Sequence analysis and transcriptional organization of the Rhodopseudomonas viridis cytochrome c2 gene

Abstract

The cytochrome c2 gene (cycA) of the purple nonsulfur bacterium Rhodopseudomonas viridis was isolated from a genomic library by using two degenerate oligonucleotides containing all possible DNA sequences predicted from the published amino acid sequence of this protein (Ambler et al., Proc. Natl. Acad. Sci. USA 73:472-475, 1976). Cloning and sequence analysis of the cytochrome c2 gene indicated the presence of a typical procaryotic 20-residue signal peptide, suggesting that this periplasmic protein in synthesized in vivo as a precursor. In addition, four amino acids were found to be different by comparing the published sequence of the mature protein with that deduced from the isolated cycA gene (Lys-14----Leu, Ser-46----Ala, Ile-84----Val, Leu-97----Ile). Northern (RNA) blot analysis and fine mapping of the 5' and 3' ends of the cycA gene transcript from photoheterotrophically grown R. viridis cells revealed one abundant transcript of 523 to 530 nucleotides in length, with the transcription start site at position -39 relative to the coding region of cytochrome c2. A low-abundance transcript with an extended 3' end (about 600 bases in length) is thought to be processed by exonucleases, resulting in the slightly shorter main transcript.

Genetics of Paracoccus denitrificans

“…It is known that the operon is controlled in an oxygen-dependent manner by a DNA-element which is found at around position -35 [2,[134][135][136]. Grisshamer has proposed a consensus sequence for the puf-operons in several purple bacteria, which seems responsible for the oxygen-dependent regulation [137]. As will be shown below, this result may well be extended to other genes and species.…”

“…Below a compilation of these ORE sequences is given separately. References: Pd/UEII [52]; Brj/hemA [126]; Rm/nodH [115]; Rhv/cycA [137]; Rhc/bchC [118]; Rhv/pufl [119]; Rhs/cycA [143]; Rhs/pue [132]; Rhc/pufQ [133,141]; Rhc/fbc [121]; Pd/fbc [31]; Rhr/fbc [122]; Rhv/fbc [123]; Pd/moxF [71]; Pd/Orf4 [53]; Brj/orf4 [65]; Pd/cycA [62]; Pd/ctaDI [53]; Pd/ctaDII [62]. Rhv = Rhodopseudomonas ciridis, Rhc = Rhodobacter capsulatus, Rhr = Rhodospirillium rubrum, Rhs = Rhodobacter sphaeroides, Pd = Paracoccus denitrificans, Brj = Bradyrhizobium japonicum, Rm = Rhizobium meliloti.…”

“…This has been confirmed when in the course of cloning the Paracoccus cycM gene the Rh. ciridis cyt c 2 gene [137] was successfully expressed in Paracoccus (Gerhus et al, in preparation). Chemotrophically grown Rh.…”

Synthesis of the Rhodopseudomonas viridis holo-cytochrome c2 in Paracoccus denitrificans

“…Paracoccus denitrificans was grown at 32°C on rich medium (tryptone/yeast extract/NaC1, 10 : 5 : 5 g 1-1), either aerobically with good rotary agitation, or anaerobically as a static culture under a layer of paraffin oil with the addition of 1% KNO 3. Plasmid-containing strains were supplemented with streptomycin, 25/xg ml For cell fractionation, the procedure of Witholt [13] was applied, which uses a brief lysozyme treatment in Tris-HCl buffer followed by a mild osmotic shock to liberate periplasmic proteins, After sedimentation of the spheroplasted cells and complete osmotic lysis, the soluble cytoplas- [9]) was ligated into the multiple cloning site (mcs) of the broad host range plasmid pEG400 and transferred into P. denitrificans PD1222 [15] by triple mating, as described earlier [14] to yield strain G480, For control purposes, strain G400 was constructed which contains the parental plasmid lacking any insert. mic fraction and the membrane fraction were separated by ultracentrifugation.…”

“…The gene coding for cytochrome c 2 from Rhodopseudomonas ~'iridis (cycA) has been sequenced and its transcripts mapped. The DNAderived amino acid sequence revealed the pres-cncc of a typical prokaryotic signal peptide, followed by the 107-residue stretch of the mature polypeptide [9]. As a prerequisite for site-directed mutagenesis experiments, the gene has been expressed in Escherichia coli under anaerobic conditions, and was found to be associated with the membrane fraction as a precursor form without attached heine moiety [10].…”