Environmental and Intestinal Phylum Firmicutes Bacteria Metabolize the Plant Sugar Sulfoquinovose via a 6-Deoxy-6-sulfofructose Transaldolase Pathway

Abstract

Bacterial degradation of the sugar sulfoquinovose (SQ, 6-deoxy-6-sulfoglucose) produced by plants, algae, and cyanobacteria, is an important component of the biogeochemical carbon and sulfur cycles. Here, we reveal a third biochemical pathway for primary SQ degradation in an aerobic Bacillus aryabhattai strain. An isomerase converts SQ to 6-deoxy-6-sulfofructose (SF). A novel transaldolase enzyme cleaves the SF to 3-sulfolactaldehyde (SLA), while the non-sulfonated C₃-(glycerone)-moiety is transferred to an acceptor molecule, glyceraldehyde phosphate (GAP), yielding fructose-6-phosphate (F6P). Intestinal anaerobic bacteria such as Enterococcus gilvus, Clostridium symbiosum, and Eubacterium rectale strains also express transaldolase pathway gene clusters during fermentative growth with SQ. The now three known biochemical strategies for SQ catabolism reflect adaptations to the aerobic or anaerobic lifestyle of the different bacteria. The occurrence of these pathways in intestinal (family) Enterobacteriaceae and (phylum) Firmicutes strains further highlights a potential importance of metabolism of green-diet SQ by gut microbial communities to, ultimately, hydrogen sulfide.

Keywords: Microbial Metabolism; Microbiology.

Graphical abstract

Figure 1

Pathways for Primary SQ Degradation in Bacteria Illustration of (A) the two known SQ-degradation pathways in Escherichia coli K12 and Pseudomonas putida SQ1, respectively, and (B) the third SQ-degradation pathway identified in this study in aerobic Bacillus aryabhattai SOS1 and in strictly anaerobic, SQ-fermenting bacteria such as Clostridium symbiosum LT0011. (A) E. coli uses an SQ-Embden-Meyerhof-Parnas (sulfo-EMP) pathway for acquisition of carbon and energy from SQ under both aerobic (Denger et al., 2014) and fermentative growth conditions (Burrichter et al., 2018). It employs a 6-deoxy-6-sulfofructose-1-phosphate (SFP) aldolase (indicated in orange), and it excretes 2,3-dihydroxypropanesulfonate (DHPS) as degradation product during growth with SQ. P. putida uses an SQ-Entner-Doudoroff (sulfo-ED) pathway (Felux et al., 2015). It employs a 2-keto-3,6-dideoxy-6-sulfogluconate (KDSG) aldolase (yellow), and it excretes 3-sulfolactate (SL) as degradation product during growth with SQ. See the main text (Introduction) for more detailed descriptions of the pathways and of the abbreviations used for intermediates and enzymes (roman numerals). (B) In this study, a third pathway for SQ was discovered in B. aryabhattai SOS1, which excretes SL during SQ degradation. It employs a newly discovered 6-deoxy-6-sulfofructose (SF) transaldolase enzyme (red) that derives fructose-6-phosphate (F6P) for growth (or sedoheptulose-7-phosphate [S7P]; not depicted in Figure 1B) through transfer of the non-sulfonated C3-(glycerone) moiety of SF onto GAP as acceptor molecule (or erythrose-4-phosphate [E4P]; not depicted in Figure 1B). The pathway was identified by differential proteomics to be present also in SQ-fermenting, human gut bacteria, for example, in C. symbiosum LT0011, which excretes DHPS during SQ fermentation.

Figure 2

Proteomic Analysis of B. aryabhattai SOS1 Cells Identified a Single Gene Cluster for SQ Degradation (A) Proteins in cell extracts of SQ- or glucose-grown cells were separated via 2D gel electrophoresis (2D-PAGE), and all prominent protein spots visible only for SQ-grown cells were excised and submitted to peptide fingerprinting-mass spectrometry. These spots are labeled according to their IMG gene locus tag numbers (A) and are encoded in the same gene cluster (illustrated in C). The results were replicated once when starting from an independent growth experiment. (B) Relative abundances of the proteins encoded in this gene cluster as observed by total proteomic analysis in SQ-versus glucose-grown cells. For comparison, the relative abundances of constitutively expressed proteins (GroL, RpoB, AtpA) are also shown, as well as of glucose-inducible enzymes (Entner-Doudoroff/pentose phosphate pathway enzymes). Data from an independent growth experiment and proteomic analysis are shown (n = 1). (C) Illustration of the identified SQ degradation gene cluster in B. aryabhattai SOS1. The locus tag numbers and gene annotations shown refer to the IMG-draft genome annotation of strain SOS1; the IMG locus tag prefix is indicated. These genes were termed sftATXGIFDE (6-deoxy-6-sulfofructose transaldolase pathway gene cluster; SFT pathway). The blue coloration indicates genes identified by proteomics (A and/or B).

Figure 3

HPLC Mass Spectrometry Confirmed a Transaldolase Reaction in Cell Extracts of SQ-grown B. aryabhattai SOS1 Using Fully ¹³C-Labeled SQ as Substrate and Erythrose-4-Phosphate as Acceptor Molecule In the presence of unlabeled erythrose-4-phosphate (E4P) and of ¹³C₆-SQ, a transient formation of ¹³C₆-SF and ¹³C₃-SLA, and an accumulation of [1,2,3-¹³C₃]-sedoheptulose-7-phosphate (S7P) and of ¹³C₃-SL, was detected. The ¹³C₃-SL formation resulted from an SLA dehydrogenase reaction because of the additional presence of NAD⁺ in the reaction mixture. The reaction contained 2 mM ¹³C₆-SQ, 6 mM E4P (with G6P as impurity; not shown), and 6 mM NAD⁺ and was sampled before addition of the gel-filtered cell extract (exclusion, >5 kDa) and after 10, 30, and 240 min of incubation. For the organosulfonates, the total-ion chromatograms (TIC) of the MS/MS-fragmentation of the quasi-molecular ions ([M-H]^-) are shown. For the sugar phosphates E4P and [1,2,3-¹³C₃]-S7P, the MS/MS-ion traces of the phosphate group ([H₂PO₄]^-, m/z = 97) are shown. Note that ¹³C₃-SLA was separated by HPLC as trident peak. The results were replicated twice when starting from independent growth experiments. When E4P was exchanged by GAP as the acceptor, [1,2,3-¹³C₃]-F6P/G6P accumulation was detected (see Figure S3). MS/MS fragmentation mass spectra are shown as Supplementary files for [1,2,3-¹³C₃]-S7P (Figure S2), ¹³C₆-SF and ¹³C₆-SQ (Figure S5), ¹³C₃-SLA (Figure S6), ¹³C₃-SL (Figure S7), and E4P (Figure S13).

Figure 4

In Vitro Reconstitution of the SQ Transaldolase Pathway by Three Recombinant Enzymes For the organosulfonates, the total-ion chromatograms (TICs) of the MS/MS fragmentation of the quasi-molecular ions ([M-H]^-) are depicted, and for the sugar phosphates F6P and G6P, the characteristic ion traces of the phosphate group ([H₂PO₄]^-, m/z = 97). The reaction mixture initially contained 2 mM ¹³C₆-SQ and 12 mM GAP (0 min). SQ isomerase SftI (protein 1323) was added (100 μg/mL) and the reaction sampled after 10 min. Then, the SF transaldolase SftT (protein 1320) (50 μg/mL) was added and the reaction sampled after 1 min. Finally, SLA dehydrogenase SftD (protein 1325) (100 μg/mL) and 6 mM NAD⁺ was added and the reaction sampled after 17 h. Note that GAP and NAD⁺ were added in excess (12 and 6 mM) because the SLA dehydrogenase oxidizes also GAP in the presence of NAD⁺; therefore, the SQ conversion was incomplete. Furthermore, the reaction mixture converted the [1,2,3-¹³C₃]-F6P to [1,2,3-¹³C₃]-G6P, owing to an activity of the SQ-isomerase also with F6P. The results were replicated twice using independently produced enzyme preparations. A reaction sequence with E4P instead of GAP as the acceptor is shown in the Supplementary files, Figure S8. A chromatogram depicting the HPLC separation of G6P and F6P in more detail is shown in Figure S11, and MS/MS fragmentation mass spectra for G6P and [1,2,3-¹³C₃]-G6P, and for F6P and [1,2,3-¹³C₃]-F6P, are shown in Figures S12 and S13, respectively.

Figure 5

Homologous SF-Transaldolase Pathway Gene Clusters are Found Widespread Particularly in Genomes of Aerobic and Anaerobic (Phylum) Firmicutes (Class) Bacilli and Clostridia Shown are selected gene-cluster architectures as retrieved from the IMG Ortholog Neighborhood Viewer when using the transaldolase SftT as query. In total, 183 orthologous gene clusters were identified in IMG. The gene clusters shown encode also candidate genes for SQ-glyceride hydrolases (alpha-glucosidases), transport systems (import of SQ and excretion of SL or DHPS), and regulation. Importantly, most of the genes clusters of the strictly anaerobic Firmicutes encode SLA reductase candidate genes (termed SftR) homologous to YihU of E. coli, instead of SLA dehydrogenases (SftD) genes, suggesting that these strains most likely catalyze an SQ-fermentation pathway that results in DHPS instead of SL as degradation product (see text). Indicated by underlined letters are three representative strains that were examined in this study for their ability to ferment SQ, excrete SL or DHPS, and produce the SFT pathway proteins specifically during growth with SQ, as confirmed by proteomics for all three strains (Figure 6).

Figure 6

Fermentation of SQ by Human Intestinal Bacteria via the SF-transaldolase Pathway (A and B) Fermentation of SQ to SL or DHPS by human intestinal bacterial strains Enterococcus gilvus DSM15689, Clostridium symbiosum LT0011, and Eubacterium rectale DSM17629 (A) and proteomic confirmation of a strong, inducible expression of the Sft-proteins during growth with SQ (B). (A) E. gilvus produced SL during SQ fermentation and C. symbiosum and E. rectale produced DHPS but not SL. The used mineral salts medium had to be supplemented with yeast extract (0.1% w/v) for observing growth, and no complete turnover of the SQ provided (10 mM) was obtained, presumably due to depletion of supplements, except for E. gilvus. Triplicates (n = 3) are shown; the error bars indicate standard deviations. From replicate cultures, cellular biomass was collected at the end of the growth phase and submitted to total proteomic analyses in comparison to glucose-fermenting cells. (B) Differential proteomics confirmed a strongly inducible expression of the Sft-pathway genes during fermentation of SQ but not when the strains were grown with glucose. Each result was replicated at least once when starting from independent growth experiments. Abbreviations used: SftA, predicted SQ importer; SftG, SQG hydrolase (alpha-glucosidase); SftI, SQ isomerase; SftT, SF transaldolase; SftR, SLA reductase; SftD, SLA dehydrogenase; SftX, DUF4867; SftE, predicted SL/DHPS exporter; GroL, chaperon; TF, translation factor; EF, elongation factor; Fba, fructose bisphosphate aldolase; Tpi, triosephosphate isomerase; Gap, GAP dehydrogenase; Pyk, pyruvate kinase; n.d., not detected; n/e, not encoded in the respective sft-gene cluster (see Figure 5).