Identification of the omega4400 regulatory region, a developmental promoter of Myxococcus xanthus.

Omega4400 is the site of a Tn5 lac insertion in the Myxococcus xanthus genome that fuses lacZ expression to a developmentally regulated promoter. Cell-cell interactions that occur during development, including C signaling, are required for normal expression of Tn5 lac omega4400. The DNA upstream of the omega4400 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 omega4400 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 beta-galactosidase production in cells containing Tn5 lac omega4400. 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 or sigC gene did not affect Tn5 lac omega4400 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 omega4400 promoter does contain the sequence 5'-CATCCCT-3' centered at -49 and the C-signal-dependent omega4403 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.

The Rhodobacter sphaeroides cytochrome c2 signal peptide is not necessary for export and heme attachment.

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.

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

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.

Expression of the Rhodobacter sphaeroides cytochrome c2 structural gene.

A Rhodobacter sphaeroides mutant (CYCA1) lacking cytochrome c2 (cyt c2) was previously constructed (T. J. Donohue, A. G. McEwan, S. Van Doren, A. R. Crofts, and S. Kaplan, Biochemistry, 27: 1918-1924, 1988) by a combination of in vivo and in vitro molecular genetic techniques. CYCA1 was incapable of photosynthetic growth (PS-); in this presentation, we show that chemoheterotrophically grown CYCA1 contained significant quantities of a high potential soluble c-type cytochrome(s) with an alpha band of approximately 554 nm which had previously gone undetected under these physiological conditions in wild-type cells. In addition, the PS- phenotype of CYCA1 can be complemented in trans with stable low-copy-number (approximately 5 to 9 per R. sphaeroides genome) broad-host-range plasmids containing the wild-type cyt c2 structural gene (cycA) and upstream regulatory sequences. cyt c2 and cycA-specific mRNA levels were elevated in both the wild type and CYCA1 derivatives harboring intact cycA genes in trans, presumably as a result of increased gene dosage. Although photosynthetically grown wild-type cells contained approximately twofold more cycA-specific transcripts than chemoheterotrophically grown cells, there was an approximately four- to sevenfold increase in cyt c2 levels under photosynthetic conditions. Similarly, complemented CYCA1 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 discussed in terms of possible posttranscriptional control over cyt c2.