Cytochromes c(550), c(552), and c(1) in the electron transport network of Paracoccus denitrificans: redundant or subtly different in function?

Paracoccus denitrificans strains with mutations in the genes encoding the cytochrome c(550), c(552), or c(1) and in combinations of these genes were constructed, and their growth characteristics were determined. Each mutant was able to grow heterotrophically with succinate as the carbon and free-energy source, although their specific growth rates and maximum cell numbers fell variably behind those of the wild type. Maximum cell numbers and rates of growth were also reduced when these strains were grown with methylamine as the sole free-energy source, with the triple cytochrome c mutant failing to grow on this substrate. Under anaerobic conditions in the presence of nitrate, none of the mutant strains lacking the cytochrome bc(1) complex reduced nitrite, which is cytotoxic and accumulated in the medium. The cytochrome c(550)-deficient mutant did denitrify provided copper was present. The cytochrome c(552) mutation had no apparent effect on the denitrifying potential of the mutant cells. The studies show that the cytochromes c have multiple tasks in electron transfer. The cytochrome bc(1) complex is the electron acceptor of the Q-pool and of amicyanin. It is also the electron donor to cytochromes c(550) and c(552) and to the cbb(3)-type oxidase. Cytochrome c(552) is an electron acceptor both of the cytochrome bc(1) complex and of amicyanin, as well as a dedicated electron donor to the aa(3)-type oxidase. Cytochrome c(550) can accept electrons from the cytochrome bc(1) complex and from amicyanin, whereas it is also the electron donor to both cytochrome c oxidases and to at least the nitrite reductase during denitrification. Deletion of the c-type cytochromes also affected the concentrations of remaining cytochromes c, suggesting that the organism is plastic in that it adjusts its infrastructure in response to signals derived from changed electron transfer routes.

Regulation of expression of terminal oxidases in Paracoccus denitrificans.

In order to study the induction of terminal oxidases in Paracoccus denitrificans, their promoters were fused to the lacZ reporter gene and analysed in the wild-type strain, in an FnrP-negative mutant, in a cytochrome bc1-negative mutant, and in six single or double oxidase-negative mutant strains. The strains were grown under aerobic, semi-aerobic, and denitrifying conditions. The oxygen-sensing transcriptional-regulatory protein FnrP negatively regulated the activity of the qox promoter, which controls expression of the ba3-type quinol oxidase, while it positively regulated the activity of the cco promoter, which controls expression of the cbb3-type cytochrome c oxidase. The ctaDII and ctaC promoters, which control the expression of the aa3-type cytochrome c oxidase subunits I and II, respectively, were not regulated by FnrP. The activities of the latter two promoters, however, did decrease with decreasing oxygen concentrations in the growth medium, suggesting that an additional oxygen-sensing mechanism exists that regulates transcription of ctaDII and ctaC. Apparently, the intracellular oxygen concentration (as sensed by FnrP) was not the only signal to which the oxidase promoters responded. At given extracellular oxygen status, both the qox and the cco promoters responded to mutations in terminal oxidase genes, whereas the ctaDII and ctaC promoters did not. The change of electron distribution through the respiratory network, resulting from elimination of one or more oxidase genes, may have changed intracellular signals that affect the activities of the qox and cco promoters. On the other hand, the re-routing of electron distribution in the respiratory mutants hardly affected the oxygen consumption rate as compared to that of the wild-type. This suggests that the mutants adapted their respiratory network in such a way that they were able to consume oxygen at a rate similar to that of the wild-type strain.

The reduction state of the Q-pool regulates the electron flux through the branched respiratory network of Paracoccus denitrificans.

In this work we demonstrate how the reduction state of the Q-pool determines the distribution of electron flow over the two quinol-oxidising branches in Paracoccus denitrificans: one to quinol oxidase, the other via the cytochrome bc1 complex to the cytochrome c oxidases. The dependence of the electron-flow rate to oxygen on the fraction of quinol in the Q-pool was determined in membrane fractions and in intact cells of the wild-type strain, a bc1-negative mutant and a quinol oxidase-negative mutant. Membrane fractions of the bc1-negative mutant consumed oxygen at significant rates only at much higher extents of Q reduction than did the wild-type strain or the quinol oxidase-negative mutant. In the membrane fractions, dependence on the Q redox state was exceptionally strong corresponding to elasticity coefficients close to 2 or higher. In intact cells, the dependence was weaker. In uncoupled cells the dependence of the oxygen-consumption rates on the fractions of quinol in the Q-pool in the wild-type strain and in the two mutants came closer to that found for the membrane fractions. We also determined the dependence for membrane fractions of the wild-type in the absence and presence of antimycin A, an inhibitor of the bc1 complex. The dependence in the presence of antimycin A resembled that of the bc1-negative mutant. These results indicate that electron-flow distribution between the two quinol-oxidising branches in P. denitrificans is not only determined by regulated gene expression but also, and to a larger extent, by the reduction state of the Q-pool.

Staphylococcus aureus small colony variants are induced by the endothelial cell intracellular milieu.

Recent studies have reported that Staphylococcus aureus small colony variants (SCVs) can cause highly persistent infections in humans and in cultured endothelial cells. To understand the process by which SCVs of S. aureus appear in subjects who have not received antibiotic treatment, bovine endothelial cells were coincubated with a wild S. aureus strain for 72 h in the presence of lysostaphin. Intracellular bacteria were harvested and screened for stable SCVs. Intracellular bacteria developed the SCV phenotype at a greater rate than control bacteria not exposed to endothelial cells: The intracellular induction rate was approximately 10(-3) versus a spontaneous rate of <10(-7). This observation suggest that SCVs are induced by the intracellular milieu and suggest a possible mechanism for the intriguing pathophysiology of tissue persistence of staphylococci.