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.
Brandner
J P
JP
Department of Bacteriology, University of Wisconsin, Madison 53706.
McEwan
A G
AG
Kaplan
S
S
Donohue
T J
TJ
D003001:Cloning, Molecular; D003574:Cytochrome c Group / Q000096:biosynthesis / Q000235:genetics*; D045362:Cytochromes c2; D004274:DNA, Recombinant / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D012242:Rhodobacter sphaeroides / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D003574:Cytochrome c Group; D004274:DNA, Recombinant; D012333:RNA, Messenger; D045362:Cytochromes c2