Interplay between Sulfur Assimilation and Biodesulfurization Activity in Rhodococcus qingshengii IGTS8: Insights into a Regulatory Role of the Reverse Transsulfuration Pathway

Abstract

Biodesulfurization is a process that selectively removes sulfur from dibenzothiophene and its derivatives. Several natural biocatalysts harboring the highly conserved desulfurization operon dszABC, which is significantly repressed by methionine, cysteine, and inorganic sulfate, have been isolated. However, the available information on the metabolic regulation of gene expression is still limited. In this study, scarless knockouts of the reverse transsulfuration pathway enzyme genes cbs and metB were constructed in the desulfurizing strain Rhodococcus sp. strain IGTS8. We provide sequence analyses and report the enzymes' involvement in the sulfate- and methionine-dependent repression of biodesulfurization activity. Sulfate addition in the bacterial culture did not repress the desulfurization activity of the Δcbs strain, whereas deletion of metB promoted a significant biodesulfurization activity for sulfate-based growth and an even higher desulfurization activity for methionine-grown cells. In contrast, growth on cysteine completely repressed the desulfurization activity of all strains. Transcript level comparison uncovered a positive effect of cbs and metB gene deletions on dsz gene expression in the presence of sulfate and methionine, but not cysteine, offering insights into a critical role of cystathionine β-synthase (CβS) and MetB in desulfurization activity regulation. IMPORTANCE Precise genome editing of the model biocatalyst Rhodococcus qingshengii IGTS8 was performed for the first time, more than 3 decades after its initial discovery. We thus gained insight into the regulation of dsz gene expression and biocatalyst activity, depending on the presence of two reverse transsulfuration enzymes, CβS and MetB. Moreover, we observed an enhancement of biodesulfurization capability in the presence of otherwise repressive sulfur sources, such as sulfate and l-methionine. The interconnection of cellular sulfur assimilation strategies was revealed and validated.

Keywords: biocatalysis; cystathionine; cystathionine β-synthase; cystathionine γ-lyase; cysteine; genetic engineering; methionine; reverse transsulfuration; sulfur metabolism.

FIG 1

Bacterial sulfur metabolism. (A) Overview of standard methionine and cysteine biosynthesis and interconversion routes in bacteria as part of the sulfur assimilation pathway (APS, adenylylsulfate; PAPS, 3′-phosphoadenylyl sulfate; OAS, O-acetyl-l-serine; OSHS, O-succinyl-l-homoserine; OAHS, O-acetyl-l-homoserine). Asterisks indicate pathway interconnection points. (B) Canonical reactions of sulfur metabolism catalyzed by CβS and MetB (C-γS/L) in the order Corynebacteriales.

FIG 2

Properties of cbs-metB genetic loci and proteins. (A) Scheme of the cbs-metB gene cluster. (B) Bacterial promoter predicted sequence. −35 and −10 boxes are displayed, and the arrow indicates the predicted transcription initiation site (+1). (C) Schematic diagram of CβS and MetB (C-γS/L) domain distribution. Cys Met Meta PP, cysteine/methionine metabolism-related PLP-binding domain. See the text for details.

FIG 3

Multiple-sequence alignments of CβS and C-γS/L, displaying only conserved residues configuring the active sites. (A) Comparison of R. qingshengii IGTS8 CβS with M. tuberculosis cystathionine β-synthase (UniProt accession no. P9WP51), B. subtilis MccA (UniProt accession no. O05393), human CβS (UniProt accession no. P35520-1), and S. cerevisiae CβS (UniProt accession no. P32582). (B) Comparison of R. qingshengii IGTS8 MetB with M. tuberculosis C-γS/L (UniProt accession no. P9WGB7), C. glutamicum CγS (UniProt accession no. Q79VD9), E. coli cystathionine γ-synthase (UniProt accession no. P00935), S. cerevisiae cystathionine γ-lyase (UniProt accession no. P31373), and human cystathionine γ-lyase (UniProt accession no. P32929). All multiple-sequence alignments were done using ClustalO. Asterisks indicate fully conserved residues, colons denote strongly conserved residues, and dots show weakly conserved residues. Residues in yellow boxes participate in PLP binding. Blue boxes indicate residues involved in substrate binding (22, 57).

FIG 4

(A) Effect of different carbon sources (0.055 M glucose, 0.110 M glycerol, and 0.165 M ethanol) on growth (biomass) and biodesulfurization activity (units per milligram of cells [dry weight] [DCW]) of R. qingshengii IGTS8. DMSO at a concentration of 1 mM was used as the sole sulfur source. (B) Statistical analysis of biodesulfurization results shown in panel A. One-way ANOVA with Tukey’s multiple-comparison test was performed (for more details, see Materials and Methods).

FIG 5

Effect of DMSO as the sole sulfur source on growth (biomass) and biodesulfurization activity (units per milligram of cells [dry weight] [DCW]) of wt (A), Δcbs (B), and ΔmetB (C) strains grown on CDM in the presence of low (0.1 mM) and high (1 mM) DMSO concentrations. Ethanol (0.165 M) was the sole carbon source in the culture medium.

FIG 6

Effect of sulfate as the sole sulfur source on growth (Biomass; g/L) and biodesulfurization activity (units/mg of cells [dry weight] [DCW]) of wt (A), Δcbs (B), and ΔmetB (C) strains grown on CDM in the presence of low (0.1 mM) and high (1 mM) sulfate concentrations. Ethanol (0.165 M) was the sole carbon source in the culture medium.

FIG 7

Effect of methionine as the sole sulfur source on growth (biomass) and biodesulfurization activity (units per milligram of cells [dry weight] [DCW]) of wt (A), Δcbs (B), and ΔmetB (C) strains grown on CDM in the presence of low (0.1 mM) and high (1 mM) methionine concentrations. Ethanol (0.165 M) was the sole carbon source in the culture medium.

FIG 8

Effect of cysteine as the sole sulfur source on growth (biomass) and biodesulfurization activity (units per milligram of cells [dry weight] [DCW]) of wt (A), Δcbs (B), and ΔmetB (C) strains grown on CDM in the presence of low (0.1 mM) and high (1 mM) cysteine concentrations. Ethanol (0.165 M) was the sole carbon source in the culture medium.

FIG 9

Comparison of dszA, dszB, dszC, cbs, and metB transcriptional levels for the wt and the Δcbs and ΔmetB isogenic strains in the presence of 1 mM (A) DMSO, (B) sulfate, (C) methionine, or (D) cysteine. Samples were collected from mid-log-phase cultures, and expression levels relative to the calibrator sample are reported. A logarithmic scale was used for dszABC. For details, see Materials and Methods. Ethanol (0.165 M) was the sole carbon source in the culture medium. AU, arbitrary units.

FIG 10

Proposed model illustrating the role of CβS and MetB (acting as CγL) in the regulation of desulfurization activity in Rhodococcus qingshengii IGTS8. Sulfate or methionine addition in the culture medium most likely necessitates reverse transsulfuration metabolic reactions as the primary route for cysteine biosynthesis. Fine-tuning of sulfur assimilation via intracellular cysteine levels is a common theme in bacterial species, where it seems to have evolved as a cellular mechanism to control gene expression appropriately, based on the available sulfur source type and abundancy. Narrow alterations in the free cysteine pool are suspected to exert an effect (directly or indirectly) on dszABC gene expression, leading to lack of biodesulfurization activity. Deletion of cbs or metB abolishes dsz repression in the presence of selected sulfur sources, such as sulfate and methionine, thus leading to detectable transcript levels and biodesulfurization activity.