Transcriptional activation of the Rhodobacter sphaeroides cytochrome c2 gene P2 promoter by the response regulator PrrA

被引:47
作者
Comolli, JC [1 ]
Carl, AJ [1 ]
Hall, C [1 ]
Donohue, T [1 ]
机构
[1] Univ Wisconsin, Dept Bacteriol, Madison, WI 53706 USA
关键词
D O I
10.1128/JB.184.2.390-399.2002
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
Anoxygenic photosynthetic growth of Rhodobacter sphaeroides, a member of the a subclass of the class Proteobacteria, requires the response regulator PrrA. PrrA and the sensor kinase PrrB are part of a two-component signaling pathway that influences a wide range of processes under oxygen-limited conditions. In this work we characterized the pathway of transcription activation by PrrB and PrrA by purifying these proteins, analyzing them in vitro, and characterizing a mutant PrrA protein in vivo and in vitro. When purified, a soluble transmitter domain of PrrB (cPrrB) could autophosphorylate, rapidly transfer phosphate to PrrA, and stimulate dephosphorylation of phospho-PrrA. Unphosphorylated PrrA activated transcription from a target cytochrome c(2) gene (cycA) promoter, P2, which contained sequences from -73 to +22 relative to the transcription initiation site. However, phosphorylation of PrrA increased its activity since activation of cycA P2 was enhanced up to 15-fold by treatment with the low-molecular-weight phosphodonor acetyl phosphate. A mutant PrrA protein containing a single amino acid substitution in the presumed phosphoacceptor site (PrrA-D63A) was not phosphorylated in vitro but also was not able to stimulate cyA P2 transcription. PrrA-D63A also had no apparent in vivo activity, demonstrating that aspartate 63 is necessary both for the function of PrrA and for its phosphorylation-dependent activation. The cellular level of wild-type PrrA was negatively autoregulated so that less PrrA was present in the absence of oxygen, conditions in which the activities of many PrrA target genes increase. PrrA-D63A failed to repress expression of the prrA gene under anaerobic conditions, suggesting that this single amino acid change also eliminated PrrA function in vivo.
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页码:390 / 399
页数:10
相关论文
共 54 条
[1]  
AIBA H, 1989, J BIOL CHEM, V264, P8563
[2]   Expression of the fixR-nifA operon in Bradyrhizobium japonicum depends on a new response regulator, RegR [J].
Bauer, E ;
Kaspar, T ;
Fischer, HM ;
Hennecke, H .
JOURNAL OF BACTERIOLOGY, 1998, 180 (15) :3853-3863
[3]   Autophosphorylation, phosphotransfer, and DNA-binding properties of the RegB RegA two-component regulatory system in Rhodobacter capsulatus [J].
Bird, TH ;
Du, SY ;
Bauer, CE .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (23) :16343-16348
[4]   The Bacillus subtilis response regulator SpoOA stimulates transcription of the spoIIG operon through modification of RNA polymerase promoter complexes [J].
Bird, TH ;
Grimsley, JK ;
Hoch, JA ;
Spiegelman, GB .
JOURNAL OF MOLECULAR BIOLOGY, 1996, 256 (03) :436-448
[5]   In vitro activation and repression of photosynthesis gene transcription in Rhodobacter capsulatus [J].
Bowman, WC ;
Du, SY ;
Bauer, CE ;
Kranz, RG .
MOLECULAR MICROBIOLOGY, 1999, 33 (02) :429-437
[6]  
CHAMBERLIN M, 1983, METHOD ENZYMOL, V101, P540
[7]  
DAHL MK, 1992, J BIOL CHEM, V267, P14509
[8]   PLASMIDS RELATED TO THE BROAD HOST RANGE VECTOR, PRK290, USEFUL FOR GENE CLONING AND FOR MONITORING GENE-EXPRESSION [J].
DITTA, G ;
SCHMIDHAUSER, T ;
YAKOBSON, E ;
LU, P ;
LIANG, XW ;
FINLAY, DR ;
GUINEY, D ;
HELINSKI, DR .
PLASMID, 1985, 13 (02) :149-153
[9]   Regulated expression of a highly conserved regulatory gene cluster is necessary for controlling photosynthesis gene expression in response to anaerobiosis in Rhodobacter capsulatus [J].
Du, SY ;
Kouadio, JLK ;
Bauer, CE .
JOURNAL OF BACTERIOLOGY, 1999, 181 (14) :4334-4341
[10]   DNA binding characteristics of RegA -: A constitutively active anaerobic activator of photosynthesis gene expression in Rhodobacter capsulatus [J].
Du, SY ;
Bird, TH ;
Bauer, CE .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1998, 273 (29) :18509-18513