Isolation and characterization of Rhodobacter capsulatus mutants affected in cytochrome cbb3 oxidase activity

Abstract

The facultative phototrophic bacterium Rhodobacter capsulatus contains only one form of cytochrome (cyt) c oxidase, which has recently been identified as a cbb3-type cyt c oxidase. This is unlike other related species, such as Rhodobacter sphaeroides and Paracoccus denitrificans, which contain an additional mitochondrial-like aa3-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 cyt cbb3 oxidase. Sequence analysis of these mutants and of the plasmids complementing them revealed that ccoNOQP 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 cyt cbb3 oxidase, and unlike in Bradyrhizobium 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 cyt cbb3 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 cyt cbb3 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 cbb3 oxidase of R. capsulatus.

FIG. 1

Physical and genetic map of the plasmids p4A and p5T, complementing the R. capsulatus mutants M4 and M7G, respectively. The plasmids p4A1V, derived from p4A, and p5TΔH and pMG1, derived from p5T (see Materials and Methods), are also shown. The locations of ccoNO and ccoOQP are indicated. B, BamHI; Bg, BglII; Bs, BstEII; H, HindIII; X, XhoI; Sp, SphI.

FIG. 2

(A) TMBZ-stained SDS-PAGE analysis of c-type cyts from various R. capsulatus NADI⁻ mutants grown on enriched MPYE medium (100 μg of membrane proteins was loaded per lane). (B) Western blot analysis with anti-CcoN (subunit I) antibodies. After SDS-PAGE (10 μg of membrane proteins per lane) and electrophoretic transfer onto an Immobilon-P membrane. CcoN was detected with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G with NiCl₂-enhanced 3,3′-diaminobenzidine as the substrate. Cyts c_p and c_o are the subunits II and III of cyt cbb₃ oxidase, and cyts c₁ and c_y correspond to the cyt c₁ subunit of the bc₁ complex and the membrane-attached electron carrier c_y, respectively.

FIG. 3

Physical and genetic map of plasmid pOX15 carrying ccoNOQP. pOX15 was constructed by ligation of the 1.3-kb BamHI fragment of pMG1 into the BamHI site of p4AIV as described in Materials and Methods. The orientation of ORF277 and ccoNOQP and the location of the mutations in different EMS-induced or constructed NADI⁻ mutants of R. capsulatus are also indicated. Km, Tn5, Δ, and TGA correspond to the kanamycin resistance gene, transposon Tn5, deletion, and stop codon, respectively; arrowheads refer to the orientation of the insertion mutations.

FIG. 4

Comparison of the genetic organization of the cco(fix)NOQP operons and their flanking regions in different organisms. FNR indicates the presence of possible Fnr-binding sites in the intergenic regions upstream and downstream of the cco(fix)NOQP operons. The hisAFE genes are involved in histidine biosynthesis, and fnrL and fnrP are fnr-like genes most likely involved in oxygen- or redox-regulated expression of cyt cbb₃ oxidase. fixJ, fixK, and fixL code for oxygen response elements in B. japonicum. hemZ and hemN correspond to the genes encoding the anaerobic coporphyrinogen III oxidase. R.c., R. capsulatus; R.s., R. sphaeroides; P.d., P. denitrificans; B.j., B. japonicum. The bottom region (not labeled) represents R. meliloti.