2014). The DsrtA mutant of each strain was confirmed by PCR and DNA sequencing using primers flanking the srtA deletion target ( Table 2).…”