Identification of Lactobacillus reuteri genes specifically induced in the mouse gastrointestinal tract

Abstract

Lactobacilli are common inhabitants of the gastrointestinal tracts of mammals and have received considerable attention due to their putative health-promoting properties. Little is known about the traits that enhance the ability of these bacteria to inhabit the gastrointestinal tract. In this paper we describe the development and application of a strategy based on in vivo expression technology (IVET) that enables detection of Lactobacillus reuteri genes specifically induced in the murine gut. A plasmid-based system was constructed containing 'ermGT (which confers lincomycin resistance) as the primary reporter gene for selection of promoters active in the gastrointestinal tract of mice treated with lincomycin. A second reporter gene, 'bglM (beta-glucanase), allowed differentiation between constitutive and in vivo inducible promoters. The system was successfully tested in vitro and in vivo by using a constitutive promoter. Application of the IVET system with chromosomal DNA of L. reuteri 100-23 and reconstituted lactobacillus-free mice revealed three genes induced specifically during colonization. Two of the sequences showed homology to genes encoding xylose isomerase (xylA) and peptide methionine sulfoxide reductase (msrB), which are involved in nutrient acquisition and stress responses, respectively. The third locus showed homology to the gene encoding a protein whose function is not known. Our IVET system has the potential to identify genes of lactobacilli that have not previously been functionally characterized but which may be essential for growth of these bacteria in the gastrointestinal ecosystem.

FIG. 1.

Selection of genes exhibiting elevated expression in the gastrointestinal tract of mice. Genes displaying elevated levels of expression in the intestinal tract were selected on the basis of their ability to drive the expression of a gene that is essential for colonization. Lactobacilli cannot become established in the guts of RLF mice treated with lincomycin administered in the drinking water. Colonization can occur only if an active promoter is inserted upstream of the promoterless ermGT gene. Recovery of strains from the gut after selection results in the detection of promoters that are either constitutive or ivi promoters. To distinguish between these two types of promoters, a promoterless marker gene (′bglM) is transcriptionally fused to ′ermGT, which enables screening for promoter activity in vitro. BglM⁻ strains (no halo after agar plates containing lichenin are flooded with Congo red) or strains with low β-glucanase activity contain fusions to promoters activated in response to the gastrointestinal tract. ′ermGT, erythromycin resistance determinant; ′bglM, β-glucanase gene; cat, chloramphenicol resistance determinant; rrnBT₁T₂, E. coli rho-independent transcription terminators; repA, Lactobacillus replication gene; oriL, Lactobacillus origin of replication; oriE, E. coli origin of replication (pUC29); ORF5, ORF originating from pGT232.

FIG. 2.

Promoter sequences of the ivi genes of L. reuteri 100-23. RBS and putative −10 and −35 sequences are underlined. The start of the genes is indicated. Inverted repeats are indicated by opposing arrows. (A) Promoter of the gene coding for a conserved hypothetical protein (Ivi127, Ivi146, and 148). (B) msrB promoter region. (C) Promoter of the xylA gene. Two putative cis-acting elements, operators 1 and 2, are shown together with the corresponding sequences of L. pentosus.

FIG. 3.

Populations of ivi clones and control strains in the different compartments of the gastrointestinal tract of RLF mice treated with lincomycin. The data are the means and standard errors for three animals. Solid bars, forestomach; open bars, jejunum; grey bars, cecum. The lower detection limit was 10³ CFU/g. Pre1 is a BglM⁻ strain obtained from the initial nonselected pools of transformants. For other strains of L. reuteri see Tables 1 and 2.