Figure 1.
Comparison of the distribution of GHs and CBM between R. gnavus E1 and ATCC 29149.
GHs and CBMs are represented by red boxes for R. gnavus E1 and by blue boxes for R. gnavus ATCC 29149. CBMs associated with GH are represented by plain boxes, with the GH family indicated inside the box. CBMs not associated with GH are represented by striped boxes.
Figure 2.
Growth curves of R. gnavus E1 and ATCC29149 with different carbohydrates as sole carbon source.
For each sugar and each strain, the growth curve represent the average growth, measured at OD600(Black, Glc; Red, GlcNAc; Green, Gal; Orange, Fuc; Blue, 2′FL and Pink, 3FL).
Table 1.
Growth rate and density of R. gnavus E1 and ATCC 29149 growth supplemented with different carbohydrates.
Figure 3.
H1 NMR analysis of R. gnavus ATCC 29149 culture supernatant.
YCFA medium supplemented with2′FL (A), 3FL (B), 3′SL (C) or pPGM (D) were analysed by H1 NMR before (control) or after 8 h or 23 h of growth of R. gnavus ATCC 29149 to assess substrate utilization. Peaks were assigned by using the appropriate sugar standards and based on literature.
Figure 4.
Quantification of propanol and propionate produced by R. gnavus ATCC 29149.
The amount of propanol (A) and propionate (B) in the YCFA medium supplemented with different sugars has been quantified by 1H NMR before (control, white box) and after (grey box) growth of R. gnavus ATCC 29149. At least 3 replicates have been performed in each condition (except YCFA+2′FL control). For each sugar (except for 2′FL where there were insufficient number of replicates), a Mann-Whitney test was performed to compare the concentration of propanol or propionate in the medium before and after R. gnavus ATCC 29149 growth. Only the production of propanol by R. gnavus ATCC 29149 grown on pPGM was significant (*, p<0.05) but R. gnavus ATCC 29149 also seemed to produce both propanol and propionate when grown with Fuc as sole carbon source, and propanol when grown with 3FL as sole carbon source (#, p = 0.06). n/a: Not applicable.
Table 2.
Enzymatic activity of R. gnavus ATCC 29149 supernatant grown on mucin and 3′SL on substrate 4-MU-Neu5Ac.
Figure 5.
Relative level of transcription of R. gnavus ATCC 29149 nan genes.
Fold change in gene transcription has been determined by qRT-PCR for the nan genes when R. gnavus ATCC 29149 was grown in presence of pPGM (white box) or 3′SL (grey box) compared to Glc as sole carbon source. The results showed averages of two biological replicates, each performed in 3 technical replicates. Data were analysed using 1-way ANOVA. For each gene, a post-hoc test (Dunnett's) was used to examine if there were any significant differences in each condition (versus Glc). The transcription of nanH was significantly increased when R. gnavus ATCC 29149 was grown with either pPGM or 3′SL compared to Glc. The transcription of both nanK and nanA was also significantly increased when ATCC 29149 was grown with 3′SL compared to Glc. *: p<0.05; **, p<0.01.
Figure 6.
The nan locus in R. gnavus ATCC 29149.
(A) Schematic representation of the nan genetic organization. Each block arrow indicates an ORF; the length of the arrow is proportional to the length of the predicted ORF. RUMGNA_02702, 02701, 02700, 02699, 02698, 02697, 02695 and 02690 are shown in block arrow to
, respectively. Circles above thick vertical lines indicate potential stem-loop structures that might act as Rho-independent transcriptional terminators. The free energy of the thermodynamic ensemble is given on top, expressed as kcal.mol−1. The inset shows the DNA sequence of the promoter located upstream of the putative RUMGNA_02701 gene (
). The putative −35 and −10 regions and ribosome-binding site (RBS) are underlined. (B) Confirmation of the nan operonic structure. The PCR products obtained following RT-PCR of RNA extracted from R. gnavus ATCC 29149 grown on pPGM were obtained using primers set spanning the SAT2 to NanK ORFs and analysed by electrophoresis on agarose gel. PCR from RT negative control (RT−) was performed to confirm the absence of genomic DNA contamination of the RNA sample prior to RT. PCR negative (−) and positive (+) controls were carried out with water or ATCC 29149 genomic DNA as template, respectively. The positions of the primers are shown in panel A and their sequences are provided in Table S1. M, DNA ladder size marker (with increments indicated in base pairs).
Figure 7.
1H NMR analysis of sialylated substrates incubated with spent media of R. gnavus ATCC 29149.
R. gnavus ATCC 29149 was grown in YCFA supplemented with pPGM or 3′SL for 9 h and the cells removed by centrifugation. 3′SL was incubated with spent media supplemented with pPGM (A) and 3′SL (B). 4-MU-Neu5Ac was incubated with spent media supplemented with pPGM (C) and 3′SL (D). The control media without inoculation with R. gnavus are shown in the lower trace of each panel. Abbreviations; 3′SL-3′-sialyllactose, Lac-lactose, A- 2,7-anhydro-Neu5Ac, pPGM-purified porcine gastric mucin, med-unidentified media component.