A Pathway for Isethionate Dissimilation in Bacillus krulwichiae
ABSTRACT
Hydroxyethyl sulfonate (isethionate) is widely distributed in the environment as an industrial pollutant and as a product of microbial metabolism. It is used as a substrate for growth by metabolically diverse environmental bacteria. Aerobic pathways for isethionate dissimilation in Gram-negative bacteria involve the cytochrome c-dependent oxidation of isethionate to sulfoacetaldehyde by a membrane-bound flavoenzyme (IseJ), followed by C-S cleavage by the thiamine pyrophosphate (TPP)-dependent enzyme sulfoacetaldehyde acetyltransferase (Xsc). Here, we report a bioinformatics analysis of Xsc-containing gene clusters in Gram-positive bacteria, which revealed the presence of an alternative isethionate dissimilation pathway involving the NAD+-dependent oxidation of isethionate by a cytosolic metal-dependent alcohol dehydrogenase (IseD). We describe the biochemical characterization of recombinant IseD from the haloalkaliphilic environmental bacterium Bacillus krulwichiae AM31DT and demonstrate the growth of this bacterium using isethionate as its sole carbon source, with the excretion of sulfite as a waste product. The IseD-dependent pathway provides the only mechanism for isethionate dissimilation in Gram-positive species to date and suggests a role of the metabolically versatile Bacilli in the mineralization of this ubiquitous organosulfur compound.
IMPORTANCE Isethionate of biotic and industrial sources is prevalent. Dissimilation of isethionate under aerobic conditions is thus far only known in Gram-negative bacteria. Here, we report the discovery of a new pathway in Gram-positive Bacillus krulwichiae. Isethionate is oxidized by a cytosolic metal-dependent alcohol dehydrogenase (which we named IseD), with NAD+ as the electron acceptor, generating sulfoacetaldehyde for subsequent cleavage by Xsc. This work highlights the diversity of organisms and pathways involved in the degradation of this ubiquitous organosulfonate. The new pathway that we discovered may play an important role in organosulfur mineralization and in the sulfur cycle in certain environments.
INTRODUCTION
The C2 organosulfonate isethionate is widespread in the environment and originates from both biotic and industrial sources (1, 2). For example, isethionate is found in certain red algae (3) and is present in mammalian tissues as a by-product of the metabolism of aminoethylsulfonate (taurine) by gut bacteria (4). Isethionate also serves as a polar head group of common surfactants used in personal care products and is discharged into certain habitats as a pollutant (5). Diverse bacteria have been reported to obtain energy through the dissimilation of isethionate. These include aerobic bacteria, which oxidize the organic carbon but excrete the sulfur as sulfate or sulfite (2), and anaerobic sulfate- and sulfite-reducing bacteria (SSRB), which use sulfonate-derived sulfite as a terminal electron acceptor to generate H2S (1, 6).
The aerobic dissimilation of isethionate has been studied in Gram-negative bacteria isolated from both terrestrial (Cupriavidus necator H16) and marine (Silicibacter pomeroyi DSS-3) environments (2), leading to the proposed pathway shown in Fig. S1 in the supplemental material. In this pathway, isethionate is imported into the cytosol by a major facilitator superfamily (MFS) transporter, IseU, in C. necator, and a tripartite ATP-independent periplasmic (TRAP) transporter, IseKLM, in S. pomeroyi. Isethionate is then oxidized intracellularly to sulfoacetaldehyde, catalyzed by the membrane-bound flavoenzyme IseJ, a member of the glucose-methanol-choline (GMC) oxidoreductase family, with cytochrome c (CytC) as the electron acceptor. The sulfoacetaldehyde product then undergoes C-S cleavage to form sulfite and acetyl phosphate, catalyzed by the thiamine pyrophosphate (TPP)-dependent enzyme sulfoacetaldehyde acetyltransferase (Xsc). Acetyl phosphate is then converted to acetyl coenzyme A (acetyl-CoA) by phosphate acetyltransferase (Pta) for further oxidation via the tricarboxylic acid (TCA) cycle. In C. necator, sulfite is exported by the TauE transporter and oxidized by the periplasmic sulfite dehydrogenase (7) (see Fig. S1).
Xsc is the only enzyme known to catalyze C-S cleavage in aerobic C2 sulfonate dissimilation pathways (8). In certain anaerobic pathways, C-S cleavage is catalyzed by the recently discovered O2-sensitive isethionate sulfo-lyase IslA, a member of the glycyl radical enzyme superfamily (9). Apart from its involvement in the dissimilation of isethionate, Xsc is also involved in the dissimilation of taurine through two proposed pathways shown in Fig. S2A and S2B in the supplemental material. In C. necator, taurine is oxidized to sulfoacetaldehyde accompanied by the release of ammonium, catalyzed by the membrane-bound flavoenzyme TauXY, with CytC as the electron acceptor (7). In S. pomeroyi (10) and the Gram-positive bacterium Rhodococcus opacus (11), taurine is converted to sulfoacetaldehyde by the soluble cytosolic enzyme taurine-pyruvate aminotransferase (Tpa). Pyruvate, required by Tpa as the amine acceptor, is regenerated by alanine dehydrogenase (AlaDH) (12).
Pathways for isethionate and taurine dissimilation that rely on the CytC-dependent IseJ and TauXY (see Fig. S1 and S2) are so far only known in Gram-negative bacteria belonging to the metabolically versatile phylum Proteobacteria, and it is not known whether similar enzymes can function with the membrane-localized electron acceptors in Gram-positive bacteria. Pathways for taurine dissimilation that rely on Tpa are present in both Gram-positive (e.g., Rhodococcus opacus) and Gram-negative (e.g., S. pomeroyi) bacteria (Fig. S2B) (12). To date, isethionate is not known to be dissimilated by aerobic Gram-positive bacteria.
The critical role of Xsc in aerobic C2 sulfonate dissimilation allows it to be used as a marker to search for new pathways for sulfonate degradation through bioinformatics. Here, we report the bioinformatics-aided discovery and biochemical confirmation of a new pathway for isethionate dissimilation in the haloalkaliphilic Gram-positive bacterium Bacillus krulwichiae strain AM31DT, involving a cytosolic NAD+-dependent isethionate dehydrogenase belonging to the metal-dependent alcohol dehydrogenase (M-ADH) family, which we named IseD (Fig. 1).
FIG 1
RESULTS AND DISCUSSION
A gene cluster for isethionate degradation in Bacillus krulwichiae.
To explore the diversity of Xsc-dependent pathways in different bacteria, we examined the sequences in the Xsc family (1,453 sequences in the InterPro family to date; accession number IPR017820). Apart from SSRB and Gram-negative bacteria in the phylum Proteobacteria, which have been previously explored (8), Xsc is also present in several Gram-positive bacteria in the metabolically versatile class Bacilli (61 sequences), which were not previously known to dissimilate C2 sulfonates. We, therefore, chose to focus our study on Bacilli.
The genome neighborhoods of Bacilli Xsc sequences were analyzed, and representative gene clusters are shown in Fig. 1A. Many of the Xsc gene clusters contain a putative Tpa and/or AlaDH, suggesting a pathway for taurine dissimilation resembling that in S. pomeroyi (see Fig. S2B in the supplemental material). However, a number of them instead contain an M-ADH (IseD), suggesting a new pathway for isethionate dissimilation (Fig. 1B). We focused our investigation on a gene cluster from B. krulwichiae, which encodes a complete putative pathway for isethionate degradation. The hypothesized pathway involves import of isethionate by the TRAP transporter, oxidation to sulfoacetaldehyde by IseD, and then cleavage to sulfite and acetyl-phosphate by Xsc. Sulfite is exported by TauE, while acetyl-phosphate is converted to acetyl-CoA by Pta (Fig. 1B).
The proposed reaction for IseD, NAD+-dependent isethionate oxidation, is thermodynamically reversible and has been detected in vitro for several sulfoacetaldehyde reductases that function in various taurine degradation pathways. These include Klebsiella pneumoniae IsfD, belonging to the short-chain ADH family, and Bilophila wadsworthia SarD (9, 13) and Bifidobacterium kashiwanohense TauF (14), belonging to the M-ADH family. IseD is distantly related to the two M-ADH enzymes (34.2% and 33.8% identity with SarD and TauF, respectively). The isethionate oxidation reaction is thermodynamically unfavorable and was only observed under forcing reaction conditions (high substrate concentrations and pH) (14, 15). Nevertheless, we hypothesized that the subsequent irreversible Xsc-catalyzed reaction might suffice to drive the pathway forward.
Biochemical characterization of IseD.
To investigate the proposed reaction and pathway, IseD was cloned, recombinantly produced, and biochemically characterized. Recombinant His6-tagged IseD was purified to near homogeneity through Co(II)-affinity chromatography (see Fig. S3 in the supplemental material). IseD catalyzed the NAD+-dependent oxidation of isethionate, with an optimal reaction pH of 10.0 (see Fig. S4A in the supplemental material). The rate of reaction was directly proportional to IseD added (see Fig. S5 in the supplemental material). IseD exhibited Michaelis-Menten kinetics for both substrates (kcat = 1.1 s−1; Km for isethionate = 3.6 mM; Km for NAD+ = 0.3 mM) (Fig. 2A and B). The reaction product sulfoacetaldehyde was detected by derivatization with 2,4-dinitrophenylhydrazine (DNPH), followed by liquid chromatography-mass spectrometry (LC-MS) (Fig. 3A to C). IseD also catalyzed the reverse reaction, NADH-dependent reduction of sulfoacetaldehyde, with an optimal reaction pH of 8.0 (see Fig. S4B) and Michaelis-Menten kinetics for both substrates (kcat = 9.9 s−1; Km for sulfoacetaldehyde = 1.0 mM; Km for NADH = 0.1 mM) (Fig. 2C and D).
FIG 2
FIG 3
To investigate the nature of the catalytic divalent metal required for M-ADH activity, chelation and metal reconstitution experiments were conducted. Activity of as-purified IseD was abolished upon chelation with EDTA (Fig. 3D). Upon reconstitution with divalent metals, the highest activity for isethionate oxidation was obtained with Mn2+ (Fig. 3D). Although the highest activity for the physiologically relevant sulfoacetaldehyde reduction by B. kashiwanohense TauF was achieved with Zn2+ (14), the highest activity of isethionate oxidation was also observed with Mn2+ (our unpublished data), suggesting a common mechanism of rate acceleration with Mn2+. Nearly all M-ADH enzymes studied to date use either Fe2+ or Zn2+ as their metal cofactor (16). An exception is a recently reported extremophilic brine pool M-ADH that exhibits the highest activity with Mn2+ as a cofactor (17). Mismetallation of recombinantly produced proteins is a common occurrence (18), and the determinants of in vivo metal loading of M-ADH are unknown (16); thus, further investigations are required to determine the physiologically relevant metal cofactor of IseD.
Growth of B. krulwichiae with isethionate as the sole carbon source.
Growth of B. krulwichiae was supported in defined medium with 20 mM glucose or 60 mM isethionate, but not taurine, as the sole carbon source (Fig. 4A). Growth was accompanied by the production of sulfite, as detected by fuchsin assay (Fig. 4B), showing that sulfite is exported and not oxidized to sulfate, as observed in C. necator (7). Transfer of cells from rich medium to defined isethionate medium was accompanied by the induction of two prominent protein bands with molecular masses of 63.4 and 47.1 kDa (Fig. 4C), identified by mass spectrometry as Xsc and isocitrate lyase (UniProt accession numbers A0A1X9M8K4 and A0A1X9MHX3) (see Data Set S1 and S2 in the supplemental material). The induction of Xsc demonstrates its involvement in isethionate degradation, while the induction of isocitrate lyase suggests activation of an anaplerotic pathway, needed for biosynthesis and replenishment of TCA cycle intermediates for subsequent metabolism of isethionate-derived acetyl-CoA. In the isethionate-containing defined medium, added glucose repressed the expression of Xsc and isocitrate lyase (Fig. 4C, lane 4), and no secretion of sulfite was observed under such growth conditions (Fig. 4B).
FIG 4
In the new IseD-dependent pathway, the electron acceptor for isethionate oxidation is NAD+ (ε0′ = −0.32 V). The energetics of this step are markedly less favorable than those of the IseJ-dependent pathway, where the electron acceptor is cytochrome c (ε0′ = +0.25 V). Isethionate is a relatively unreactive alcohol due to the electron-withdrawing nature of the sulfonate group. Our experiments demonstrate that such a pathway is nevertheless viable in supporting bacterial growth with isethionate as a sole carbon source. This may be facilitated by the strong induction of Xsc, which catalyzes the irreversible subsequent step upon transition to isethionate medium.
In conclusion, the growth of B. krulwichiae on isethionate, together with the biochemical characterization of the isethionate dehydrogenase IseD, supports the proposed pathway for isethionate dissimilation (Fig. 1B). Our study demonstrates that, in addition to Gram-negative bacteria, the ability to dissimilate isethionate is also present in Gram-positive bacteria belonging to the metabolically versatile class Bacilli, which are ubiquitous in terrestrial environments and could play an important role in sulfur recycling.
MATERIALS AND METHODS
General.
Lysogeny broth (LB) medium was prepared with yeast extract and tryptone purchased from Oxoid (Hampshire, UK). Methanol and acetonitrile used for LC-MS were high-purity solvents from Concord Technology (MN, USA). Formic acid was purchased from Merck (NJ, USA). Water used in this work was ultrapure deionized water from Millipore Direct-Q. Talon cobalt resin was purchased from Clontech Laboratories, Inc. (CA, USA). Protein purification chromatographic experiments were performed on an Äkta pure fast protein liquid chromatography (FPLC) machine equipped with appropriate columns in a 4°C cold cabinet. Data were analyzed and plotted using GraphPad Prism 5.
Bioinformatics.
Gene syntheses and cloning.
The codon-optimized gene fragment of iseD (UniProt accession number A0A1X9M8Q0) was synthesized by General Biosystems (Anhui, China) and inserted into pET-28a-HT at the SspI restriction site. The resulting plasmid HT-IseD contains, in tandem, a His6 tag and a tobacco etch virus (TEV) protease cleavage site, followed by iseD (21).
Expression and purification of IseD.
HT-IseD was transformed into Escherichia coli BL21(DE3) cells. The transformant was grown in LB containing 50 μg/ml kanamycin in flasks in a 37°C incubator with shaking at 220 rpm. When optical density at 600 nm (OD600) reached ∼0.8, the temperature was decreased to 18°C and isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.3 mM. Cells were harvested by centrifugation (8,000 × g for 10 min at 4°C) after 16 h.
Cells (∼1 g wet weight) were suspended in 5 ml of lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mM phenylmethanesulfonyl fluoride [PMSF], 0.2 mg/ml lysozyme, 25 μg/ml DNase I, 0.03% Triton X-100). The cell suspension was frozen in a −80°C freezer and then thawed and incubated at room temperature (RT) for 40 min to allow lysis. A total of 6% streptomycin sulfate and glycerol were added to the cell lysate (final concentrations of 1% streptomycin sulfate, 10% glycerol [vol/vol]) followed by gentle mixing and then centrifugation (20,000 × g for 10 min at 4°C) to remove nucleic acid. The supernatant was filtered and loaded onto a 5-ml Talon cobalt column pre-equilibrated with buffer A (40 mM HEPES, pH 7.5, 5 mM β-mercaptoethanol [BME], 0.5 M KCl, and 10% glycerol). The column was washed with 10 column volumes (CV) of buffer A and eluted with 10 CV of buffer A containing 150 mM imidazole. The eluate (∼20 ml) was dialyzed against 2 liters of buffer A at 4°C overnight, frozen in aliquots in liquid N2, and stored at −80°C. The purified IseD (ε280 = 23,380 M−1 cm−1) was analyzed on a 10% SDS-PAGE gel.
Assay for isethionate oxidation by IseD.
To determine the optimal pH for isethionate oxidation, a 200-μl reaction solution containing 40 mM buffer [either Tris-HCl, 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonate (CAPSO) or 3-(cyclohexylamino)-1-propanesulfonate (CAPS) in the pH range of 7.5 to 11.0], 0.5 M KCl, 150 mM isethionate, and 5 mM NAD+ was premixed in a 96-well plate at RT. The reaction was initiated by addition of 700 nM IseD. The absorbance at 340 nm was monitored using a Tecan M200 plate reader at 10-s intervals for 1.5 min. In all subsequent assays, the reaction buffer used was 40 mM CAPSO at the optimal pH of 10.0.
To ascertain that the rate of reaction was directly proportional to IseD added, the assay was repeated with varying concentrations of IseD (180, 360, 720 nM). To determine the kinetic parameters for isethionate oxidation by IseD, the concentration of one substrate was fixed (at 100 mM isethionate or 5 mM NAD+), while varying the concentration of the other substrate.
Assay for sulfoacetaldehyde reduction by IseD.
The substrate sulfoacetaldehyde was introduced as a bisulfite adduct using the method reported by Denger et al. (22). To determine the optimal pH for sulfoacetaldehyde reduction, a 200-μl reaction solution containing 40 mM buffer [either 2-(N-morpholino)ethanesulfonic acid (MES), Tris-HCl, CAPSO, or CAPS, pH in a range of 5.5 to 11.0], 0.5 M KCl, 2 mM sulfoacetaldehyde, and 0.4 mM NADH was premixed in a 96-well plate at RT. The reaction was initiated by the addition of 300 nM IseD. The absorbance at 340 nm was monitored at 10-s intervals for 1.5 min. In all subsequent assays, the reaction buffer used was 40 mM Tris-HCl at the optimal pH of 8.0.
To determine the kinetic parameters for sulfoacetaldehyde reduction by IseD, the concentration of one substrate was fixed (at 3 mM sulfoacetaldehyde or 0.4 mM NADH) while varying the concentration of the other substrate.
LC-MS detection of sulfoacetaldehyde formation.
A 200-μl reaction mixture containing 40 mM CAPSO, pH 10.0, 1.1 μM IseD, 100 mM isethionate, and 5 mM NAD+ was incubated for 10 min at 30°C. Two negative controls, omitting either IseD or isethionate, were also prepared. The sulfoacetaldehyde product was detected by derivatization with DNPH (J&K, Beijing, China) (23). After the enzyme reaction, 100 μl of reaction solution was mixed with 1.1 ml of 0.73 M sodium acetate buffer, pH 5.0, followed by 800 μl of freshly prepared DNPH solution (0.04%). The mixture was incubated at 50°C for 1 h and then filtered prior to LC-MS analysis. LC-MS analysis was performed exactly as previously described (15).
Growth of B. krulwichiae in isethionate medium.
B. krulwichiae strain AM31DT (JCM11691T) (24) was purchased from Japan Collection of Microorganisms (JCM), Riken BioResource Center (Saitama, Japan). The rich medium was prepared by dissolving 10 g glucose, 5 g peptone, 5 g yeast extract, 1 g K2HPO4, and 0.2 g MgSO4·7H2O in 900 ml distilled water and autoclaving. A total of 100 ml of 10% Na2CO3 was filtered with a 0.22-μm filter and added to the autoclaved medium to adjust the pH to 10.0. The defined medium used in growth studies was a modified DSMZ medium 1208, omitting yeast extract and containing 20 mM glucose, 60 mM isethionate/taurine, or 20 mM glucose plus 60 mM isethionate as the carbon source. B. krulwichiae cells were inoculated into rich medium and grown overnight in a 30°C incubator with shaking at 230 rpm. Then 100-μl portions of the culture were transferred into 20 ml of defined medium containing the different carbon sources. The OD600 was then monitored over 2 days.
Fuchsin assay for sulfite detection.
Sulfite produced during growth of B. krulwichiae was detected using a colorimetric assay involving the formation of a colored complex between sulfite and fuchsin dye in acidic solution (22). The media were sampled prior to cell inoculation and after cells had grown to an OD600 of ∼1 and centrifuged at 8,000 × g for 10 min. The supernatant was used either directly for the fuchsin assay or after dilution to a suitable concentration with distilled water. Serial dilutions of sodium sulfite (20, 10, 5, 2.5, 1.25, 0 μM) were used to establish a standard curve as a reference. The fuchsin reagent (0.8 M H2SO4, 0.08% fuchsin, and 1.6% formaldehyde, mixed 7:2:1) was freshly prepared. A 10-μl portion of sample was mixed with 190 μl of fuchsin reagent and incubated for 10 min at RT, and the absorbance at 580 nm was recorded.
Protein identification by SDS-PAGE and mass spectrometry.
Cells were harvested by centrifugation, lysed by boiling in Laemmli loading buffer, and analyzed on a 10% SDS-PAGE gel. Prominent protein bands induced by growth on isethionate were manually excised and sent to Shanghai Applied Protein Technology Co. Ltd. for analysis. After in-gel digestion and extraction, the peptide mixtures were loaded onto a Q Exactive Orbitrap liquid chromatography-tandem mass spectrometry (LC-MS/MS) system (Thermo Fisher) equipped with an Easy-nLC 1000 nanoflow liquid chromatograph (Thermo Fisher). The peptide hits were searched against B. krulwichiae GCA_002109385.1 in the NCBI database by MASCOT. Protein identifications were performed based on probability-based molecular weight search (MOWSE) scoring algorithm with a confidence level of 95%.
ACKNOWLEDGMENTS
We thank the instrument analytical center of the School of Pharmaceutical Science and Technology at Tianjin University for providing the LC-MS analysis and Zhi Li and Xiangyang Zhang for the helpful discussion.
This work was supported by the National Science Foundation of China, grants 31870049 and 31570060 (Y.Z.), and the Agency for Science, Research and Technology of Singapore Visiting Investigator Program grant 1535j00137 (H.Z.).
We declare no conflict of interest.
Y.T. designed and carried out biochemistry experiments and was involved in writing the manuscript. Y.W. designed and carried out bioinformatics experiments and was involved in conceptualizing the project and writing the manuscript. Y.H. was involved in molecular cloning. E.L.A., H.Z., and Y.Z. were involved in conceptualizing the project, getting grants for the project, overall supervision of the project, and writing the manuscript.
Supplemental Material
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Information & Contributors
Information
Published In
Applied and Environmental Microbiology
Volume 85 • Number 15 • 1 August 2019
eLocator: e00793-19
Editor: Rebecca E. Parales, University of California, Davis
PubMed: 31126948
Copyright
© 2019 American Society for Microbiology. All Rights Reserved.
History
Received: 5 April 2019
Accepted: 20 May 2019
Published online: 18 July 2019
Keywords
Contributors
Editor
Rebecca E. Parales
Editor
University of California, Davis
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2019.
A Pathway for Isethionate Dissimilation in Bacillus krulwichiae. Appl Environ Microbiol
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