Abstract
We report the DNA sequence and mutational analysis of a novel cluster of six Bradyrhizobium japonicum genes of which at least three (designated cycV, cycW, and cycX) are essential for the formation of all cellular c-type cytochromes. Mutants having insertions in these genes were completely devoid of any soluble (periplasmic) or membrane-bound c-type cytochromes; even the apo form of cytochrome c1 was not detectable, neither in the membrane nor in the soluble fraction. As a consequence, the mutants had pleiotropic phenotypes such as defects in nitrate respiration, H2 oxidation, electron transport to cytochrome alpha alpha 3, and microaerobic respiration during symbiosis. A fourth open reading frame (ORF132) encoded a protein that might also be concerned with cytochrome c formation, but perhaps only indirectly. The other two open reading frames did not appear to function in this process. The predicted amino acid sequences of the cycW and cycX gene products suggested that these proteins were membrane-bound. The cycV gene product showed extensive similarity to the ATP-binding subunit of a superfamily of membrane-associated transport systems. The predicted ORF132 product was strikingly similar to bacterial thioredoxins and eukaryotic protein disulfide isomerase. Based on these findings it is possible that these proteins are members of a complex transport system involved in the biogenesis of all cytochromes c.
Highlights
The C-type cytochromes consist of an apoprotein to which a heme cofactor is covalently bound
We report the discovery of three novel bacterial genes cycV, cycW, and cycX that are functionally involved in a related process, the biogenesis of C-type cytochromes
Given the fact that cytochromes c must be coupled with heme and translocated from the bacterial cytoplasm to the periplasm, or from the mitochondrial matrix to theintermembrane space, the involvement of additional components in these processes was not unexpected, yet, therewas little evidence for that until now
Summary
Bacterial Strains-B. japonicum llOrifl5 [22], a rifampicin-resistant derivative of strain 3Ilb110 For the construction of mutant 93 (Fig. l), an exonuclease III/mung bean nuclease-generated deletion plasmid called pRJ2630 was used It contained a 1396-bp long B. japonicurn DNA fragment (nucleotides 1-1396 in Fig. 2) in the Bluescript vector pKS+. Mung bean nuclease-generated deletion plasmid, pRJ2613, which contained a 2202-bp B. japonicum DNA fragment (nucleotides 2992500 in Fig. 2) was used. The resulting, mutagenized plasmids were called pRJ2664 and pRJ2665 Both plasmids were digested with BamHI and Asp718, which cut within the multiple cloning site of the Bluescript vector releasing the mutated B. japonicum fragments. For the construction of mutant 98 (Fig. l),another exonuclease III/mung bean nuclease-generated deletion clone, called pRJ2670, was used I phosphatase (Bio-Rad).The bands were made visible using a color-developingreagent containing 5-bromo-4-chloro-3-indolylphosphate andnitroblue tetrazolium (Bio-Rad)
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