The PTS transporters of Lactobacillus gasseri ATCC 33323

Abstract

BackgroundLactobacilli can utilize a variety of carbohydrates which reflects the nutrient availability in their respective environments. A common lactobacilli in the human gastrointestinal tract, Lactobacillus gasseri, was selected for further study. The currently available annotation of the L. gasseri ATCC 33323 genome describes numerous putative genes involved in carbohydrate utilization, yet the specific functions of many of these genes remain unknown.ResultsAn enzyme I (EI) knockout strain revealed that a functional phosphotransferase transporter system (PTS) is required to ferment at least 15 carbohydrates. Analysis of the L. gasseri ATCC 33323 genome identified fifteen complete (containing all of the necessary subunits) PTS transporters. Transcript expression profiles in response to various carbohydrates (glucose, mannose, fructose, sucrose and cellobiose) were analyzed for the fifteen complete PTS transporters in L. gasseri. PTS 20 was induced 27 fold in the presence of sucrose and PTS 15 was induced 139 fold in the presence of cellobiose. No PTS transporter was induced by glucose, fructose or mannose. Insertional inactivation of PTS 15 and PTS 20 significantly impaired growth on cellobiose and sucrose, respectively. As predicted by bioinformatics, insertional inactivation of PTS 21 confirmed its role in mannose utilization.ConclusionsThe experiments revealed the extensive contribution of PTS transporters to carbohydrate utilization by L. gasseri ATCC 33323 and the general inadequacy of the annotated sugar specificity of lactobacilli PTS transporters.

Identification of lactose phosphotransferase systems in Lactobacillus gasseri ATCC 33323 required for lactose utilization

“…First, an API 50 CH assay revealed that NCK334 could utilize lactose, while the EI knockout strain could not. Second, growth curves using sMRS supplemented with 1 % lactose confirmed the API 50 CH results (Francl et al, 2010). From these results, we concluded that PTS(s) are required for the utilization of lactose by NCK334.…”

“…On: Wed, 27 Mar 2019 22:11:11 of the PTS and potential b-galactosidase genes, as previously described (Francl et al, 2010). Relative fold change values were compared with either glucose or fructose.…”

“…Two-step RT-PCR was performed to carry out the relative quantification as described previously (Francl et al, 2010). Primers were designed for the IIC subunit of the 15 complete PTSs which were reported previously (Francl et al, 2010). The primers for the seven potential b-galactosidase genes in L. gasseri were determined using Clone Manager 9 and are shown in Table 2.…”

“…Typically, 100 ng RNA ml 21 was treated with TURBO DNA-free according to the supplier's instructions (Ambion) in a 50 ml reaction volume. Two-step RT-PCR was performed to carry out the relative quantification as described previously (Francl et al, 2010). Primers were designed for the IIC subunit of the 15 complete PTSs which were reported previously (Francl et al, 2010).…”

“…Briefly, an EI knockout strain (MJM75) was evaluated for its ability to grow on lactose using two different growth assays (Francl et al, 2010). PTSs require a functional EI to import carbohydrates (Cvitkovitch et al, 1995).…”