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 ...

Cytochrome c2 is a periplasmic redox protein involved in both the aerobic and photosynthetic electron transport chains of Rhodobacter sphaeroides. The process of cytochrome c2 maturation has been analyzed in order to understand the protein sequences involved in attachment of the essential heme moiety to the cytochrome c2 polypeptide and localization of the protein to the periplasm. To accomplish this, five different translational fusions which differ only in the cytochrome c2 fusion junction were constructed between cytochrome c2 and the Escherichia coli periplasmic alkaline phosphatase. All five of the fusion proteins are exported to the periplasmic space. The four fusion proteins that contain the NH2-terminal site of covalent heme attachment to cytochrome c2 are substrates for heme binding, suggesting that the COOH-terminal region of the protein is not required for heme attachment. Three of these hybrids possess heme peroxidase activity, which indicates that they are functional as electron carriers. Biological activity is possessed by one hybrid protein constructed five amino acids before the cytochrome c2 COOH terminus, since synthesis of this protein restores photosynthetic growth to a photosynthetically incompetent cytochrome c2-deficient derivative of R. sphaeroides. Biochemical analysis of these hybrids has confirmed CycA polypeptide sequences sufficient for export of the protein (A. R. Varga and S. Kaplan, J. Bacteriol. 171:5830-5839, 1989), and it has allowed us to identify regions of the protein sufficient for covalent heme attachment, heme peroxidase activity, docking to membrane-bound redox partners, or the capability to function as an electron carrier.

Rhodobacter sphaeroides cytochrome c2 (cyt c2) is a member of the heme-containing cytochrome c protein family that is found in the periplasmic space of this gram-negative bacterium. This exported polypeptide is made as a higher-molecular-weight precursor with a typical procaryotic signal peptide. Therefore, cyt c2 maturation is normally expected to involve precursor translocation across the cytoplasmic membrane, cleavage of the signal peptide, and covalent heme attachment. Surprisingly, synthesis as a precursor polypeptide is not a prerequisite for cyt c2 maturation because deleting the entire signal peptide does not prevent export, heme attachment, or function. Although cytochrome levels were reduced about threefold in cells containing this mutant protein, steady-state cyt c2 levels were significantly higher than those of other exported bacterial polypeptides which contain analogous signal peptide deletions. Thus, this mutant protein has the unique ability to be translocated across the cytoplasmic membrane in the absence of a signal peptide. The covalent association of heme with this mutant protein also suggests that the signal peptide is not required for ligand attachment to the polypeptide chain. These results have uncovered some novel aspects of bacterial c-type cytochrome biosynthesis.

Ω4400 is the site of a Tn5 lac insertion in theMyxococcus xanthus genome that fuses lacZexpression to a developmentally regulated promoter. Cell-cell interactions that occur during development, including C signaling, are required for normal expression of Tn5 lac Ω4400. The DNA upstream of the Ω4400 insertion has been cloned, the promoter has been localized, and a partial open reading frame has been identified. From the deduced amino acid sequence of the partial open reading frame, the gene disrupted by Tn5 lac Ω4400 may encode a protein with an ATP- or GTP-binding site. Expression of the gene begins 6 to 12 h after starvation initiates development, as measured by β-galactosidase production in cells containing Tn5 lacΩ4400. The putative transcriptional start site was mapped, and deletion analysis has shown that DNA downstream of −101 bp is sufficient for C-signal-dependent, developmental activation of this promoter. A deletion to −76 bp eliminated promoter activity, suggesting the involvement of an upstream activator protein. The promoter may be transcribed by RNA polymerase containing a novel sigma factor, since a mutation in the M. xanthus sigB orsigC gene did not affect Tn5 lac Ω4400 expression and the DNA sequence upstream of the transcriptional start site did not match the sequence of any M. xanthus promoter transcribed by a known form of RNA polymerase. However, the Ω4400 promoter does contain the sequence 5′-CATCCCT-3′ centered at −49 and the C-signal-dependent Ω4403 promoter also contains this sequence at the same position. Moreover, the two promoters match at five of six positions in the −10 regions, suggesting that these promoters may share one or more transcription factors. These results begin to define the cis-acting regulatory elements important for cell-cell interaction-dependent gene expression during the development of a multicellular prokaryote.

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