Functional Analysis ofluxSin the Probiotic StrainLactobacillus rhamnosusGG Reveals a Central Metabolic Role Important for Growth and Biofilm Formation

Abstract

Quorum sensing is involved in the regulation of multicellular behavior through communication via small molecules. Given the high number and diversity of the gastrointestinal microbiota, it is postulated that members of this community communicate to coordinate a variety of adaptive processes. AI-2 is suggested to be a universal bacterial signaling molecule synthesized by the LuxS enzyme, which forms an integral part of the activated methyl cycle. We have previously reported that the well-documented probiotic strain Lactobacillus rhamnosus GG, a human isolate, produces AI-2-like molecules. In this study, we identified the luxS homologue of L. rhamnosus GG. luxS seems to be located in an operon with a yxjH gene encoding a putative cobalaminindependent methionine synthase. In silico analysis revealed a methionine-specific T box in the leader sequence of the putative yxjH-luxS operon. However, transcriptional analysis showed that luxS is expressed mainly as a monocistronic transcript. Construction of a luxS knockout mutant confirmed that the luxS gene is responsible for AI-2 production in L. rhamnosus GG. However, this mutation also resulted in pleiotropic effects on the growth of this fastidious strain. Cysteine, pantothenate, folic acid, and biotin could partially complement growth, suggesting a central metabolic role for luxS in L. rhamnosus GG. Interestingly, the luxS mutant also showed a defect in monospecies biofilm formation. Experiments with chemically synthesized (S)-4,5-dihydroxy-2,3-pentanedione, coculture with the wild type, and nutritional complementation suggested that the main cause of this defect has a metabolic nature. Moreover, our data indicate that suppressor mutations are likely to occur in luxS mutants of L. rhamnosus GG. Therefore, results of luxS-related studies should be carefully interpreted.

Functional Analysis of Lactobacillus rhamnosus GG Pili in Relation to Adhesion and Immunomodulatory Interactions with Intestinal Epithelial Cells

“…Positive knockout mutants, wherein successful double homologous recombination and allelic replacement have taken place, were selected according to tetracycline resistance and erythromycin sensitivity. Complete allelic replacement was confirmed by PCR using the Pro-249, Pro-250, and HR 5=-end and 3=-end primers (Table 2) as described before (22). The ⌬welE ⍀spaC double mutant CMPG5365 is derived from the welE mutant CMPG5351 by integration of plasmid pCMPG10102, which insertionally inactivates the spaC gene (19).…”

“…PCR primers were designed to amplify two ca.-1,000-bp-long homologous regions (HRs) that flank the 5= and 3= ends of the target gene(s) ( Table 2). Each of the HR-containing PCR fragments were subsequently ligated into the respective multiple cloning sites (MCSs) upstream and downstream of the rhamnosus GG by using a method described previously (22). Positive knockout mutants, wherein successful double homologous recombination and allelic replacement have taken place, were selected according to tetracycline resistance and erythromycin sensitivity.…”

“…Knockout mutants of L. rhamnosus GG were constructed according to strategies described previously (22). Briefly, open reading frames (ORFs) encoding putative adhesins targeted for inactivation were identified in the L. rhamnosus GG genome sequence (19).…”

The Use of Starter Cultures in Traditional Meat Products

“…Some reports have also described the genes responsible for quorum sensing, adhesion, and biofilm formation [147][148][149][150].…”

Computational Analysis of Cysteine and Methionine Metabolism and Its Regulation in Dairy Starter and Related Bacteria

“…Enzyme group 1 consisted of homoserine dehydrogenase (hom; EC 1.1.1.3; COG0460E, K00003, PF03447, and PF00742 [7,44,54] [38,58).…”