Genetic evidence for the role of isocytochrome c2 in photosynthetic growth of Rhodobacter sphaeroides Spd mutants

Abstract

In Rhodobacter sphaeroides, cytochrome c2 (cyt c2)-deficient mutants are photosynthetically incompetent (PS-). However, mutations which suppress the photosynthetic deficiency (spd mutations) of cyt c2 mutants increase the levels of a cyt c2 isoform, isocyt c2. To determine whether isocyt c2 was required for photosynthetic growth of Spd mutants, we used TnS mutagenesis to generate a PS-mutant (TP39) that lacks both cyt c2 and isocyt c2. DNA sequence analysis of wild-type DNA that restores isocyt c2 production and photosynthetic growth to TP39 indicates that it encodes the isocyt c2 structural gene, cycL. The Tn5 insertion in TP39 is -1.5 kb upstream of cycI, and our results show that it is polar onto cycL. The cycI gene has been physically mapped to a region of chromosome I that is -700 kb from the R. sphaeroides photosynthetic gene cluster. Construction of a defined cycI null mutant and complementation of several mutants with the cycI gene under the control of the cyt c2 promoter region indicate that an increase in the levels of isocyt c2 alone is necessary and sufficient for photosynthetic growth in the absence of cyt c2. The data are discussed in terms of the obligate role of isocyt c2 in cyt c2-independent photosynthesis of R. sphaeroides. sphaeroides DNA (10). The concentration of tetracycline used with pRK415-derived plasmids (15) was increased to 10 ,ug/ml for E. coli only. Trimethoprim, suspended in dimethylformamide, was used at 30 ,g/ml for R. sphaeroides and 100 ,ug/ml for E. coli. Spectinomycin was used at 25 ,g/ml for both R. sphaeroides and E. coli. For maintenance of pUC-derived plasmids, ampicillin was added to E. coli cultures at 50 ,ug/ml. R sphaeroides was grown in Sistrom's minimal medium A (16, 31) as previously described (8). Cell growth was measured turbidimetrically (35), and cultures were harvested during the exponential phase (6 x 108 to 1.5 x 109 cells per ml) (25). For photosynthetic growth on solid media, GasPak (BBL Microbiology Systems) jars with H2-CO2 generators were used.Transposon mutagenesis with pSUPTP5. Because the strain used for TnS mutagenesis contained a genomic kan gene (25), the use of wild-type TnS was not practical. Thus, a TnS Tpr derivative (28) was cloned as an -5.5-kb BamHI fragment into pSUP202 (30) to generate pSUPTP5. Control experiments showed that this derivative transposed with approximately the same frequency as wild-type TnS and that a spectrum of auxotrophic and pigment mutants could be obtained (lla).To isolate an isocyt c2 mutant, pSUPTP5 was mobilized into R sphaeroides CYCA65R7 (25) to generate independent transposition events. Briefly, -40 ml of an exponentialphase CYCA65R7 culture was harvested by centrifugation, and on top of this, -40 ml of a saturated S17-1(pSUPTP5) culture was collected. The cells were gently suspended in 4 ml of Sistrom's medium, and 120 RI was spread onto a series of LB plates. After incubation for 3 h at 32°C, the cells were replica plated to Sistrom's medium containing trimethoprim and incubated under aerobic conditions. Fro...

Mobile Cytochrome c 2 and Membrane-Anchored Cytochrome c y Are Both Efficient Electron Donors to the cbb 3 - and aa 3 -Type Cytochrome c Oxidases during Respiratory Growth of Rhodobacter sphaeroides

“…The data indicated that R. sphaeroides mutants carrying a cycA knockout allele had about 1/10 of the 550-nm absorption peak found in a wild-type strain. The absorption maximum of the remaining ascorbate-reducible ctype cyt was shifted to 552 nm, consistent with it being isocyt c 2 (33). Moreover, supernatants of the cyt bc 1 Ϫ mutant BC17 contained about threefold more cyt c 2 than those of the wildtype strain Ga.…”

“…On the other hand, in R. sphaeroides, the Ps electron transport pathway can be mediated only by a mobile electron carrier like cyt c 2 (8) or its functional analogues such as isocyt c 2 (33) (Fig. 1).…”

Organization and expression of the Rhodobacter sphaeroides cycFG operon

“…1B). Therefore, cycFG is at least 480 kb from the photosynthetic gene cluster which contains the gene for cyt c 2 (42) and ϳ190 kb from the operon that encodes isocytochrome c 2 (38). Genes encoding respiratory complex subunits for the cytochrome a/a 3 and b/c 1 complexes (42) are more than 800 kb from cycFG.…”

Link between the Membrane-Bound Pyridine Nucleotide Transhydrogenase and Glutathione-Dependent Processes in Rhodobacter sphaeroides

“…This screen identified three independent mutants, two of which (MS21 and MS65) are analyzed in this study (Table 2). Pulsed-field electrophoresis mapped the Tn5 insertions in MS21 and MS65 to the same region (coordinate ϳ2970) of R. sphaeroides chromosome (data not shown), which is ϳ200 kb away from the GSH-FDH structural gene (39). Assays for GSH-FDH activity showed that both MS21 and MS65 contained wild-type levels of this enzyme (Table 3).…”