Isolation and Characterization of Rhodobacter capsulatus Mutants Affected in Cytochrome cbb ₃ Oxidase Activity

Abstract

The facultative phototrophic bacterium Rhodobacter capsulatus contains only one form of cytochrome (cyt)c oxidase, which has recently been identified as acbb3 -type cyt c oxidase. This is unlike other related species, such as Rhodobacter sphaeroides and Paracoccus denitrificans, which contain an additional mitochondrial-likeaa 3-type cyt c oxidase. An extensive search for mutants affected in cyt c oxidase activity in R. capsulatus led to the isolation of at least five classes of mutants. Plasmids complementing them to a wild-type phenotype were obtained for all but one of these classes from a chromosomal DNA library. The first class of mutants contained mutations within the structural genes (ccoNOQP) of the cytcbb 3 oxidase. Sequence analysis of these mutants and of the plasmids complementing them revealed thatccoNOQP in R. capsulatus is not flanked by the oxygen response regulator fnr, which is located upstream of these genes in other species. Genetic and biochemical characterizations of mutants belonging to this group indicated that the subunits CcoN, CcoO, and CcoP are required for the presence of an active cytcbb 3 oxidase, and unlike inBradyrhizobium japonicum, no active CcoN-CcoO subcomplex was found in R. capsulatus. In addition, mutagenesis experiments indicated that the highly conserved open reading frame 277 located adjacent to ccoNOQP is required neither for cytcbb 3 oxidase activity or assembly nor for respiratory or photosynthetic energy transduction in R. capsulatus. The remaining cyt c oxidase-minus mutants mapped outside of ccoNOQP and formed four additional groups. In one of these groups, a fully assembled but inactive cytcbb 3 oxidase was found, while another group had only extremely small amounts of it. The next group was characterized by a pleiotropic effect on all membrane-bound c-type cytochromes, and the remaining mutants not complemented by the plasmids complementing the first four groups formed at least one additional group affecting the biogenesis of the cyt cbb 3oxidase of R. capsulatus.

Ornithine lipid is required for optimal steady‐state amounts of c‐type cytochromes in Rhodobacter capsulatus

“…Rhodobacter capsulatus Nadimutants that are not complemented with ccoNOQP-ccoGHIS Screening of Nadi -(i.e. cyt c oxidase deficient) mutants that are not complemented with the ccoNOQP-ccoGHIS gene clusters, known to affect the cbb3-Cox activity, defined a new group of mutants, including MR2, IJ1 (Koch et al, 1998) and AYG4 (Table 1). These mutants exhibited complex phenotypic characteristics (Fig.…”

“…Next, ccoN::lacZ (pXG2, Koch et al, 1998) andcycY::lacZ (pHM12, Myllykallio et al, 1997) or cycY::phoA (pHM11, Myllykallio et al, 1997) fusions were used to probe whether or not the absence of the c-type cyts was Abbreviations of antibiotic resistances are as follows: Amp, ampicillin; Kan, kanamycine; Rif, rifampicin; Spe, spectinomycin; Tet, tetracycline.…”

“…All R. capsulatus strains, except Y262, are derived from MT1131. c. MT1131/pOX15 overproduces the wild-type cbb3-Cox by about fivefold (Koch et al, 1998). due to general transcription-translation or secretion defects. The b-galactosidase and alkaline phosphatase activities exhibited by appropriate wild-type or mutant transconjugants carrying these fusions were similar when cells were grown in MPYE under respiratory conditions ( Fig.…”

Cooperation between two periplasmic copper chaperones is required for full activity of the cbb3‐type cytochrome c oxidase and copper homeostasis in Rhodobacter capsulatus

“…Inverted cytoplasmic membranes from wild-type R. capsulatus (MT1131) and its mutants were prepared as described previously (Koch et al, 1998a;Pawlik et al, 2010). For Blue Native (BN) PAGE analyses, ICMs (100 mg of total protein) were incubated at 258C for 15 min with continuous shaking (550 rpm Eppendorf Thermomixer), in lysis buffer (50 mM NaCl, 50 mM imidazole/HCl, pH 7.0, 5 mM 6aminohexanoic acid) containing 1% n-dodecylmaltoside (Thermo Scientific Inc.).…”

Regulators of nonsulfur purple phototrophic bacteria and the interactive control of CO2assimilation, nitrogen fixation, hydrogen metabolism and energy generation

“…As would be predicted, anaerobic expression of cbb 3 oxidase was increased in the regB mutant, however, the expression level was 2-fold higher than that of the wild-type [98]. This discrepancy was explained when it was shown that FnrL positively regulate the ccoNOQP operon under anaerobic photosynthetic and semiaerobic conditions in both R. capsulatus and R. sphaeroides, possibly acting at a consensus FNRbinding sequence that is centered approximately 100 bp upstream of the ccoN transcription start in R. capsulatus [98,99] and 73.5 and 41.5 bp upstream of the FnrL-dependent P2 N transcription start in the R. sphaeroides cco promoter [37]. In an interesting twist, FnrL was shown to negatively regulate the R. capsulatus cydAB operon under semi-aerobic conditions leading to the speculation that in R. capsulatus, cbb 3 oxidase has a higher affinity for O 2 than quinol oxidase and is therefore, preferentially expressed under low O 2 conditions [98].…”