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

Summary 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.

INTRODUCTION 6-Deoxy-6-sulfoglucose (sulfoquinovose, SQ) is the polar head group of the plant sulfolipids sulfoquinovosyl diacylglycerols (SQDGs) (Benson, 1963). These lipids are constituents of photosynthetically active membranes in, essentially, all phototrophic organisms (Benning, 1998). Furthermore, certain archaea contain SQ in their surface-layer glycoproteins (Meyer et al., 2011). More on the role of SQ in the biosphere can be found in a recent review (Goddard-Borger and Williams, 2017). Microbial degradation of the large amounts of SQ produced by phototrophs is an important yet largely understudied part of the biogeochemical sulfur cycle . SQ is introduced into the digestive systems of all herbivores and omnivores through their green-vegetable diet and, thus, SQ may be utilized as a substrate also by intestinal microbes .
Complete degradation (mineralization) of the SQ into, ultimately, inorganic sulfate  or hydrogen sulfide  can be achieved in two tiers by bacterial communities through metabolite cross-feeding. In the first tier, bacteria catalyze a primary degradation of SQ to C 3 -organosulfonates, either 3-sulfolactate (SL) or 2,3-dihydroxypropane-1-sulfonate (DHPS). Such a primary SQdegradation pathway is the subject of this study. In the second tier, SL-and DHPS-mineralizing bacteria release the inorganic sulfur. Notably, specialized anaerobic bacteria can utilize these SQ degradation intermediates (SL and DHPS) for sulfite respiration (e.g., some Desulfovibrio and Bilophila spp.) producing potentially harmful hydrogen sulfide (H 2 S) , which implies that a complete, anaerobic two-step degradation of SQ to H 2 S may be a trait of the intestinal microbiota that is relevant to human health (see ref. Burrichter et al., 2018 and Discussion).
We have previously described two types of biochemical pathways (depicted in Figure 1A) for a primary degradation of SQ to DHPS or SL, in Escherichia coli K-12 and Pseudomonas putida SQ1, respectively .

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  and fermentative growth conditions . 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 . It employs a 2-keto-3,6dideoxy-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.

Isolation and Examination of an SQ-Degrading Bacillus aryabhattai Strain
Sulfo-EMP and sulfo-ED pathway gene clusters can be found frequently in genomes of Proteobacteria but not in Gram-positive (phylum Firmicutes) bacteria. Therefore, we started new aerobic enrichment cultures with SQ as sole carbon and energy source  and using soil samples, pond water, or plant leaves as inocula. Replicate soil inocula were pasteurized in order to aid in the enrichment of spore-forming Firmicutes. All enrichments grew, degraded SQ as confirmed by HPLC, and were sub-cultivated until pure cultures were obtained, which were identified by 16S rRNA gene sequencing. From the unpasteurized samples, proteobacterial strains of Pseudomonas sp. (plant leaf), Rhanella sp. (non-pasteurized soil), and Aeromonas sp. (pond) were obtained but not examined further, as they degrade SQ most likely via one of the previously investigated SQ pathways ( Figure 1A). However, one of the unpasteurized enrichments (maple leaf) yielded a Bacillus sp. strain (termed strain SOS1) with a 16S rRNA gene sequence identity of 99.9% to the B. aryabhattai type strain. Two more Bacillus sp. strains (strains AF1 and AF2) were isolated from the pasteurized soil enrichment cultures. They utilized the substrate SQ concomitantly with stoichiometrical SL excretion, as determined by HPLC during growth experiments (a linearized growth plot is shown for strain SOS1 in the Supplementary files, Figure S1), but they did not excrete DHPS or sulfate.
We chose B. aryabhattai SOS1 (deposited as DSM 104036) as our model organism for further examination of its SQ-degradation pathway. Cell extracts of SQ-grown cells were examined for enzyme activities and metabolites of the two known SQ-degradation pathways, under the reaction conditions used previously . Upon addition of SQ, formation of SF was detectable by HPLC-MS (see below), indicative of an SQ isomerase activity. However, addition of known co-substrates (see Figure 1A, ATP and/or NAD + ) yielded no traces of the key intermediate(s) of the sulfo-EMP pathway (SFP; Figure 1A)  nor of the sulfo-ED pathway (SG, KDSG; Figure 1A) . We concluded that strain SOS1 may harbor a different pathway that branches from the sulfo-EMP pathway at the level of SF as first intermediate. Furthermore, the SQ isomerase activity was not detectable in cell extract of glucose-grown cells, suggesting that also this SQ pathway is inducibly expressed .
Identification of a Metabolic Gene Cluster that Is Highly Expressed during SQ Degradation in B. aryabhattai SOS1 An annotated draft-genome sequence of strain SOS1 was generated for proteomics using Illumina HiSeq sequencing and JGI's Integrated Microbial Genomes (IMG) annotation pipeline ; the annotation is available at IMG (GOLD Analysis Project Id, Ga0111075). Two-dimensional protein gel electrophoresis (2D-PAGE) as well as total proteomics were performed ( Figure 2) for extract of cells grown with SQ in comparison with cells grown with glucose, in order to identify the inducible pathway enzymes/genes (e.g., . For the 2D-PAGE (Figure 2A), all major protein spots visible only for SQ-grown cells, indicative of abundant proteins specifically induced during growth with SQ, were excised and identified by peptide fingerprinting-mass spectrometry. These results were confirmed by the total proteomic analyses ( Figure 2B).
Six identified, strongly produced proteins ( Figure 2A) were encoded in the same cluster ( Figure 2C), for which the predicted proteins/genes had been auto-annotated by the IMG pipeline as follows (gene numbers; the IMG locus tag prefix, Ga0111075_1003, is omitted): 1319 as predicted MFS-type sugar:cation symporter; 1320 as predicted transaldolase; 1321 as protein domain of unknown function (DUF4867) gene; 1322 as glucoside hydrolase (alpha-glucosidase); 1323 as aldose/ketose isomerase; 1324 as transcriptional regulator (GntR family); 1325 as aldehyde dehydrogenase; and 1326 as predicted sulfite/organosulfonate exporter (TauE-type) ( Figure 2C). The total proteomic analysis ( Figure 2B) confirmed a strong expression of these six proteins (Figure 2A), and identified an additional protein (1319) encoded in the same gene cluster, for a predicted glycoside symporter in SQ-grown B. aryabhattai SOS1.

Identification of a 6-Deoxy-6-sulfofructose Transaldolase Enzyme in Cell Extracts
The identified, predicted transaldolase 1320 (termed SftT) showed 41.7% amino acid identity (full length) to a characterized fructose-6-phosphate transaldolase (from Thermoplasma acidophilum; Lehwess-Litzmann et al., 2011a; Lehwess-Litzmann et al., 2011b) but much lower identity to characterized fructose-6-phosphate aldolases (e.g., from E. coli, 25.2%;ref. Schurmann and Sprenger, 2001), suggesting that an acceptor molecule, such as glyceraldehyde-3-phosphate (GAP) or erythrose-4-phosphate (E4P), may be crucial for catalysis. We refined our enzyme reaction conditions (see Transparent Methods) and used gel-filtered cell extract, in which potential co-substrates and acceptor molecules had been removed (size exclusion, >1-5 kDa). Gel-filtered cell extract and SQ alone produced only SF, but a conversion of the SQ via SF further to SLA was detectable after addition of GAP or E4P to the assay, i.e., as acceptor molecules for a transaldolase reaction. Furthermore, with addition of NAD + for a predicted SLA dehydrogenase reaction, also a formation of SL was detectable. The transfer of a dihydroxyacetone moiety of SF onto an acceptor molecule by a transaldolase enzyme (glyceronetransferase; EC 2.2.1.2) concomitant with SLA formation (see Figure 1B), as strongly suggested by the results described above, was unequivocally confirmed when we used fully isotopically ( 13 C)-labelled SQ ( 13 C 6 -SQ) as substrate and when we observed the corresponding mass shifts of the intermediates in MS/MS while the HPLC retention times were unaffected in comparison with authentic, unlabeled standards. As shown in Figure 3, the 13 C 6 -SQ was converted to 13 C 6 -SF, and through the presence of unlabeled E4P, accumulation of [1,2,3-13 C 3 ]-SLA and of [1,2,3-13 C 3 ]-sedoheptulose-7-phosphate (S7P) was detectable, and in presence of NAD + , [1,2,3-13 C 3 ]-SL accumulated. Hence, the mass shifts occurring for the molecular-ion of S7P ( Figure 3) and its fragmentation products (see Figure S2) identified 13 C 6labeled SF as the origin of the transferred C 3 -(dihydroxyacetone) moiety. When unlabeled GAP instead of E4P was used together with NAD + , the reaction products were 13 C 3 -SLA and 13 C 3 -SL, and [1,2,3-13 C 3 ]-hexose phosphates but not [1,2,3-13 C 3 ]-S7P ( Figure S3), confirming that also GAP served as acceptor for the SF transaldolase reaction.
From the results of these enzyme tests with cell extract (Figures 2, S2, and S3) in combination with the proteomic and genomic results (Figures 2A-2C), we concluded that the SQ catabolic pathway in strain SOS1 involves only three inducible enzymes, as depicted in Figure Figure S3). The F6P/S7P powers energy conservation and growth of B. aryabhattai SOS1 and serves for regeneration of the corresponding acceptor molecule (as depicted in Figure 1B). The SLA is oxidized to SL (Figures 3 and S3) by the co-encoded, co-induced NAD + -dependent dehydrogenase 1325 ( Figure 2). , 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).
The enzyme catalyzed the SF-cleavage also with E4P as acceptor, forming 13 C 3 -SLA and [1,2,3-13 C 3 ]-S7P (see Figure S8), but not with F6P as acceptor. Thus, protein SftT is an SF-cleaving, SLA-forming, GAP/ E4P-dependent transaldolase; it showed activity also as sedoheptulose-7-phosphate transaldolase (see Figure S9). Finally, after addition of protein 1325 (SftD) and NAD + to the reaction mixture, the 13 C 3 -SLA was converted to 13 C 3 -SL (Figure 4), as compared with authentic SL standard ( Figure S7). Thus, protein . The reaction mixture initially contained 2 mM 13 C 6 -SQ and 12 mM GAP (0 min). SQ isomerase SftI (protein 1323) was added (100 mg/mL) and the reaction sampled after 10 min. Then, the SF transaldolase SftT (protein 1320) (50 mg/mL) was added and the reaction sampled after 1 min. Finally, SLA dehydrogenase SftD (protein 1325) (100 mg/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 [1,2,3-13 C 3 ]-F6P to [1,2,3-13 C 3 ]-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 [1,2,3-13 C 3 ]-G6P, and for F6P and [1,2,3-13 C 3 ]-F6P, are shown in Figures S12 and S13, respectively.

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iScience 23, 101510, September 25, 2020 7 iScience Article SftD is another NAD + -dependent SLA dehydrogenase, in addition to the one identified in the P. putida sulfo-ED pathway ( Figure 1A); SftD oxidized also GAP and E4P as substrates but showed no activity with SL and DHPS, as tested in photometrical assays. Note that specific activities for SLA dehydrogenase could not be determined, because no authentic SLA was available as substrate; the SLA for the enzyme tests was generated in a coupled assay with E. coli SLA-reductase (YihU), converting DHPS with NAD + to SLA . Finally, in reaction mixtures in which GAP served as the acceptor (Figure 5), the F6P formed by the SF transaldolase was converted further to G6P, and SQ isomerase SftI was responsible for this activity (see Figure S10).

Enterococcus gilvus, Clostridium symbiosum, and Eubacterium rectale Strains Employ the SFT Pathway for SQ Fermentation
We searched for bacterial genomes that harbor sft-gene clusters using IMG's (https://img.jgi.doe.gov) Ortholog Neighborhood Viewer and Cluster Scout (Hadjithomas et al., 2017) and retrieved a total of 189 candidate clusters, predominantly in members of the classes Bacilli and Clostridia (phylum Firmicutes), e.g., in members of the genera Bacillus, Enterococcus, Lactobacillus, Butyrivibrio, Pseudobutyrivibrio, Eubacterium, and Clostridium, and also in individual genomes of members of the phyla Fusobacteria, Chloroflexi, Actinobacteria, Spirochaetes, and Thermotogae. Representative gene-cluster architectures comprising the key genes for enzymes SftITD as well as for SQ-glyceride cleavage of the sulfolipid (SftG, alpha-glucosidases), and predicted genes for transport systems (import of SQ and excretion of SL or DHPS), regulation and metabolism of glycerol, if present (Discussion), are illustrated in Figure 5. For a list of all genomes retrieved via IMG (as of 3 July 2020) that contain candidate SFT-gene clusters, see Table  S2. For example, we found candidate gene cluster in approximately 1% of all Bacillus genomes; hence, the gene cluster may be descriptive of specific ecotypes, e.g., plant or intestine associated, but it is not part of the core genome (pan-genome) of Bacillus species, in contrast to the sulfo-EMP pathway gene cluster in E. coli .
Interestingly, the gene clusters of the strictly anaerobic Clostridia encoded, instead of an SLA dehydrogenase (SftD; dark green in Figures 5 and 1A), exclusively an NADH-dependent DHPS-forming SLA reductase (SftR, indicated in light green in Figures 5 and 1A), such as the characterized SLA reductases of E. coli (YihU; ref. Sharma et al., 2020) and of Desulfovibrio sp. DF1 (DhpA, physiologically catalyzing the reverse reaction; see ref. . This strongly suggested that these strains catalyze an SQ fermentation pathway that may result in DHPS as degradation product, instead of SL, hence, as an additional fermentation step to recover NAD + , as with the sulfo-EMP pathway of E. coli ( Figure 1A; ref. . Hence, we additionally confirmed as part of this study for three strictly anaerobic, human-gut Firmicutes strains with sft-gene clusters, that they (1) indeed are capable of growing through SQ fermentation and (2) excrete DHPS or SL ( Figure 6A) and (3) produce the Sft proteins specifically during growth with SQ, as observed by differential proteomics ( Figure 6B). Enterococcus gilvus DSM15689 (order Lactobacillales)  and Eubacterium rectale (Agathobacter rectalis) DSM17629 (order Clostridiales) (Duncan and Flint, 2008) were examined, and Clostridium symbiosum LT0011, which we isolated from human feces by streaking on SQ-containing agar plates and picking of colonies into SQ-containing liquid medium. Strain LT0011 (deposited as DSM108250) was genome sequenced (IMG Genome ID, 2802428835) for the proteomic analysis ( Figure 6B); SQ fermentation was confirmed also for C. symbiosum type-strain DSM934 (Kaneuchi et al., 1976). For growth of the strains in anaerobic liquid culture, a carbonate-buffered minerals salts medium with Ti(III)NTA as reducing agent  supplemented with SQ (10 mM) and yeast extract (0.1% w/v) was used.
As shown in Figure 6A, E. gilvus produced SL but not DHPS during fermentation of SQ under our growth conditions, whereas C. symbiosum and E. rectale produced DHPS but not SL. The predicted inducible expression of the SFT pathway genes during SQ fermentation was confirmed by the differential proteomics results, as shown in Figure 6B.

DISCUSSION
In this study, we established the first primary SQ-degradation pathway known in the ecologically and industrially important group of Gram-positive (phylum Firmicutes) bacteria, by using a newly isolated environmental B. aryabhattai strain as model system, by differential proteomics, by in vitro reconstruction of the iScience Article pathway with recombinant enzymes, and by identification of all intermediates using fully 13 C-labeled SQ as substrate. Surprisingly, homologous pathway gene cluster can be found widespread particularly in genomes of aerobic and anaerobic (phylum) Firmicutes, (class) Bacilli and Clostridia, and also in individual genomes of members of other important bacterial phyla, for example, Fusobacteria, Actinobacteria, Spirochaetes, Chloroflexi, and Thermotogae. For three representative strains of the strictly anaerobic and 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).

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iScience 23, 101510, September 25, 2020 9 iScience Article important human-gut (class) Bacilli and Clostridia, we confirmed their predicted ability to grow with SQ and that they express the pathway enzymes inducibly during fermentative growth with SQ.
Our previous additions to the known glycolytic (monosaccharide degradation) pathways in bacteria were the two ''sulfoglycolytic'' pathways  for catabolism of the 6-sulfonated-6-deoxy hexose SQ, which are analogous to the Embden-Meyerhof-Parnas (e.g., Kresge et al., 2005;Bara nskaa et al., 2007) and the Entner-Doudoroff (Entner and Doudoroff, 1952) pathways for glucose-6-phosphate (G6P) ( Figure 1A), given the structural similarity of the substrates SQ and G6P. All these pathways employ aldolases (aldehyde lyases, EC 4.1.2.x) for cleavage of the C 6 -monosaccharides into C 3 fragments. The third bacterial pathway for primary SQ degradation uncovered in this study reminds somewhat of another glycolytic pathway for G6P, the pentose-phosphate pathway (Gunsalus et al., 1955;Horecker, 2002), in that a transaldolase enzyme (aldehyde/ketone transferase, EC 2.2.1.x) is involved in 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 sftgene cluster (see Figure 5).

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OPEN ACCESS iScience 23, 101510, September 25, 2020 iScience Article acquisition of monosaccharide carbon, as depicted in Figure 1B. The SF transaldolase (proposed systematic name, 6-deoxy-6-sulfofructose:D-glyceraldehyde-3-phosphate glyceronetransferase) excises a sulfonated C 3 fragment (SLA), and it acquires a non-sulfonated C 3 fragment from SQ by transferring a glycerone-moiety onto an acceptor molecule, GAP or E4P, yielding F6P or S7P (Figures 3, 4, S3, and S8). The F6P and S7P is used for regeneration of the acceptor and as carbon and energy source for cellular biomass formation ( Figure 1B). Differences of the newly discovered SQ pathway to the pentose phosphate pathway, however, are absence of both NADH generation and decarboxylation reaction and, thus, that not a pentose intermediate is cleaved.
The biochemical and genetic identity of this third bacterial pathway for SQ catabolism, as established in this study, is adding to a surprisingly high, but yet mostly underappreciated, diversity of microbial pathways for utilization of organosulfonates, as has probably first been noted for taurine (e.g., Cook and Denger, 2006; see also METACYC [https://biocyc.org], taurine degradation I-IV, and Peck et al., 2019). The genetic and biochemical identification of this third SQ pathway is also adding valuable insight, as well as future experimental access, to important questions regarding physiology, enzymology, and pathway energetics of SQ degradation, regarding the environmental microbiology of SQ degradation and its contributions to the biogeochemical carbon and sulfur cycles, and also regarding its potential roles in intestinal microbiomes and for human health.
The glycolytic pathways for G6P are optimized according to their overall thermodynamic and biochemical properties of the pathway, necessity of investing in enzymes (protein) to maintain sufficient pathway fluxes, and their molar yields of energy equivalents (ATP) and reducing equivalents (NADH) relative to the electron acceptors used (e.g., Bar-Even et al., 2012;Flamholz et al., 2013). Given the structural similarity of SQ and G6P, similar energetic and ecophysiological constraints have obviously led to the evolution of also several biochemical strategies for degradation of SQ. However, a common theme for all known SQ-degradation pathways is that the ''waste product'' SLA is employed either as additional electron donor or as additional electron acceptor (Figure 1). One of the many interesting questions arising is whether the benefits of this type of SLA utilization, either as electron donor or acceptor, might in many environmental settings outweigh the benefit of further catabolizing the SLA-carbon (for example, by desulfonation of SL to pyruvate through sulfolactate sulfo-lyase, SuyAB; ref.  and whether this might be an explanation for the division of labor (Tsoi et al., 2018;Giri et al., 2019) observed thus far in complete SQ degradation, that is, primary SQ degradation and cross-feeding of the DHPS or SL to different microorganisms that mediate the desulfonation reactions  for closing the sulfur cycle. Further interesting questions are arising around the different forms non-sulfonated C 3 -carbon acquired through the three different SQ pathways, that is DHAP by the sulfo-EMP pathway, pyruvate by the sulfo-ED pathway, and a C 3 -(glycerone)-moiety incorporated into F6P/S7P by the SFT pathway ( Figure 1). Although the yields of reducing equivalents and ATP of the different pathways appear well traceable (see below), the particular benefits and costs of funneling the SQ-carbon into the anabolic reactions and fermentations at these different levels is unclear. For example, the particular advantages of the different SQ-pathway strategies may become fully visible only if degradation of the sulfolipid metabolite SQ-glyceride (SQG) is explored further as substrate, instead of only SQ: every SQ-degradation gene cluster examined thus far co-encodes an SQG glucosidase (sulfoquinovosidase) homolog (SftG in Figures 2 and 5; see also refs.  for cleaving SQG to SQ and glycerol (Speciale et al., 2016), and every SQ-degrading strain examined thus far by proteomics strongly co-induced this sulfoquinovosidase (Figures 2 and 6B ;refs. Denger et al., 2014;Li et al., 2020). Furthermore, E. coli is able to grow with SQG (Abayakoon et al., 2018, and our own observation) as well as P. putida SQ1, B. aryabhattai SOS1 (our own observations), and SQ-degrading Rhizobium leguminosarum SRDI565 (Li et al., 2020). We suggest that for the SFT pathway, the glycerol acquired from SQG cleavage may directly feed into generation of the acceptor molecule GAP for the SF transaldolase, through glycerol kinase and glycerol-3-phosphate dehydrogenase, some of which are co-encoded in the predicted gene clusters (see Figure 5). This needs to be addressed in future, as well as the directly related questions whether indeed SQG rather than SQ might be the more environmentally relevant substrate for SQ-catabolic microorganisms (Abayakoon et al., 2018) and whether the SQG glucosidase gene may be a universal genetic marker for SQ(G) metabolism across all types of pathways.
Overall, the now three known pathways for SQ with either SLA oxidation to SL or its reduction to DHPS, seem to reflect adaptations to the aerobic, facultatively anaerobic, or strictly anaerobic ecophysiology ll OPEN ACCESS iScience 23, 101510, September 25, 2020 iScience Article of the different environmental and intestinal microorganisms. SQ utilization by the SL-producing, aerobic P. putida SQ1 through the sulfo-ED pathway inclusive of the SLA dehydrogenase reaction ( Figure 1A) , is represented by the equation SQ + 2 NAD + / pyruvate + SL + 2 NADH +2 H + .
The pyruvate-carbon is assimilated and oxidized for energy generation through aerobic respiration, and the two NADH are also driving respiration. SQ utilization by DHPS-producing, facultatively anaerobic E. coli K12 through the sulfo-EMP pathway including the SLA reductase reaction , and including substrate-level phosphorylation of the DHAP released (see Figure 1A), is represented by the equation SQ + ADP + P i / pyruvate + DHPS + ATP.
The pyruvate-carbon is also assimilated and oxidized for energy generation through aerobic respiration , or it is metabolized and reduced as electron acceptor in mixed-acid fermentation , as has been discussed previously . The SQ utilization by the SL-producing, aerobic B. aryabhattai SOS1 through the oxidative SFT pathway including the SLA dehydrogenase reaction, as described in this study, and including of the regeneration of the transaldolase-acceptor molecule and substrate-level phosphorylation of the DHAP acquired from SQ (see Figure 1B), is represented by the equation SQ + 2 NAD + + ADP + P i / pyruvate + SL + 2 NADH + ATP +2 H + .
Hence, as with the sulfo-ED pathway in P. putida, this pathway generates NADH for respiration; however, in addition, also one ATP is conserved from DHAP further downstream (see Figure 1B), as with the sulfo-EMP pathway of E. coli. Furthermore, the acquisition of the SQ-carbon requires only two enzymes in the SFT pathway, in comparison with the sulfo-ED pathway with four enzymes and the sulfo-EMP pathway with three enzymes (Figure 1). Hence, explanations on the particular advantages of the different SQ-pathway strategies may be provided not only based on pathway energetics and the ATP and NADH yields in relation to the electron acceptor used but also on the level of investment in protein synthesis. Finally, the SQ utilization through the reductive SFT pathway by the fermenting C. symbiosum and E. rectale strains that encode (Figure 5) and express SLA reductases ( Figure 6B) and excrete DHPS ( Figure 6A) is represented by the equation SQ + ADP + Pi / pyruvate + DHPS + ATP, thus, as with the sulfo-EMP pathway of E. coli. However, why Enterococcus gilvus DSM15689 is excreting SL instead of DHPS during SQ fermentation under our cultivation conditions ( Figure 6A), which is counterintuitive, remains yet unresolved. Note that its gene cluster ( Figure 5) encodes both SLA dehydrogenase (SftD) and reductase (SftR) candidates and that both are expressed ( Figure 6B). Thus, E. gilvus may be able to switch between SL and DHPS production depending on its growth conditions, e.g., in presence of alternative electron acceptors or in dependence on a metabolic partner organism. In addition, future work will also have to corroborate the yet only tentatively identified non-sulfonated fermentation products formed, that is, for E. gilvus apparently acetate and formate, and for C. symbiosum and E. rectale apparently acetate and H 2 , but not formate, butyrate, or butanol. Their identification and thorough quantification relative to total biomass formation will establish carbon and electron balances (e.g.,  and, thus, allow for an overall quantitative, stoichiometrical comparison of the SQ fermentations employing the SFT pathway ( Figure 6) relative to that of E. coli via the sulfo-EMP pathway . Such detailed examination might also provide answers to the intriguing questions why the sulfo-EMP pathway is especially widespread among Enterobacteriaceae , the sulfo-ED pathway across (other) Proteobacteria , and why the (oxidative and reductive) SFT pathway(s) are found so prominently across genomes of aerobic as well as anaerobic Gram-positive (phylum Firmicutes) bacteria ( Figure 5).
Environmental studies of SQ-degrading microorganisms, as well as of SL-or DHPS-degrading microorganisms, are needed to fully reveal their diversity, ecological niches, and significance in the carbon and sulfur cycles in all habitats where SQ is produced and/or degraded. The sulfo-ED pathway genes are found in marine-or saline-associated bacteria (e.g., Halomonas, Salinarimonas), freshwater-, soil-, and plant rhizosphere-associated bacteria (e.g. Microvirga, Ensifer, Herbaspirillum, Rhizobium strains), and also in ll OPEN ACCESS iScience 23, 101510, September 25, 2020 iScience Article human-gut symbionts (e.g., Hafnia, Leminorella, and Serratia strains) and/or potential pathogens (Vibrio, Plesiomonas, and Halomonas strains). The sulfo-EMP pathway gene cluster is a feature of the E. coli core genome and also of other Enterobacteriaceae genomes (e.g., Salmonella; Rhanella sp. isolated in this study) as trait relevant for their intestinal as well as environmental lifestyles. The SFT pathway gene cluster is found ( Figure 5) in Firmicutes of the soil and plant environment (e.g., Bacillus and Lactobacillus strains) and also among strains of the human intestinal tract and rumen of livestock (e.g., Clostridium, Eubacterium, Enterococcus, Butyrivibrio, and Faecalibacterium strains). Given SQ is a relevant constituent of the greendiet of all herbivores and omnivores Goddard-Borger and Williams, 2017) and given the wide distribution of SQ degradation gene clusters in phylogenetically diverse intestinal bacteria, SQ and its degradation products DHPS and/or SL might represent yet unappreciated substrates for the intestinal microbiota of humans and animals. For the primary degradation (fermentation) of SQ, it is probably most interesting to explore whether commensal or pathogenic Enterobacteriaceae, or whether intestinal Firmicutes, utilize the dietary SQ. The genetic signatures of these metabolic features are now available, e.g., for their assessments using omics methods. Importantly, organosulfonates such as the SL and DHPS produced from primary SQ degradation can be substrates for sulfite respiration and sulfide (H 2 S) production by the intestinal pathobiont Bilophila wadsworthia and by Desulfovibrio spp. Peck et al., 2019). H 2 S is a key microbial and host-cell-derived metabolite in the intestine and has many detrimental as well as beneficial contributions to human health and disease (e.g., Attene- Ramos et al., 2010;Carbonero et al., 2012;Singh and Lin, 2015;Ijssennagger et al., 2016). Thus, the genetic and biochemical information provided in this study will help to disentangle also effects of diet-derived SQ on the microbiota composition, and on H 2 S homeostasis in the gut, each contributing to an improved understanding of the complex interactions between diet, microbiota, and host health (Heintz-Buschart and Wilmes, 2018).

Limitations of the Study
The insights gained via our approach using pure bacterial cultures and classical microbiological and biochemical methods must be complemented with empirical data on SQ degradation processes examined directly in the environment and gut microbiomes, e.g., by omics methods. Hence, environmental studies of SQ-degrading microorganisms, as well as of SL-or DHPS-degrading microorganisms, are urgently needed to fully reveal their diversity, ecological niches, and significance in the carbon and sulfur cycles in all habitats where SQ is produced and/or degraded. A thorough determination of carbon and electron balances and, thus, an overall stoichiometric and energetic comparison of, e.g., the SQ fermentations via the SFT pathway relative to that of E. coli via the sulfo-EMP pathway, must also await future, more detailed studies, as well as further descriptions of the enzymes and their kinetic parameters once authentic substrates are available. In summary, our study highlights the importance of establishing the genetic identity of physiological and biochemical features in isolated bacterial strains in the laboratory, for evaluating their role and importance directly in the environment by omics methods. In this case, for adding valuable insight to important questions regarding the environmental microbiology of SQ degradation and its contribution to the biogeochemical carbon and sulfur cycles, and also regarding its potential roles in intestinal microbiomes and for human health.

Resource Availability Lead Contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Prof. David Schleheck (david.schleheck@uni-konstanz.de).

Materials Availability
Data related to this paper may be requested from the lead author.

DECLARATION OF INTERESTS
The authors declare they have no competing interests.       For two-dimensional (2D) protein gel electrophoresis and total proteomics, cell pellets were resuspended in Tris-HCl buffer (pH 7.5) containing 25 μg/ml DNAse and 2 mM MgCl2. The cells were disrupted by five passages through a chilled French Pressure Cell (SLM Instruments, USA) at 140 MPa and unbroken cells and debris was removed by centrifugation (10 min, 15,000 x g, 4°C) to obtain cell extracts. Samples of cell extract were submitted to total proteomic analysis (see below). For 2D-protein gel electrophoresis, membrane fragments were removed by ultracentrifugation (1 h, 70,000 x g, 4°C; Beckman Optima TL) to obtain soluble protein fraction, which was desalted using PD-10 columns (GE Healthcare) prior to 2D-protein gel electrophoresis (see below). Protein concentrations in the extracts were determined through Bradford assay (Bradford 1976) against BSA as standard.
For enzyme assays, the cell pellets were resuspended and washed twice with (NH4)2CO3 buffer (50 mM, pH 9.0) by centrifugation (15 min, 5000 x g, 4°C); after the washing, 1 U/ml DNase was added. The lysis was performed by five passages through a chilled French pressure cell at 140 MPa. Cell debris was removed by centrifugation (15 min, 21.380 x g, 4°C) and small molecules were removed by gel filtration if appropriate (exclusion size 1-5 kDa; illustra NAP-10 gel filtration columns, GE Healthcare Life Sciences).
Draft-genome sequencing and proteomic analysis of B. aryabhattai SOS1. Genomic DNA of strain SOS1 was prepared by the CTAB-protocol (William et al., 2012) including RNase treatment. Whole genome shotgun sequencing of strain SOS1 was performed by GATC Biotech (Konstanz, Germany; now Eurofins) using an Illumina HiSeq 2500 platform and a 2x125bp paired-end library, which generated 11,92 million reads. The program Trimmomatic v0.33 (Bolger et al., 2014) was used to remove adapters and filter the reads by quality with default settings, as well as to discard sequences shorter than 50 nucleotides. The filtered reads were assembled de novo using the program SOAPdenovo v2.04 (Luo et al., 2012) with a k-mer size of 67 and setting the minimum contig length at 200 bp. The de novo assembly procedure resulted in 383 sequences (N50: 0.808 Mb) that were further scaffolded by the reference-guided algorithm implemented in Ragout 2.0b (Kolmogorov et al., 2014)  2D-protein gel electrophoresis (isoelectric focusing and SDS gel electrophoresis) of the soluble protein fraction of strain SOS1 was conducted as described previously .
Total proteomics of cell extracts, and identification of proteins in spots excised from the SDS gels, was done as described previously , Schmidt et al., 2013 at the Proteomics Centre of the University of Konstanz (https://www.biologie.unikonstanz.de/proteomics-centre/) with the exception that each sample was analyzed twice on a Orbitrap Fusion with EASY-nLC 1200 (Thermo Fisher Scientific), and tandem mass spectra were searched against the protein database using Mascot (Matrix Science) and Proteom Discoverer V1.3 (Thermo Fisher Scientific) with "Trypsin" enzyme cleavage, static cysteine alkylation by chloroacetamide, and variable methionine oxidation (Peck et al., 2019).
HPLC for determination of substrate and product concentrations in cultures. SQ, DHPS and SL in samples of culture supernatant (see above) were separated by hydrophilic interaction chromatography (HILIC) using an HPLC apparatus (Prominence LC-20A System; Shimadzu), and were detected by an Evaporative Light Scattering Detector (ELSD) (model ZAM 3000; Schambeck SFD GmbH, Germany). A SeQuant ZIC-HILIC HPLC Column (Merck, Germany) as described previously  was used for the separation. Samples of culture supernatants (0.6 ml) were mixed with acetonitrile (0.3 ml) in HPLC-vials. The injection volume was 10 μl. The column temperature was set at 30°C. The eluents were (A) 0.1 M ammonium acetate in water supplemented with 10% acetonitrile, and (B) 100% acetonitrile.
The flow rate was 0.75 ml/min. The HPLC gradient was set from 90% B to 65% B in 25 min; hold at 65% B for 10 min; gradient to 90% B in 0.5 min; hold (reequilibration) at 90% B for 9.5 min. SQ, DHPS and SL were identified by their specific retention time against authentic standards (SQ, 25.9 -26.4 min; DHPS, 17.6 -17.7 min; SL, 27.2 -27.6 min) and quantified by peak area integration against authentic standards (see above). SL was quantified also by ion chromatography with suppression under conditions described previously (Styp von Rekowski et al., 2005); SL eluted after 6.1 min. Tschech and Pfennig 1984;vitamin solution, ref. Pfennig 1978) with 10 mM SQ and yeast extract (0.1% w/v) as supplement. The cultures were incubated in serum bottles enclosed with butyl-rubber stoppers under a N2/CO2 (80:20) gas atmosphere. Inoculation and sampling was done through the rubber stoppers with syringe and needle. Samples were taken at intervals for monitoring growth (OD580) (see above) and for HPLC analysis of SQ, DHPS and SL (see above). Short chain fatty acids and alcohols against authentic standards were analyzed on a HPLC system (system 10A, Shimadzu) with an Aminex column (HPX-87H, BioRad) and a refractive index detector (RID-10A, Shimadzu) at 60°C. The eluent was 5 mM H2SO4 at an isocratic flow of 0.6 ml/min. Under these conditions, the fermentation product acetate eluted at 11.7 min. This Aminex HPLC-RID method generally separates and detects carbohydrates, carboxylic acids, short-chain fatty acids, alcohols, ketones and other metabolites; no other products (peaks) than acetate were detected in any growth experiment. Hydrogen production was tested with a Peak Perfomer 1 (Peak Laboratories) trace gas analyzing gas chromatography system with a reducing compound photometer (RCP) detector; N2 was used as carrier gas. For harvesting of the cultures, the serum bottles were opened and the cell suspension centrifuged under air atmosphere; the cell were disrupted, the cell extracts prepared, and the total proteomic analyses were done as, described above in the appropriate sections for B. aryabhattai SOS1.