Mutations resulting in a defective mini-F replicon in the presence of both origins, oriI and oriII.

Within the region of the origin of replication (oriII) of plasmid mini-F, we introduced a set of deletions around the XhoI site (45.080F) and an insertion of 4 nucleotides at the Bg/II site (45.213F). These alterations had the effect that replication from oriII as well as from the alternative origin, oriI, was abolished. The analysis suggests that an AT-rich region which may function as entry site for DnaB-DnaC protein is an essential structural requirement for replication of plasmid mini-F.

Synthesis of the Rhodopseudomonas viridis holo-cytochrome c2 in Paracoccus denitrificans.

The gene encoding the Rhodopseudomonas viridis cytochrome c2 (cycA) has been introduced on a broad host range vector into Paracoccus denitrificans, leading to high-level expression of the holo-cytochrome with the heme moiety covalently attached to the apoprotein. The cytochrome was demonstrated to reside in the periplasmic space of the host cell. In contrast to R. viridis, aerobic rather than anaerobic growth conditions led to higher production levels of the holo-cytochrome in P. denitrificans. This heterologous expression system provides a suitable genetic background for the functional expression and mutagenesis of polypeptides involved in bacterial photosynthesis, offering the possibility of detailed structural and functional investigation.

Paracoccus denitrificans mutants deleted in the gene for subunit II of cytochrome c oxidase also lack subunit I.

As a prerequisite to site-directed mutagenesis on cytochrome c oxidase, two different mutants are constructed by inactivating the cta gene locus encoding subunits II and III (ctaC and ctaE) of the Paracoccus denitrificans oxidase. Either a short fragment encoding part of the putative copper binding site near the C terminus of subunit II, or a substantial fragment, comprising parts of the coding region for both subunits and all of the intervening three open reading frames, are removed and replaced by the kanamycin resistance gene. Each construct, ligated into a suicide vector, is mated into Paracoccus, and mutants originating from double homologous recombination events are selected. We observe complete loss of alpha-type heme and of oxidase subunits, as well as a substantial decrease in the cytochrome c oxidase activity. Upon complementation with the ctaC gene (plus various lengths of downstream sequence extending into the operon), subunit II gets expressed in all cases. Wild-type phenotype, however, is only restored with the whole operon. Using smaller fragments for complementation gives interesting clues on roles of the open reading frames for the assembly process of the oxidase complex; two of the open reading frame genes most likely code for two independent assembly factors. Since homologous genes have been described not only for other bacterial oxidases, but their gene products shown to participate also in the assembly of the yeast enzyme, they seem to constitute a group of evolutionary conserved proteins.

The cytochrome c reductase/oxidase respiratory pathway of Paracoccus denitrificans: genetic and functional studies.

Data are presented on three components of the quinol oxidation branch of the Paracoccus respiratory chain: cytochrome c reductase, cytochrome c552, and the a-type terminal oxidase. Deletion mutants in the bc1 and the aa3 complex give insight into electron pathways, assembly processes, and stability of both redox complexes, and, moreover, are an important prerequisite for future site-directed mutagenesis experiments. In addition, evidence for a role of cytochrome c552 in electron transport between complex III and IV is presented.

Paracoccus denitrificans cytochrome c1 gene replacement mutants.

We describe the construction and characterization of gene replacement mutants for the respiratory chain component cytochrome c1 in the bacterium Paracoccus denitrificans. Its structural gene (fbcC) was inactivated by insertion of the kanamycin resistance gene, introduced into a suicide vector, and conjugated into Paracoccus; chromosomal mutants obtained by homologous recombination were selected by antibiotic resistance screening and further characterized biochemically. They showed the complete spectral, enzymatic, and immunological loss of the fbcC gene product together with a serious defect in the assembly of the two other gene products of the fbc operon, cytochrome b and the FeS protein. A possible role of the cytochrome c1 in the assembly process for the enzyme complex is discussed. A functional restoration to wild-type phenotype was achieved by complementing in trans with a newly constructed broad-host-range vector carrying the fbcC gene cassette. When the complete fbc operon was present on this vector, overexpression of complex III subunits was observed. Apart from their physiological significance, such mutants are a prerequisite for probing structure-function relationships by site-directed mutagenesis in order to understand molecular details of electron transport and energy transduction processes of this respiratory enzyme in bacteria and in mitochondria.