Hypersensitivity of Escherichia coli Delta(uvrB-bio) mutants to 6-hydroxylaminopurine and other base analogs is due to a defect in molybdenum cofactor biosynthesis

Abstract

We have shown previously that Escherichia coli and Salmonella enterica serovar Typhimurium strains carrying a deletion of the uvrB-bio region are hypersensitive to the mutagenic and toxic action of 6-hydroxylaminopurine (HAP) and related base analogs. This sensitivity is not due to the uvrB excision repair defect associated with this deletion because a uvrB point mutation or a uvrA deficiency does not cause hypersensitivity. In the present work, we have investigated which gene(s) within the deleted region may be responsible for this effect. Using independent approaches, we isolated both a point mutation and a transposon insertion in the moeA gene, which is located in the region covered by the deletion, that conferred HAP sensitivity equal to that conferred by the uvrB-bio deletion. The moeAB operon provides one of a large number of genes responsible for biosynthesis of the molybdenum cofactor. Defects in other genes in the same pathway, such as moa or mod, also lead to the same HAP-hypersensitive phenotype. We propose that the molybdenum cofactor is required as a cofactor for an as yet unidentified enzyme (or enzymes) that acts to inactivate HAP and other related compounds.

FIG. 1

(A) The bio-uvrB region of the E. coli chromosome. The genes and transposon insertion sites relevant to this study are presented. The indicated extent of the uvrB-bio deletion is based on data from Cleary et al. (5). (B) Map of the moeAB operon. The locations of the moeA120(Am) and moeA121::mini-Tn10cam mutations are indicated. Plasmid pMoeA1 used for complementation assays contained the AvaI-StuI fragment (−77 to 1226) (see Materials and Methods).

FIG. 2

Spot tests for determination of HAP sensitivity. The test was performed as described in Materials and Methods. The strains used were KA796 (upper left), NR10107 [Δ(uvrB-bio)] (upper right), NR11958 (mut-1) (lower left), and NR12383 (moeA121::mini-Tn10cam) (lower right). See the text for the renaming of the mut-1 allele as moeA120(Am).

FIG. 3

HAP sensitivities of various mol mutants defective in molybdenum cofactor synthesis. The central filter disc contained HAP (100 μg) (left plate) or no HAP (right plate). Suspensions of eight different strains were applied in a series of spots radiating out from the central disc. The strains, arranged in isogenic pairs, are NR13044 (moe⁺) (1), NR13043 (moeA5) (2), NR13036 (moa⁺) (3), NR13035 (moaA1) (4), NR13040 (moa⁺) (5), NR13039 (moaA18) (6), NR13032 (mod⁺) (7), and NR13031 (modC4) (8). See Table 1 and Materials and Methods for strain details.