Coxiella burnetii superoxide dismutase gene: cloning, sequencing, and expression in Escherichia coli.
A superoxide dismutase (SOD) gene from the obligate intracellular bacterium Coxiella burnetii has been cloned, and its DNA sequence has been determined and expressed in Escherichia coli. The gene was identified on pSJR50, a pHC79-derived genomic clone, by using the polymerase chain reaction with degenerate oligonucleotide primers corresponding to conserved regions of known SODs. Sequences resembling conventional E. coli ribosomal and RNA polymerase-binding sites preceded the C. burnetii 579-bp SOD open reading frame. An E. coli SOD-deficient double mutant (sodA sodB) that carried pSJR50 had growth and survival responses similar to those of the wild type when the transformant was challenged with 0.05 mM paraquat and 5 mM hydrogen peroxide, respectively. These observations indicated that the C. burnetii gene was functionally expressed in E. coli. Staining of native polyacrylamide gels for SOD activity demonstrated that pSJR50 insert DNA codes for an SOD that comigrates with an SOD found in C. burnetii cell lysates. The enzyme was inactivated by 5 mM hydrogen peroxide, which is indicative of an iron-containing SOD. Additionally, the predicted amino acid sequence was significantly more homologous to known iron-containing SODs than to manganese-containing SODs. Isolation of the C. burnetii SOD gene may provide an opportunity to examine its role in the intracellular survival of this rickettsia.
Heinzen
R A
RA
Department of Microbiology, Washington State University, Pullman 99164-4233.
Frazier
M E
ME
Mallavia
L P
LP
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D016997:Coxiella burnetii / Q000201:enzymology* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D006861:Hydrogen Peroxide / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D013482:Superoxide Dismutase / Q000235:genetics*
D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase