Genetic analysis of disulfide isomerization in Escherichia coli: expression of DsbC is modulated by RNase E-dependent mRNA processing.
We designed a selection strategy for the isolation of Escherichia coli mutants exhibiting enhanced protein disulfide isomerase activity. The folding of a variant of tissue plasminogen activator (v-tPA), a protein containing nine disulfide bonds, in the bacterial periplasm is completely dependent on the level of disulfide isomerase activity of the cell. Mutations that increase this activity mediate the formation of catalytically active v-tPA, which in turn cleaves a p-aminobenzoic acid (PABA)-peptide adduct to release free PABA and thus allows the growth of an auxotrophic strain. Following chemical mutagenesis, a total of eight E. coli mutants exhibiting significantly higher disulfide isomerization activity, not only with v-tPA but also with two other unrelated protein substrates, were isolated. This phenotype resulted from significantly increased expression of the bacterial disulfide isomerase DsbC. In seven of the eight mutants, the upregulation of DsbC was found to be related to defects in RNA processing by RNase E, the rne gene product. Specifically, the genetic lesions in five mutants were shown to be allelic to rne, while an additional two mutants exhibited impaired RNase E activity due to lesions in other loci. The importance of mRNA stability on the expression of DsbC is underscored by the short half-life of the dsbC transcript, which was found to be only 0.8 min at 37 degrees C in wild-type cells but was two- to threefold longer in some of the stronger mutants. These results (i) confirm the central role of DsbC in disulfide bond isomerization in the bacterial periplasm and (ii) suggest a critical role for RNase E in regulating DsbC expression.
Zhan
Xiaoming
X
Institute for Cell and Molecular Biology, University of Texas Austin, Austin, Texas 78712, USA.
Gao
Junjun
J
Jain
Chaitanya
C
Cieslewicz
Michael J
MJ
Swartz
James R
JR
Georgiou
George
G
D004220:Disulfides / Q000378:metabolism*; D004722:Endoribonucleases / Q000235:genetics / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D007536:Isomerism; D019704:Protein Disulfide-Isomerases / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism*
D004220:Disulfides; D012333:RNA, Messenger; D004722:Endoribonucleases; C022537:ribonuclease E; D019704:Protein Disulfide-Isomerases