Additional characterization is needed to identify which PTS transporters are involved in the utilization of β-glucosides. Conclusions PTS transporters were confirmed to be largely important in the carbohydrate utilization potential of L. gasseri ATCC 33323. The PTS transporters were identified in various

lactobacilli species using bioinformatic analysis. Comparative carbohydrate utilization assays were used to analyze the PTS content with carbohydrate utilization capability of three L. gasseri strains. The PTS carbohydrate specificity of transporters in L. gasseri ATCC 33323 was characterized by studying the transcript expression profiles in response to different carbohydrates. Lastly, the growth activity of selected PTS knockouts confirmed PTS transporter specificity predictions based on bioinformatics and transcript Alectinib nmr expression profiles. Our results confirm the importance of combining bioinformatics, transcript expression profiles and gene inactivation in identifying carbohydrate specificity of PTS transporters. Methods Bioinformatic Analysis The genomes of Lactobacillus acidophilus NCFM, L. brevis ATCC 367, L. casei ATCC 334, L. delbrueckii ssp. bulgaricus ATCC 11842, L. delbrueckii ssp. bulgaricus ATCC BAA-365, L. gasseri ATCC 33323, L. johnsonii NCC 533, Birinapant order L. plantarum WCFS1, L. reuteri F275,

L. sakei ssp. sakei 23 K and L. salivarius ssp. salivarius UCC118 were analyzed using Concise Protein BLAST [40]. The PTS transporters of these strains were compared based on sequence similarity and function. PTS transporters were placed in the same cluster based on reciprocal Bay 11-7085 best-hit blastP scores. Homologs were defined as PTS transporters that were in the same cluster. The number

of complete and incomplete PTS transporters present was determined for each species through bioinformatic analysis of the genomes. A complete PTS transporter was defined as having complete EIIA, EIIB and EIIC domains, which are required for PTS functionality [25]. An incomplete PTS transporter (also known as an orphan PTS) was defined as lacking in at least one of these domains. The sequential numbering of PTS transporters was based on their location in each respective genome. In order to identify non-PTS transporters with a PTS IIA domain, the conserved domain database was searched for PTS IIA domains [21, 41]. Bacterial Strains, Plasmids and Growth Conditions The bacterial strains and plasmids used in this study are listed in Table 5. L. gasseri strains were grown at 37°C, in deMan, Rogosa, Sharpe (MRS) broth (Difco, Sparks, MD) or on MRS supplemented with 1.5% agar (Fisher, Fair Lawn, NJ). Agar plates were incubated anaerobically in a Coy anaerobic chamber (Grass Lake, MI) with a gas composition of 90% nitrogen, 5% hydrogen and 5% carbon dioxide. When necessary, erythromycin (Fisher) was added at a concentration of 2.5 μg/mL, and chloramphenicol (Fisher) was added at a concentration of 5 μg/mL. For the real-time PCR studies, L.