Functional and comparative genomic analyses of an operon involved in fructooligosaccharide utilization by Lactobacillus acidophilus

Abstract

Lactobacillus acidophilus is a probiotic organism that displays the ability to use prebiotic compounds such as fructooligosaccharides (FOS), which stimulate the growth of beneficial commensals in the gastrointestinal tract. However, little is known about the mechanisms and genes involved in FOS utilization by Lactobacillus species. Analysis of the L. acidophilus NCFM genome revealed an msm locus composed of a transcriptional regulator of the LacI family, a four-component ATP-binding cassette (ABC) transport system, a fructosidase, and a sucrose phosphorylase. Transcriptional analysis of this operon demonstrated that gene expression was induced by sucrose and FOS but not by glucose or fructose, suggesting some specificity for nonreadily fermentable sugars. Additionally, expression was repressed by glucose but not by fructose, suggesting catabolite repression via two cre-like sequences identified in the promoter-operator region. Insertional inactivation of the genes encoding the ABC transporter substrate-binding protein and the fructosidase reduced the ability of the mutants to grow on FOS. Comparative analysis of gene architecture within this cluster revealed a high degree of synteny with operons in Streptococcus mutans and Streptococcus pneumoniae. However, the association between a fructosidase and an ABC transporter is unusual and may be specific to L. acidophilus. This is a description of a previously undescribed gene locus involved in transport and catabolism of FOS compounds, which can promote competition of beneficial microorganisms in the human gastrointestinal tract.

Fig. 1.

Operon layout. The start and stop codons are shaded, the putative ribosome binding site is boxed, and the cre-like elements are underlined. Terminators are indicated by hairpin structures.

Fig. 2.

Sugar induction and repression. (A) Transcriptional induction of the msmE and bfrA genes, monitored by RT-PCR (Upper) and RNA slot blots (Lower). Cells were grown on glucose (Glc), fructose (Fru), sucrose (Suc), FOS GF_n, and FOS F_n. Chromosomal DNA was used as a positive control for the probe. (B) Transcriptional repression analysis of msmE and bfrA by variable levels of Glc and Fru: 0.1% (5.5 mM), 0.5% (28 mM), and 1.0% (55 mM), in the presence of 1% F_n. Cells were grown in the presence of F_n until OD₆₀₀ approximated 0.5–0.6, glucose was added, and cells were propagated for an additional 30 min.

Fig. 3.

Growth curves. The two mutants, bfrA (Upper) and msmE (Lower), were grown on semisynthetic medium supplemented with 0.5% wt/vol carbohydrate: fructose (•), GF_n (○), F_n (▾), after one passage on F_n (▪). The lacZ mutant grown on F_n was used as control (▿).

Fig. 4.

Operon architecture analysis. (A) Alignment of the msm locus from selected bacteria. Regulators, white; α-galactosidases, blue; ABC transporters, gray; fructosidases, yellow; sucrose phosphorylases, red. (B) Alignment of the sucrose locus from selected microbes. Regulators, white; fructosidases, yellow; PTS transporters, green; fructokinases, purple; putative proteins, black.

Fig. 5.

Neighbor-joining phylogenetic trees. Lactobacillales, black; bacillales, green; clostridia, blue; thermotogae, yellow; proteobacteria, red. (A) 16S; (B) fructosidase; (C) ABC; (D) PTS; (E) regulators; (F) fructokinase. L. acidophilus proteins are boxed and shaded when encoded by the msm locus. Bars indicate scales for computed pairwise distances.