Transformation of, and heterologous protein expression in, Lactobacillus agilis and Lactobacillus vaginalis isolates from the chicken gastrointestinal tract

Abstract

Lactobacilli are naturally found in the gastrointestinal tract of chickens, and there is interest in utilizing autochthonous strains for the delivery of therapeutic proteins. Previously we identified three chicken-derived Lactobacillus strains, Lactobacillus agilis La3, Lactobacillus vaginalis Lv5, and Lactobacillus crispatus Lc9, which persist in the gastrointestinal tract of chickens fed either a commercial or high-protein diet. In the current study, we investigated the ability to electrotransform these strains, determined plasmid vector stability, and compared reporter gene expression directed by several different promoters. The La3 and Lv5 strains were reproducibly transformed with efficiencies of 10(8) and 10(6) transformants per microgram of plasmid DNA, respectively. The third strain tested, L. crispatus Lc9, was recalcitrant to all transformation protocols examined. The plasmid vectors pTRK563 and pTRKH2 were maintained over 100 generations in La3 and Lv5, respectively. The ability of La3 and Lv5 to express the heterologous reporter gene gfp was analyzed using heterologous and homologous promoters. Transformants of both La3 and Lv5 containing the La3 ldhL promoter were the most fluorescent. To our knowledge, this is the first report of successful transformation and heterologous protein expression in L. agilis and L. vaginalis. The ability of these strains to express heterologous proteins in vitro indicates their potential utility as in vivo delivery vectors for therapeutic peptides to the chicken gastrointestinal tract.

FIG. 1.

Transformation efficiency of L. agilis La3 using various plasmids and the Mason et al. transformation protocol. Plasmid pTRKH2 and derivatives replicate via the theta mechanism, while pTRK563 replicates via the sigma mechanism. The transformation data presented are from at least three replicates of three independent transformants and represent the mean transformation efficiencies, with the standard errors of the means incorporated as error bars.

FIG. 2.

L. agilis La3 and L. vaginalis Lv5 GFP expression. Fluorometry results of GFP expression in L. agilis La3 (A) and L. vaginalis Lv5 (B) using heterologous and homologous promoters, indicated by (het) and (hom), respectively. The data presented are from at least three replicates of three independent transformants and represent the mean numbers of RFU, with the standard errors of the means expressed as error bars. (C) GFP expression in La3 and Lv5 using the pTRKH2:P_ldhL:gfp:T_slpA plasmid, with the wild-type strains shown as negative controls.

FIG. 3.

Clustal W alignment of the homologous and heterologous promoters. The sequences were compared from 50 bp upstream of the −35 hexamer to 13 bp downstream of the −10 hexamer. Putative −35 and −10 hexamers, predicted using the Web-based Prokaryotic Promoter Prediction (PPP) tool, are boxed. The putative mRNA transcriptional start sites are underlined. Consensus nucleotides are in black text, while nonconsensus nucleotides are in gray text. L. agilis La3 P_ldhL contains a second putative promoter region 11 bp upstream of the promoter, which may be the reason the promoter is most efficient in both L. agilis La3 and L. vaginalis Lv5.