Role for CysJ flavin reductase in molybdenum cofactor-dependent resistance of Escherichia coli to 6-N-hydroxylaminopurine

Abstract

We have previously described a novel Escherichia coli detoxification system for the removal of toxic and mutagenic N-hydroxylated nucleobases and related compounds that requires the molybdenum cofactor. Two subpathways (ycbX and yiiM) were identified, each employing a novel molybdo activity capable of inactivating N-hydroxylated compounds by reduction to the corresponding amine. In the present study, we identify the cysJ gene product as one additional component of this system. While the CysJ protein has been identified as the NADPH:flavin oxidoreductase component of the CysJI sulfite reductase complex (CysJ(8)I(4)), we show that the role of CysJ in base analog detoxification is unique and independent of CysI and sulfite reductase. We further show that CysJ functions as a specific partner of the YcbX molybdoenzyme. We postulate that the function of CysJ in this pathway is to provide, via its NADPH:flavin reductase activity, the reducing equivalents needed for the detoxification reaction at the YcbX molybdocenter. In support of the proposed interaction of the CysJ and YcbX proteins, we show that an apparent CysJ-YcbX "hybrid" protein from two Vibrio species is capable of compensating for a double cysJ ycbX defect in E. coli.

FIG. 1.

Genetic framework for the major molybdenum cofactor (MoCo)-dependent pathways of detoxification of N-hydroxylated base analogs in E. coli (17). moaA to mogA indicate the series of genes required for MoCo biosynthesis (19, 28), while ycbX and yiiM represent the two independent subpathways identified within the MoCo-dependent pathway (17). Specifically, ycbX and yiiM produce apoenzymes that react with MoCo to form the active YcbX and YiiM proteins. The diagram also indicates the differential specificity of the two subpathways toward the model N-hydroxylated compounds used in our studies: 6-N-hydroxylaminopurine (HAP), 2-amino-HAP (AHAP), and hydroxylamine (NH₂OH). For simplicity, the diagram does not distinguish between the MPT and MGD forms of MoCo (19). As shown elsewhere (19), YcbX and YiiM likely employ the MPT form. One additional, minor pathway for HAP detoxification dependent on biotin sulfoxide reductase (an MGD-requiring enzyme) is observable only in the double ycbX yiiM-deficient background and is likewise not shown here (see reference for details).

FIG. 2.

(A) Organization of the E. coli cysJIH operon. The binding element for the CysB positive regulator of the operon is boxed. CysJp indicates the promoter of the operon. (B) HAP sensitivity of the wild-type (WT) strain (NR10836) and its cysJ (NR16043) and cysI (NR16069) derivatives on two different types of media. Spot tests are shown to reveal the HAP-induced inactivation of each of the strains. Cells were plated using a multiprong replicator device to minimal medium plates supplemented either with 0.33 mM l-cysteine or with 0.8 mM sodium thiosulfate as indicated, and 50 μg of HAP was spotted onto the center of each plate. The plates are shown as they appear after 24 h of incubation. In these assays, the edges of the inhibition zones are typically sharp and well defined. The reason for the sharpness is not fully understood but presumably reflects the precise mode of killing by HAP (not yet fully established) along with certain regulatory systems that appear to be triggered by exposure to HAP (unpublished data).

FIG. 3.

HAP sensitivity of cysB strains. Spot test results are shown for a ΔcysB strain and a cysB-constitutive (cysB^c) strain (NR16098 and NR16195, respectively), along with wild-type (NR10836) and ΔcysJ (NR16043) controls. Cells were plated using a multiprong replicator device to minimal medium plates supplemented with 0.33 mM l-cysteine, and 50 μg of HAP was spotted onto the center of each plate.

FIG. 4.

CysJ operates within the MoCo-dependent pathway of base analog detoxification (A) and within the ycbX-dependent pathway (B). Shown are results of survival tests after a 2-h exposure to the indicated concentrations of HAP in liquid medium. Strains used were NR16523 (moaE), NR16757 (cysJ moaE), NR15871 (yiiM), NR15873 (ycbX), NR16043 (cysJ), NR16087 (cysJ ycbX), and NR16085 (cysJ yiiM). See Materials and Methods for further details.

FIG. 5.

Lack of HAP sensitization by l-cysteine in moaE (NR15996) and ycbX (NR16262) mutants. In contrast, clear sensitization is observed in a yiiM strain (NR15870). Cells were plated using a multiprong replicator device to minimal medium plates supplemented with 0.8 mM sodium thiosulfate (VB) or with 0.33 mM l-cysteine, and 50 μg of HAP was spotted onto the center of each plate.

FIG. 6.

(A) Domain architecture of the E. coli YcbX and CysJ proteins and the corresponding Vibrio proteins, VCA0924 and VPA0411, which may be regarded as hybrids of YcbX and CysJ. The presented domains as obtained by Pfam database searches (; http://pfam.sanger.ac.uk/) are MOSC (MoCo sulfurase C-terminal domain, PF03473), β-barrel (MOSC N-terminal β-barrel domain, PF03476), Fer ([2Fe-2S] iron-sulfur cluster binding domain, PF00111), FAD_1 (oxidoreductase FAD-binding domain, PF00667), FAD_6 (oxidoreductase FAD-binding domain, PF00970), NAD (NAD-binding domain, PF00175), and FLD (flavodoxin, PF00258). (B) HAP sensitivities of strain NR16743 (ycbX cysJ) and its isogenic derivatives NR16920 and NR16921, which carry the VCA0924 and VPA0411 Vibrio genes, respectively, replacing the ycbX gene at its endogenous chromosomal location under expression from the ycbX promoter. See Materials and Methods for details. Cells were plated using a multiprong replicator device to minimal medium plates supplemented with 0.33 mM l-cysteine, and 100 μg of HAP was spotted onto the center of each plate.

FIG. 7.

Lack of suppression of the HAP sensitivity of a moaE mutant (NR16523) by a multicopy vector expressing the Vibrio VCA0924 gene. Note that the same plasmid fully complements the HAP sensitivity of a ycbX strain (NR16262) or a ycbX cysJ strain (not shown). Cells were plated using a multiprong replicator device to minimal medium plates supplemented with ampicillin, and 50 μg of HAP was spotted onto the center of each plate.