https://orcid.org/0000-0002-3214-6130
Figures & data
Figure 1. Increasing concentrations of ethanolamine (EA) do not confer a growth advantage to L. brevis.
(a) Growth curves of L. brevis ATCC 14869 in 1:9 diluted MRS with 150 nM adenosylcobalamin and various concentrations of EA. OD600 was measured spectrophotometrically. (b) Bacterial growth was also measured through quantification of CFU/mL after 24 hours. Error bars represent mean ± standard deviation (SD) for 4 biological replicates (one-way ANOVA complemented with Tukey’s multiple-comparison test). ****, P < .0001; ns = not significant.
Figure 2. Ethanolamine (EA) clearance during growth.
All bacteria were grown in 1:9 diluted MRS with or without 10 mM EA and 150 nM adenosylcobalamin (AdoCbl). OD600 was monitored spectrophotometrically and remaining EA in the culture supernatant was quantified via HPLC analysis. (a–b) L. brevis ATCC 14869. (c–d) E. coli CFT073. (e–f) E. coli CFT073 ∆eutR. (g–h) Salmonella enterica ssp. enterica serovar Typhimurium LT2. (i–j) L. plantarum ATCC 14917. (k–l) E. faecalis ATCC 33186. Error bars represent mean ± standard deviation (SD) for 4 biological replicates (unpaired, two-tailed t test). **, P < .01; ***, P < .001; ****, P < .0001; ns = not significant.
Figure 3. Utilization of ethanolamine as a nitrogen source reduces the growth of L. brevis ATCC 14869.
L. brevis ATCC 14869 was grown in (a–b) cfMRS, (c–d) rnMRS and (e–f) nfMRS with or without 10 mM EA and 150 nM AdoCbl. OD600 was monitored spectrophotometrically and remaining EA in the culture supernatant was quantified via HPLC analysis. Error bars represent mean ± standard deviation (SD) for 4 biological replicates (unpaired, two-tailed t test). *, P < .05; ns = not significant.
Figure 4. Transmission electron microscopy (TEM) of L. brevis ATCC 14869 reveals bacterial microcompartment formation.
For TEM, cultures of L. brevis were grown in 1:9 diluted MRS (a) with 10 mM ethanolamine (EA; b), 150 nM adenosylcobalamin (AdoCbl; c) or both metabolites (d–e). After 24 hours of growth, bacterial cells were fixed and embedded prior to sectioning and contrast staining to qualitatively observe the presence of microcompartment structures within individual cells.
Figure 5. RNA sequencing analysis of L. brevis ATCC 14869 grown with ethanolamine (EA) and adenosylcobalamin (AdoCbl).
Cultures of L. brevis ATCC 14869 were grown in 1:9 diluted MRS with or without 10 mM EA and 150 nM AdoCbl prior to RNA extraction and sequencing. (a) Venn diagram of differentially expressed genes. The number within each circle represents the number of differentially expressed genes between the different conditions relative to the control. (b–d) Volcano plots of RNA sequencing analysis comparing cultures grown in 1:9 diluted MRS with those grown with EA, AdoCbl, or both metabolites. Data points in green represent significantly upregulated genes, whereas those in red and black signify those significantly downregulated or unchanged, respectively. (e–f) Schematic representation of the eut and pdu operons found in L. brevis. Genes shown in green were found to be significantly upregulated via RNA sequencing analysis in cultures grown with EA and AdoCbl compared to the control, whereas those indicated in black were unchanged.
Figure 6. qPCR analysis of L. brevis ATCC 14869 eut, pdu, and apf genes.
qPCR was conducted during the stationary growth phase to assess the impact of ethanolamine (EA) on select L. brevis ATCC 14869 eut (a–b), apf (c) and pdu (d–e) gene expression. Cultures of L. brevis ATCC 14869 were grown in 1:9 diluted MRS with or without 10 mM EA and 150 nM adenosylcobalamin (AdoCbl). Gene expression data points are displayed as mean fold change (relative to gyrA and rpoD). Error bars represent mean ± standard deviation (SD) of 4 biological replicates (unpaired, two-tailed t test). *, P < .05; **, P < .01; ns = not significant.
Table 1. Differentially expressed transcripts by L. brevis in the presence of ethanolamine and adenosylcobalamin.
Figure 7. The influence of ethanolamine (EA) on L. brevis ATCC 14869 adhesion to abiotic factors.
(a) A MATH assay was performed using L. brevis ATCC 14869 grown in 1:9 diluted MRS with or without 10 mM EA and 150 nM adenosylcobalamin (AboCbl). Adhesion of bacteria to hydrocarbons was evaluated as the fraction partitioned to the hydrocarbon phase (FPC), calculated relative to optical density measured at 600 nm. FPC was estimated as final absorbance over initial absorbance (Af /Ao) of the bacterial suspension. Error bars represent the mean ± standard deviation (SD) of 6 biological replicates (one-way ANOVA). (b) In vitro adhesion to mucin was assessed using a plate-based assay. Percent adherence was calculated relative to the initial CFU/mL quantified prior to adhesion from overnight cultures grown in each respective condition. Quantity of recovered bacteria was calculated through quantification of CFU/mL after a 1-hour incubation followed by washing off non-adherent bacteria. Error bars represent mean ± standard deviation (SD) for 4 biological replicates (one-way ANOVA complemented with Tukey’s multiple-comparison test). *, P < .05; ns = not significant.
Figure 8. Ethanolamine (EA) influences L. brevis ATCC 14869 adhesion to human intestinal epithelial cells.
Caco-2 cells were incubated with bacteria for 3 hours. Relative adherence was calculated in relation to bacteria recovered from cultures that were grown in 1:9 diluted MRS. L. brevis was grown with or without 10 mM EA and 150 nM adenosylcobalamin (AdoCbl) and allowed to adhere Caco-2 cells. White arrows indicate adherent bacteria. Scale bars represent 24 µm. Error bars represent mean ± standard deviation (SD) of 6 biological replicates (unpaired, two-tailed t test). **, P < .01.
Figure 9. Ethanolamine (EA) enhances the ability of L. brevis ATCC 14869 to competitively exclude Salmonella Typhimurium LT2 from binding human intestinal epithelial cells.
Caco-2 cells were first incubated with L. brevis ATCC 14869 grown overnight with or without 10 mM EA and 150 nM adenosylcobalamin (AdoCbl), washed to remove non-adherent bacteria, and subsequently infected with S. Typhimurium LT2 for 30 minutes. Remaining adherent bacteria were recovered and quantified via analysis of CFU/mL. (a) L. brevis relative adherence. (b) S. Typhimurium LT2 relative adherence. Error bars represent the mean ± standard deviation (SD) of 6 biological replicates (one-way ANOVA complemented with Tukey’s multiple-comparison test). **, P < .01; ***, P < .001; ****, P < .0001; ns = not significant.
Table A1. Levilactobacillus brevis ATCC 14869 qPCR primer sequences.
Figure A1. Ethanolamine promotes in vitro aggregation of Levilactobacillus brevis ATCC 14869.
Cultures of L. brevis were grown in 1:9 diluted MRS with 150 nM adenosylcobalamin (left) or both 150 nM adenosylcobalamin and 10 mM ethanolamine (right), as previously described. After 24 hours of growth, bacterial culture tubes were imaged to qualitatively visualize cellular aggregation.
Data availability statement
Illumina RNA sequencing reads can be found in NCBI under BioProject accession number PRJNA1001957.