Isolation and characterization of new mutants of the cytochrome d terminal oxidase of Escherichia coli
The cytochrome d terminal oxidase from Escherichia coli is a two-subunit, three-heme integral membrane cytochrome. Subunit I contains the heme center $b\sb{558}$ and Subunit II, is necessary for the binding of the $b\sb{595}$ and d heme prosthetic groups. Earlier work suggests that the N-terminus of Subunit I participates in the binding of the $b\sb{595}$ and d heme centers. Mutants in the cytochrome d terminal oxidase from Escherichia coli were isolated using hydroxylamine mutagenesis. These mutants, isolated both in the chromosome and on plasmids, were characterized using reduced minus air-oxidized difference spectroscopy, Western immunoblotting, CO difference spectroscopy, and oxidase activity assays. Plasmic-encoded mutations were mapped by marker rescue and identified by DNA sequencing. Two new classes of cytochrome d terminal oxidase mutants were identified. Under aerobic growth conditions, one class produces both subunits, and binds both the $b\sb{558}$ and $b\sb{595}$ heme centers, but varying amounts of the heme d center. The mutations conferring this phenotype are in the N-terminus of Subunit II. The conclusion is that the N-terminus of Subunit II along with the N-terminus of Subunit I forms the interface between these two subunits where the $b\sb{595}$ and d heme centers bind. The second class of mutations also produces both subunits but binds only $b\sb{558}$ and $b\sb{595}$. No heme d is bound under aerobic conditions. One of these mutations was identified and is also found in the N-terminus of Subunit I confirming its involvement in the formation of the heme-binding interface. There is another phenotype associated with the above mutants. When cells containing these mutations are grown anaerobically, the stability of the mutant enzymes improved as evidenced by increased heme binding and subunit stability. During the course of this work, a technique was developed to create chromosomal replacements in E. coli. Plasmid DNA carrying the desired allele for gene replacement was transformed into a recBC sbcBC mutant of E. coli. A double cross-over event exchanges the wild-type and plasmid-encoded alleles to effect a replacement. This double cross-over event is screened for by the use of a positive drug marker linked to the replacement allele.
Biology, Molecular, Biology, Genetics, Chemistry, Biochemistry
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign