Directed chromosomal integration and expression of the reporter gene gusA3 in Lactobacillus acidophilus NCFM

Abstract

Lactobacillus acidophilus NCFM is a probiotic microbe that survives passage through the human gastrointestinal tract and interacts with the host epithelium and mucosal immune cells. The potential for L. acidophilus to express antigens at mucosal surfaces has been investigated with various antigens and plasmid expression vectors. Plasmid instability and antibiotic selection complicate the possibility of testing these constructs in human clinical trials. Integrating antigen encoding genes into the chromosome for expression is expected to eliminate selection requirements and provide genetic stability. In this work, a reporter gene encoding a β-glucuronidase (GusA3) was integrated into four intergenic chromosomal locations. The integrants were tested for genetic stability and GusA3 activity. Two locations were selected for insertion downstream of constitutively highly expressed genes, one downstream of slpA (LBA0169), encoding a highly expressed surface-layer protein, and one downstream of phosphopyruvate hydratase (LBA0889), a highly expressed gene with homologs in other lactic acid bacteria. An inducible location was selected downstream of lacZ (LBA1462), encoding a β-galactosidase. A fourth location was selected in a low-expression region. The expression of gusA3 was evaluated from each location by measuring GusA3 activity on 4-methyl-umbelliferyl-β-d-glucuronide (MUG). GusA3 activity from both highly expressed loci was more than three logs higher than the gusA3-negative parent, L. acidophilus NCK1909. GusA3 activity from the lacZ locus was one log higher in cells grown in lactose than in glucose. The differences in expression levels between integration locations highlights the importance of rational targeting with gene cassettes intended for chromosomal expression.

Fig. 1.

Intergenic locations selected for gusA3 integration into the L. acidophilus NCFM chromosome. The symbols downstream of gusA3 in NCK2136, NCK2139, and NCK2171 indicate a terminator. The figure is drawn approximately to scale.

Fig. 2.

Similarity of genomic arrangement surrounding phosphopyruvate hydratase between L. acidophilus NCFM and several closely related lactic acid bacteria. Genes shown in the same color share percent protein identity, indicated in parentheses, with the gene of the same color in L. acidophilus. Genes in blue do not show percent protein identity to the NCFM genes displayed here. The figure is drawn approximately to scale.

Fig. 3.

Activity of GusA3 from chromosomal locations compared to plasmid expression in cells grown in glucose (white bars) or lactose (gray bars). The results shown here are the averages of three independent assays ± one standard deviation.

Fig. 4.

Stability of gusA3 in each chromosomal location after 35 generations. (A) PCR analysis confirming each integration was stable after 35 generations. L, ladder; BP, base pairs; WT, wild type (NCK1909); I, integrant. Lane 1, NCK2136; 169 primers; WT amplicons, 1,566 bp; I amplicons, 3,430 bp. Lane 2, NCK2139; 1,462 primers; WT amplicons, 1,583 bp; I amplicons, 3,463 bp. Lane 3, NCK2171; 889 primers; WT amplicons,1,531 bp; I amplicons, 3,403 bp. Lane 4, NCK2190; 645 primers; WT amplicons, 1,574 bp; I amplicons, 3,395 bp. (B) GusA3 activity in glucose after seven generations (white bars) and 35 generations (gray bars). The results shown for seven generations are the averages of three independent assays ± one standard deviation. One GusA3 assay was completed after 35 generations for each integrant construct, and PCR assays were conducted to confirm the absence of wild-type amplicons. For the plasmid construct NCK1829(pTRK892), genetic stability was evaluated by plate count comparisons of Em^r versus Em^s colonies.