Role of d-Cysteine Desulfhydrase in the Adaptation of Escherichia coli to d-Cysteine

Abstract

Escherichia coli contains an enzyme activity capable of catalyzing the transformation of D-cysteine into pyruvate, H 2 S, and NH 3 (1). This enzyme was named D-cysteine desulfhydrase. A similar activity could be detected in Citrobacter freundii, Klebsiella pneumoniae, Enterobacter cloacae, Chlorella fusca, Spinacia oleracea, and various Fusobacterium species (1-4). On the other hand, no activity was found in the bacteria of genera Arthrobacter, Alcaligenes, Agrobacterium, Bacillus, Brevibacterium, Corynebacterium, Erwinia, Flavobacterium, Micrococcus, Proteus, Pseudomonas, Salmonella, Sarcina, Serratia The physiological role of bacterial D-cysteine desulfhydrase is unknown. However, recent studies indicated that E. coli growth is impaired in the presence of micromolar amounts of D-cysteine (6), suggesting that D-cysteine desulfhydrase has a detoxification function.In the present work, we report the cloning of the E. coli gene (yedO) encoding D-cysteine desulfhydrase activity. Involvement of the product of this gene in the adaptation of the cell to D-cysteine is established. Overexpression of the desulfhydrase gene protects E. coli against D-cysteine, whereas its inactivation renders the bacterium hypersensitive to the D-amino acid. Moreover, our data suggest that, similarly to L-cysteine (7), D-cysteine exerts its toxicity through an inhibition of threonine deaminase. Decomposition of D-cysteine by the desulfhydrase produces H 2 S, which the bacterium can utilize as a sulfur source. Accordingly, we establish that the presence of the yedO gene stimulates cell growth in the presence of D-cysteine as sole sulfur source. In agreement with an involvement of the desulfhydrase in sulfur metabolism, sulfate starvation induces expression of yedO. MATERIALS AND METHODSL-and D-Amino acids, ␤-chloro-D-alanine, NЈ,NЈ-dimethyl-p-phenylenediamine, 1-aminocyclopropane-1-carboxylate, and 2,4-dinitrophenylhydrazine were from Sigma. Pyridoxal phosphate and 2-oxobutyrate (sodium salt) were from Fluka. Rabbit muscle L-lactate dehydrogenase and NADH were from Roche Molecular Biochemicals. DEAE-Sephacel and Superdex 75 were from Amersham Pharmacia Biotech.Purification of D-Cysteine Desulfhydrase-Cells from E. coli strain K37 (Table I) were grown at 37°C in 8 liters of 2ϫ TY medium and harvested by centrifugation for 35 min at 3,000 ϫ g. All buffers used for the purification of D-cysteine desulfhydrase contained 10 M pyridoxal phosphate, 0.1 mM dithiothreitol, and 0.1 mM EDTA. The cell pellet was suspended in 0.1 M potassium phosphate (pH 7.0) at an optical density of 140 at 650 nm. Crude extract preparation, nucleic acid precipitation with streptomycin sulfate, and ammonium sulfate fractionation of proteins were performed as described previously (8), except that ammonium sulfate precipitations were at 35 and 55% saturation, instead of 50 and 80%, respectively. Then the protein pellet was dissolved in 50 ml of 0.1 M potassium phosphate (pH 7.0) and dialyzed overnight against 5 liters of the same buffer. The resulting solution was app...

Functional Demonstration of Reverse Transsulfuration in the Mycobacterium tuberculosis Complex Reveals That Methionine Is the Preferred Sulfur Source for Pathogenic Mycobacteria

“…We postulate that it is by controlling the intracellular pool of cysteine. With its reactive thiol group, cysteine can be highly toxic, so its intracellular concentration is tightly regulated in nature (45,46). We suggest that another enzyme that we discovered in the tubercle bacilli during this study, a cysteine desulfhydrase, has this role.…”

Hydrogen sulfide is involved in the chilling stress response in Vitis vinifera L.

“…Similar to animals, the homologs of CBS (LCD) and CSE (DCD) also are found in plants, and they are reported mainly responsible for generating H 2 S [4,36]. LCD (At3g62130) and DCD (At1g48420) genes have been isolated from Arabidopsis [2,37]. Meanwhile, the homolog genes of CDes also were isolated from B. napus and O. sativa [38].…”

Bacterial Cysteine-Inducible Cysteine Resistance Systems

“…Cells in logarithmic phase were collected, washed with a buffer containing 0.85% NaCl at 4°C, and suspended in 0.1 M potassium phosphate buffer (pH 8.0) containing 50 g ml Ϫ1 bovine serum albumin and 10 M pyridoxal phosphate. The cells were disrupted by ultrasonication and centrifuged, and the supernatant (cell extract) was analyzed for CD activity as previously described (29).…”