Mutagenesis of the gene encoding cytochrome c550 of Paracoccus denitrificans and analysis of the resultant physiological effects

Abstract

By using synthetic oligonucleotides, the gene encoding soluble cytochrome c550 was isolated from a genomic bank of Paracoccus denitrificans. The nucleotide sequence of the gene was determined, and the deduced amino acid sequence of the mature protein was found to be similar to the primary structure of purified cytochrome c550 except for the presence of seven additional amino acid residues at the C terminus. At the N terminus of the primary structure was found an additional stretch of 19 amino acid residues that had the typical features of the signal sequence of the cytochrome. Comparison of the nucleotide sequences of the upstream regions of the P. denitrificans cytochrome c550 gene and bc, operon revealed three regions with a distinct organization that showed strong similarity. Downstream of the c5. gene was found part of another gene, the deduced amino acid sequence of which showed strong homology with subunit 1 of the cytochrome aa3 oxidase. For gene replacement experiments, the suicide vector pGRPdl was constructed. The cytochrome c550 gene was inactivated by insertion of a kanamycin resistance gene, and the mutated gene was cloned into this vector. Recombination with the wild-type gene resulted in a mutant strain with an inactivated cytochrome gene. Isolated mutant strains were unable to synthesize the soluble cytochrome, as judged by spectrum analysis and analysis of periplasmic proteins by gel electrophoresis and heme staining. The mutation resulted in a 14% decrease in the growth yield during aerobic heterotrophic growth and in a 40% decrease in the maximum specific growth rate during growth on methylamine. Furthermore, a longer lag phase was observed under both growth conditions. The mutation had no effect on growth yield, maximum specific growth rate, and duration of the lag phase during anaerobic growth in the presence of nitrate. In addition, there was no accumulation of nitrite and nitrous oxide.

Cloning and Expression in Escherichia coli of the Cytochrome c 552 Gene from Thermus thermophilus HB8

“…The DNA sequence reveals that the cytochrome is synthesized in Thermus cells as a pre-protein having an N-terminal signal peptide. The sequence of this signal peptide, (M)K ϩ R ϩ TLMAFLLLGGLALA2Q, is very similar in length and composition to other known pre-apocytochrome c sequences (27,28,32,67,68), which typically have 1-2 positively charged amino acids at the N terminus followed by 14 -20 hydrophobic amino acids; proteolytic cleavage occurs prior to the first hydrophilic residue (69), in this case 2Q. Very likely, this sequence is responsible for directing the protein into the periplasm of Thermus cells (10).…”

Regulation of oxidative phosphorylation: The flexible respiratory network ofParacoccus denitrificans

“…The induction of cytochrome aa 3 under methylotrophic conditions (Table II), when the reduction level of the cytochrome c pool is expected to be relatively high, suggests that expression of this oxidase responds to the redox state of the cytochrome c pool. This might also explain the repression of cytochrome aa3 in the Ac550 mutant (Van Spanning et al, 1990a) (Table II).…”

“…This suggests that only the protontranslocating type of NADH dehydrogenase (NDH-1) is expressed in P. denitrificans, bz vitro studies have provided evidence for electron transfer between the bc~ complex and cytochrome c552 (Turba, 1994), and between cytochrome c550 and the aa3-type oxidase (Van Wielink et al, 1989;Lappalainen et aL, 1995). Together with the clustering of the corresponding cycA (Css0) and ctaDH (aa3) genes on the Paracoccus chromosome, and the finding that deletion of cycA affects the expression of cytochrome aa3 (Van Spanning et al, 1990a), this contributes to the belief that the latter cytochromes are physiological redox partners. Based on the redox midpoint potentials of the three components of the cytochrome c pool of P. denitrificans (at pH 7.0: E'o cyt.…”

“…The function of the individual enzymes was studied by means of directed mutagenesis. In this procedure, the gene encoding the redox enzyme of interest is disrupted in a gene-exchange experiment (Van Spanning et al, 1990a). Next, the characteristics of the mutated strain were analyzed in terms of the kinetics and efficiency of respiration.…”

Formation of a cytochrome c–nitrous oxide reductase complex is obligatory for N2O reduction by Paracoccus pantotrophus

“…14 In the closely related organism P. denitrificans deletion of the cytochrome c 550 gene does not compromise its ability to reduce nitrous oxide suggesting that pseudoazurin may also be a natural electron donor to N 2 OR in vivo. 15 The second domain comprises a 7-bladed propeller of b-sheets that contains the catalytic site, 7 a unique [Cu 4 S] center, called Cu Z , Figs. 1a and 1c.…”