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 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.

FIG. 1.

Construction of the pTRK935 counterselectable integration vector. ori, origin of replication of pWV01; erm, gene encoding Em resistance; lacZ′, lacZ-alpha gene from pUC19; MCS, MCS from pUC19. Only unique restriction sites are shown.

FIG. 2.

Markerless gene replacement strategy with upp as a counterselectable marker for in-frame deletion of 1,356 bp in the slpX gene. Single-crossover recombination of pTRK956 occurred in the chromosomal slpX region homologous to either fragment A or B (only recombination via fragment A is shown). Removal of Em selection for the integrated pTRK956 plasmid facilitates a second recombination and plasmid excision, generating recombinants with either a wild-type allele or a ΔslpX allele. The resulting recombinants can be selected on SDM containing 100 μg/ml of 5-FU. DNA fragments A (dotted box) and B (shaded box) represent the cloned copy of a ΔslpX allele, where fragment A consists of the 5′ region of slpX (105 bp) and the upstream flanking region and fragment B consists of the 3′ region of slpX (36 bp) and the downstream flanking region.

FIG. 3.

PCR screening for the ΔslpX genotype in random colonies recovered after 5-FU selection. (A) Gene organization of the region surrounding the slpX locus. Arrows indicate the locations of primers slpX-up and slpX-dw used for PCR analysis. The shaded region in the slpX gene represents the deletion target. Hairpin structures indicate putative rho-independent terminators. The chromosomal map is not drawn to scale. (B) Colony PCR analysis of 18 selected 5-FU^r recombinants. The expected amplicon sizes generated from wild-type and ΔslpX genotypes are approximately 3.14 kb and 1.78 kb, respectively. Lane M, DNA size marker; lanes 1 to 18, 5-FU^r isolates; lane WT, parent strain NCFM (control).

FIG. 4.

SDS-PAGE of purified S-layer proteins from L. acidophilus wild-type strain NCFM (lane WT), the NCFM slpA integrant NCK1377 (lane 1377), and the slpX deletion mutant NCK1962 (lane 1962) used for MS analysis. The S-layer profile of each strain is summarized at the bottom based on results of the MS analysis of the purified S-layer proteins. +, present; −, absent. Molecular masses of standard proteins (lane M) are indicated on the left.

FIG. 5.

Growth of L. acidophilus NCK1962 and NCK1909 in MRS medium at 37°C under ambient atmospheric conditions. Growth was assessed by determining the optical density and cell counts (CFU/ml).

FIG. 6.

Survival of NCK1962 (ΔslpX mutant) and NCK1909 (reference strain) after stress challenge assays. (A) Survival of early-log-phase cultures of NCK1909 and NCK1962 after exposure to MRS medium supplemented with 0.3% porcine bile or 2.5% oxgall bile for 2 h at 37°C under ambient atmospheric conditions. (B) Early-log-phase cultures of NCK1909 and NCK1962 were inoculated (1% inoculum) into MRS broth supplemented with SDS or Triton X-100 surfactant, and cell density was measured after 24 h of incubation at 37°C under ambient atmospheric conditions. The data are the means ± standard errors of the means for three independent replicates.