Short-Chain Fatty Acids Modulate Metabolic Pathways and Membrane Lipids in Prevotella bryantii B14

Short-chain fatty acids (SCFAs) are bacterial products that are known to be used as energy sources in eukaryotic hosts, whereas their role in the metabolism of intestinal microbes is rarely explored. In the present study, acetic, propionic, butyric, isobutyric, valeric, and isovaleric acid, respectively, were added to a newly defined medium containing Prevotella bryantii B14 cells. After 8 h and 24 h, optical density, pH and SCFA concentrations were measured. Long-chain fatty acid (LCFA) profiles of the bacterial cells were analyzed via gas chromatography-time of flight-mass spectrometry (GC-ToF MS) and proteins were quantified using a mass spectrometry-based, label-free approach. Cultures supplemented with single SCFAs revealed different growth behavior. Structural features of the respective SCFAs were identified in the LCFA profiles, which suggests incorporation into the bacterial membranes. The proteomes of cultures supplemented with acetic and valeric acid differed by an increased abundance of outer membrane proteins. The proteome of the isovaleric acid supplementation showed an increase of proteins in the amino acid metabolism. Our findings indicate a possible interaction between SCFAs, the lipid membrane composition, the abundance of outer membrane proteins, and a modulation of branched chain amino acid biosynthesis by isovaleric acid.


Ingredients
Weight/Volume Final concentration in culture  Table S2. Composition of media for growth of P. bryantii B14.

III
The cultivations are divided into Pre-and Main cultivations and with incubation time of 8 or 24 h. Pretreatments 1 and 2 represent the chronological order, followed by the main treatment. Mean and standard deviation (SD) of optical density were calculated for each condition (n=3). High SD at 24 h for iVal and Prop indicate deviation in inoculation composition or differences in adaptation time. Acidification is described as ΔpH and calculated as the difference between the pH at the start (blank) and the end of the incubation time (sample). _____pH_____ Table S3. Optical density and pH of cultivations for LCFA harvesting.

IV
The cultivation parameters like mean optical density of pre-and main culture (OD; n=3), the OD of the respective main culture and the acidification (n=3) were correlated with the LCFA type in the manner of Pearson to obtain the correlation coefficient (R).

Mean OD
Mean OD vs. LCFA type Table S5. Cultivation parameters and relative amounts of trait specific long chain fatty acids.
V Prevotella was incubated with different SCFA, for incubation times at 8 and 24 h. The incubated SCFAs were iso-valeric acid (iVal),valeric acid (Val), iso-butyric acid (iBut), butyric acid (But), propionic acid (Prop) and acetic acid (Acet). The description of the long-chain fatty acids (LCFA) is described by the possible position of methylation, the number of linear carbon atoms behind the letter C and the corresponding type iso or anteiso in brackets.  Table S6. Percentile amount of long-chain fatty acids found in lipid membrane of P. bryantii B14.

VI
Acetic acid (C2), propionic acid (C3), butyric acid (C4), isobutyric acid (iC4), valeric acid (C5) and isovaleric acid (iC5) are displayed in mM for each approach in triplicate (n=3) with one blank (n=1). An inaccuracy of ±1.5 mM can be expected. The number of blanks in the table is depending if the cultivation for 8 or 24 h was performed in one or two runs.  Table S8. SCFA measurement from supernatant of cultivation for proteomic analysis.

VIII
Duplicates are indicated by (A) or (B), each n=1. The glucose is given in milli molar and was determined enzymatically. Optical density (OD) was measured at 600 nm wavelength. Dry mass was calculated by mass(vessel) -mass(vessel + dry cells).  Proteins which were maximal expressed among all cultures were counted and grouped by their functionality using the cluster of orthologous groups (COGs). Colors indicate the number of proteins with maximal abundance within a COG and cultivation condition. The color code is illustrated at the bottom of the graph. COGs are explained on the right side. Proteins with multiple COG assignations can also be found.

COGs Min count
Post-translational modification, protein turnover, chaperone functions Figure S3. Count of least abundant COGs per culture conditions. Proteins which were least expressed among all cultivation conditions were counted and grouped by their functionality using the cluster of orthologous groups (COGs). Colors indicate the number of proteins with minimal abundance within a COG and SCFA. The color code is illustrated at the bottom of the graph. COGs are explained on the right side. Proteins with multiple COG assignations can also be found.  Figure S4. Porphyrin synthesis pathway in P. bryantii B14 with enzyme abundances. Protein abundances standardized by protein abundance of the Acet culture. Enzyme names are given with the Uniprot ID in brackets and the EC number (in the right side). A side reaction of the pathway is illustrated in the lower part. Chemical compounds are written in italic. Color code is given below the figure.  Figure S6. Similar percentage (SIMPER) analyzed proteins with >1% contribution. The listed proteins contribute with at least 1% to the differential plotting in the PCO plot of Figure 1. Label-free quantification (LFQ) values are related and standardized by the Acet culture and represented the average abundance in form of a heatmap, see color code below. The UniprotKB is followed in brackets after the protein name.  Figure S8. Acet and Val culture specific proteins. Label-free quantification (LFQ) values are related and standardized by the Acet culture and represent the average abundance in form of a heatmap, see color code below. In cases where Acet is zero, proteins from cultivation condition with the lowest abundance were set to one. Proteins written with a red font indicate proteins involved in iron transport. The UniprotKB is given in brackets after the protein name. Proteins are sorted by the major clusters of orthologous groups (COGs). COG functions: membrane and cell wall assembly (M), inorganic transport (P) and unknown function (S).

Acet 8 h Prop 8 h But 8 h iBut 8 h Val 8 h iVal 8 h
Anteiso Iso Linear Figure S9. Percentile distribution of long-chain fatty acids (LCFA) by traits of chain length and branching structure after 8 h incubation. The distribution of the trait with odd or even number of the straight chain length of carbon atoms is shown in bluish colors (upper). The chain length trait divides the fatty acid length in even for Acet, But and iVal and odd for Prop, iBut and Val. The distribution of non-branched (linear), iso-or anteiso methylated long-chain fatty acids are in yellow-greenish colors (lower). SCFAs are grouped by the branching trait in linear for Acet, Prop, But, Val and in iso for iBut and iVal. The two traits are calculated by the sum of the percentile appearance in the LCFAprofile.

Acet 24 h Prop 24 h But 24 h iBut 24 h Val 24 h iVal 24 h
Anteiso Iso Linear Figure S10. Percentile distribution of long-chain fatty acids (LCFA) by traits of chain length and branching structure after 24 h incubation. The distribution of the trait with odd or even number of the straight chain length of carbon atoms is shown in bluish colors (upper). The chain length trait divides the fatty acid length in even for Acet, But and iVal and odd for Prop, iBut and Val. The distribution of non-branched (linear), iso-or anteiso methylated long-chain fatty acids are in yellow-greenish colors (lower). SCFAs are grouped by the branching trait in linear for Acet, Prop, But, Val and in iso for iBut and iVal. The two traits are calculated by the sum of the percentile appearance in the LCFA-profile. Media composition was similar except the addition of any SCFAs. Inoculation and transfer volume: 4% (v/v). Optical density measurements were conducted in Hungate tubes at a wavelength of 600 nm. Time axis is compressed from 10 to 50 h after inoculation. Figure S13. KEGG pathway of porphyrin and chlorophyll metabolism found in all cultivation conditions. The fields with red letters represent the found enzymes from all cultivation conditions combined. The pathway is completed from L-glutamate down to protoporphyrin IX. The orange marked enzyme is the one which annotation is missing to form heme.