Expression of the Rhodobacter sphaeroides cytochrome c2 structural gene

Abstract

A Rhodobacter splaeroides mutant (CYCAl) lacking cytochrome c2 (cyt c2) was previouly comtructed (T. J. Donohue, A. G. McEwan, S. Van Doren, A. R. Crofts, and S. Kaplan, Biochemistry, 27: 1918-1924) by a combination of in vivo and in vitro moleular genetic techniques. CYCAl was inc b of photosynthetic growth (PS-); in this presentation, we show that chemoheterotrophically grown CYCAl contained si ant quantities of a high potential soluble c-type cytochrome(s) with an alpha band of -554nm which had previously gone undetected under these physiologkal conditions in wild-type cells. In addition, the PS phenotype of CYCAl can be complemented in trans with stable low-copy-number (-5 to 9 per R. spkaeroides genome) broad-host-range plamis containing the wid-type cyt c2 structural gene (cycA) and upstrem regulatory sequences. cyt c2 and cycA-specific mRNA levels were elevated in both the wfld type and CYCAl derivatives harboring intact cycA genes in rans, presumably as a result of Increased gene dosage. Although photosyntheticafly grown wid-type cells contained approximately twofold more cycA-specific tr s t chemoheterotrophicaly grown cells, there was an approximately four-to sevenfold increase in cyt c2 leels under photosynthetic conditions. Simiarly, complemented CYCAl strains contained between 1.3-and 2.3-fold more cycA mRNA under photosynthetic conditions than under chemoheterotrophic conditions and had 6-to 12-fold higher steady-state levels of cyt c2 under the same physiological conditions. These data are dicu in tenus of possible posttranscriptional control over cyt c2.Rhodobacter sphaeroides is a purple nonsulfur photosynthetic bacterium possessing the capacity to grow aerobically, anaerobically in the light (34), or anaerobically in the dark in the presence of external electron acceptors such as dimethyl sulfoxide. During aerobic growth, the R. sphaeroides cell envelope resembles that of other gram-negative bacteria (13, 31), and energy is generated by a branched aerobic respiratory chain whose components are structurally and functionally similar to those of mitochondria (1,37,38). Anaerobiosis induces differentiation of the cell membrane through a process of invagination, resulting in synthesis of the intracytoplasmic membrane (20). The intracytoplasmic membrane, which is structurally continuous with but functionally distinct from the cell membrane, contains the pigment-protein complexes and redox components necessary for the capture of light energy and its conversion to cellular energy (10,20).Cytochrome c2 (cyt c2) is a soluble electron carrier located in the periplasm of R. sphaeroides (29) which is common to both the respiratory and the cyclic photosynthetic redox chains. In aerobically grown cells, cyt c2 transfers electrons from the membrane-bound ubiquinol-cyt c2 oxidoreductase (cyt b-cl) complex (15) to a terminal cyt a-a3 oxidase similar to that of mitochondria (16). Under photoheterotrophic conditions, cyt c2 functions to complete the cyclic photosynthetic redox chain (4, 28, 30) by transferring an ...

Regions of Rhodobacter sphaeroides cytochrome c2 required for export, heme attachment, and function

“…Western blots suggest that the fusion proteins are present at higher levels than wild-type cyt c2, which is consistent with the multicopy state of the RK2 plasmid encoding these fusions (5). However, the level of heme bound to the fusion proteins or their peroxidase activity is not consistent with the protein levels detected by immunoblot analysis.…”

“…Under aerobic conditions, cells containing plasmids pC2A11 to pC2Q119 grew with average doubling times of 3.0 + 0.9 h (data not shown), which is virtually identical to the generation time of wild-type cells (5). While wild-type cells have an -3.4-h photosynthetic generation time, strains containing plasmids pC2A11 to pC2Q119 had generation times of 4.1 to 6.7 h under these conditions (Fig.…”

“…The copy number of RK2-derived plasmids has been estimated to be -5 to 10 per R. sphaeroides genome (5,9). From the immunoblot analysis with cyt c2 antiserum, it is evident that the fusion proteins are being accumulated to higher levels than is the wild-type cyt C2 encoded by the 2.4.1 chromosome.…”

“…Therefore, all of the hybrids with a fusion junction downstream of the heme attachment site are substrates for covalent heme binding in vivo. The [3H]ALA-labeled proteins in the -14-kDa region are the multiple wild-type R. sphaeroides c-type cytochromes present in aerobically grown cells (5,35).…”

“…The order and cellular location of these processes 'are unknown and must be analyzed in more detail to define the cyt c2 biosynthetic pathway in molecular terms. A detailed molecular model for cyt c2 biosynthesis is also necessary to allow a thorough examination of those systems which may regulate cyt c2 synthesis at the posttranscriptional level (5).…”