Genomic Characterization of Non-Mucus-Adherent Derivatives of Lactobacillus rhamnosus GG Reveals Genes Affecting Pilus Biogenesis

Abstract

c Lactobacillus rhamnosus GG is one of the best-characterized lactic acid bacteria and can be considered a probiotic paradigm. Comparative and functional genome analysis showed that L. rhamnosus GG harbors a genomic island including the spaCBAsrtC1 gene cluster, encoding the cell surface-decorating host-interacting pili. Here, induced mutagenesis was used to study pilus biogenesis in L. rhamnosus GG. A combination of two powerful approaches, mutation selection and next-generation sequencing, was applied to L. rhamnosus GG for the selection of pilus-deficient mutants from an enriched population. The isolated mutants were first screened by immuno-dot blot analysis using antiserum against pilin proteins. Relevant mutants were selected, and the lack of pili was confirmed by immunoelectron microscopy. The pilosotype of 10 mutant strains was further characterized by analyzing pilin expression using Western blot, dot blot, and immunofluorescence methods. A mucus binding assay showed that the mutants did not adhere to porcine intestinal mucus. Comparative genome sequence analysis using the Illumina MiSeq platform allowed us to determine the nature of the mutations in the obtained pilus-deficient derivatives. Three major classes of mutants with unique genotypes were observed: class I, with mutations in the srtC1 gene; class II, with a deletion containing the spaCBA-srtC1 gene cluster; and class III, with mutations in the spaA gene. Only a limited number of collateral mutations were observed, and one of the pilus-deficient derivatives with a deficient srtC1 gene contained 24 other mutations. This strain, PB12, can be considered a candidate for human trials addressing the impact of the absence of pili. Lactobacillus rhamnosus GG is one of the best-studied lactic acid bacteria and is marketed worldwide in probiotic products (1, 2). Initially, it was isolated from the intestine of a healthy female subject (3), and ever since, it has been used in various large and well-controlled probiotic trials that showed positive effects, i.e., symptom alleviation or inhibition of disease onset, on respiratory tract infections (4), atopic disease in children (5, 6), and also various types of diarrhea (7).The genome of L. rhamnosus GG has recently been sequenced and was found to contain a genomic island harboring a functional gene cluster coding for proteinaceous pili that are decorated by the mucus binding SpaC protein (8). The pili, located at the cell surface, were further studied and found to consist of polymers containing the major subunit SpaA and the minor pilins SpaB and SpaC (9). The major pilin SpaA forms the pilus shaft, while the minor pilin SpaB decorates the shaft and is supposedly also located at the base of the pilus structure, where it is involved in arresting pilus biosynthesis and the docking of the pilus into the peptidoglycan. The latter reaction involves the activity of the housekeeping sortase SrtA (10), and we have recently provided evidence for the involvement of the pilus-specific srtC1 gene product in the specifi...

Polymorphisms, Chromosomal Rearrangements, and Mutator Phenotype Development during Experimental Evolution of Lactobacillus rhamnosus GG

“…Mucus-binding assays of the three L. rhamnosus isolates (N1, N3, and N8), the pilus-negative strain PB12 (16), and the reference strain GG were carried out, as described previously (16). The relative binding capacities of N1, N3, N8, and PB12 were calculated compared to the capacity of the reference strain GG.…”

“…Excluding the acquisition of foreign mobile DNA, such as plasmids, conjugative transposons, or phages, bacterial genomes would mostly evolve through the loss, decay, or duplication of genes that result from either small mutational events, i.e., single nucleotide polymorphisms (SNPs) and insertions/deletions (indels), or larger genomic rearrangement events that are typically mediated by insertion sequence (IS) elements. In addition to causing large chromosomal deletions or inversions (15,16), IS elements can also have some more localized impacts on coding capacities and gene expression by inactivating or activating gene(s) (17). Thus, we identified that the activation of the pilus gene cluster spaCBA-srtC1 was caused by the insertion of an iso-IS30 element that created an active and constitutive promoter upstream of the spaC gene, from which we determined the transcriptional start site experimentally (18).…”

“…A total of 69 IS-like elements were annotated throughout the L. rhamnosus GG chromosome (20). It is noteworthy that the genomic island GGISL2 containing the pilus gene cluster spaCBA-srtC1 and numerous phosphotransferase systems (PTSs) is highly enriched in IS elements (17 IS elements within an ϳ125-kb region), explaining why this region has high variability in L. rhamnosus isolates as well as in derivatives of L. rhamnosus GG (16,21,22).…”

“…All of these adaptations result in non-genetically modified organism (GMO) derivatives (based on European Union regulations) that could be instrumental for cause-effect studies as these strains can be consumed without specific regulations. Such a non-GMO strategy has also been carried out for L. rhamnosus GG, where a set of mutations were identified that affected the production of the mucus-binding pili (16). In the present study, we focus on L. rhamnosus GG that was grown for 1,000 generations under four conditions, including the presence of various stressors, to provide insights into the genomic dynamics and plasticity and the impact of IS elements.…”

Lactobacillus strains belonging to Casei group display various adherence to enterocytes and mucus

“…They also have the ability to bind to the mucus, through the presence of the subunit SpaC mucus binding domain (Kankainen et al, 2009;Reunanen et al, 2012). Another factor differentiating ability of individual strains to adhere to the intestinal epithelium are variations in the sequences encoding the fi mbrial protein structure (Toh et al, 2013) and its expression Rasinkangas et al, 2014). The spaCBA complete coding sequence is present in L. paracasei DS6 and L. casei DS8 isolates but the protein is not expressed (Markowicz et al, 2014).…”