Differential reduction in soluble and membrane-bound c-type cytochrome contents in a Paracoccus denitrificans mutant partially deficient in 5-aminolevulinate synthase activity.
A mutant of Paracoccus denitrificans, DP104, unable to grow anaerobically with nitrate as the terminal electron acceptor or aerobically with methanol as the electron donor and staining negatively in the dimethylphenylene diamine oxidation (Nadi) test, was isolated by transposon Tn5::phoA mutagenesis. P. denitrificans DP104 grown aerobically with succinate or choline had very low levels (2 to 3% of the wild-type levels) of spectroscopically detectable soluble c-type cytochromes. In contrast, membrane cytochromes of the a, b, and c types were present at 50% of the levels found in the wild type. The apo form of cytochrome c550, at an approximately 1:1 molar ratio with the holo form, was found in the periplasm of DP104. The TnphoA element was shown to be inserted immediately upstream of the translational start of hemA, the gene coding for 5-aminolevulinate synthase, which was sequenced. Low-level expression of this gene, driven off an incidental promoter provided by TnphoA-cointegrated suicide vector DNA, is the basis of the phenotype which could be complemented by the addition of 5-aminolevulinate to growth media. Disruption of the hemA gene generated a P. denitrificans strain auxotrophic for 5-aminolevulinate, establishing that there is no hemA-independent pathway of heme synthesis in this organism. The differential deficiency in periplasmic c-type cytochromes relative to membrane cytochromes in DP104 is suggested to arise from unequal competition for the restricted supply of heme which results from the effects of the transposon insertion.
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M D
MD
Department of Biochemistry, University of Oxford, United Kingdom.
Ferguson
S J
SJ
D000624:5-Aminolevulinate Synthetase / Q000235:genetics*; D000445:Aldehyde Oxidoreductases / Q000235:genetics; D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D000622:Aminolevulinic Acid / Q000378:metabolism; D001483:Base Sequence; D002451:Cell Compartmentation; D003574:Cytochrome c Group / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010231:Paracoccus denitrificans / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D010641:Phenotype; D000623:Porphobilinogen Synthase / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012641:Selection, Genetic; D012995:Solubility
D003574:Cytochrome c Group; D008565:Membrane Proteins; D000622:Aminolevulinic Acid; C031732:cytochrome C-550; D000445:Aldehyde Oxidoreductases; C067282:glutamyl tRNA reductase; D000624:5-Aminolevulinate Synthetase; D000469:Alkaline Phosphatase; D000623:Porphobilinogen Synthase