Immunological characterization of an Escherichia coli strain which is lacking cytochrome d

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Escherichia coli mutants lacking specific cytochromes (d, a1, b558) exhibit reduced N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activity.
• Previous research identified this oxidase activity with a specific material, the function of which was not fully elucidated.

Purpose of the Study:

• To characterize the N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activity in Escherichia coli.
• To determine the molecular identity of the enzyme responsible for this oxidase activity.
• To investigate the genetic basis of the cytochrome d complex and its associated oxidase function.

Main Methods:

• Isolation of bacterial membranes from a cytochrome-deficient Escherichia coli mutant strain.
• Enzymatic assays to measure N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activity.
• Immunological characterization using antisera against the cytochrome d complex and its subunits.
• Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to analyze protein subunits.
• Genetic complementation by introducing F-prime F152 into the mutant strain.

Main Results:

• The N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activity is attributed to the two-subunit cytochrome d complex.
• The mutant strain was confirmed to lack both subunits of the cytochrome d complex.
• Complementation with F-prime F152 restored both the cytochrome d subunits and the associated enzymatic activity.

Conclusions:

• The two-subunit cytochrome d complex is the primary N,N,N',N'-tetramethyl-p-phenylenediamine oxidase in Escherichia coli.
• The genetic locus for the cytochrome d complex is essential for both its structure and enzymatic function.
• This finding clarifies the molecular basis of a key respiratory enzyme in E. coli.