Copper Stress Causes an in Vivo Requirement for the Escherichia coli Disulfide Isomerase DsbC

Abstract

In Escherichia coli, the periplasmic disulfide oxidoreductase DsbA is thought to be a powerful but nonspecific oxidant, joining cysteines together the moment they enter the periplasm. DsbC, the primary disulfide isomerase, likely resolves incorrect disulfides. Given the reliance of protein function on correct disulfide bonds, it is surprising that no phenotype has been established for null mutations in dsbC. Here we demonstrate that mutations in the entire DsbC disulfide isomerization pathway cause an increased sensitivity to the redox-active metal copper. We find that copper catalyzes periplasmic disulfide bond formation under aerobic conditions and that copper catalyzes the formation of disulfide-bonded oligomers in vitro, which DsbC can resolve. Our data suggest that the copper sensitivity of dsbC ؊ strains arises from the inability of the cell to rearrange copper-catalyzed non-native disulfides in the absence of functional DsbC. Absence of functional DsbA augments the deleterious effects of copper on a dsbC ؊ strain, even though the dsbA ؊ single mutant is unaffected by copper. This may indicate that DsbA successfully competes with copper and forms disulfide bonds more accurately than copper does. These findings lead us to a model in which DsbA may be significantly more accurate in disulfide oxidation than previously thought, and in which the primary role of DsbC may be to rearrange incorrect disulfide bonds that are formed during certain oxidative stresses.Most periplasmic Escherichia coli proteins contain at least two cysteine residues and many are stable and active only when these cysteines form their native disulfide bond pairings (1). In E. coli, a family of thioldisulfide oxidoreductases ensures that periplasmic and secreted proteins form correct disulfide bonds. DsbA is the primary disulfide oxidant in the periplasm. It rapidly donates its disulfide directly to substrate proteins and oxidizes them (2). DsbA is believed to act as a relatively nonspecific oxidant, joining any two cysteines that approach each other (3). A dsbA Ϫ strain shows several in vivo phenotypes, including attenuated virulence and loss of motility, because of the absence of disulfide bonds in proteins involved in these pathways (4, 5). DsbC, a second periplasmic thiol-disulfide oxidoreductase, appears to function as a disulfide isomerase both in vitro and in vivo. In vitro, DsbC has been shown to rearrange non-native disulfides in well studied isomerization substrates such as BPTI and RNase A (6, 7). In vivo, DsbC is required for full activity of a handful of proteins containing at least one non-consecutive disulfide bond (1). We have found that the periplasmic proteins RNase I (four disulfides, one non-consecutive) and MepA (three disulfides, two non-consecutive) require DsbC for their stability and, in the case of RNase I, in vivo activity (1). Berkmen et al. (3) recently showed that the folding of Agp (three consecutive disulfides) becomes DsbC-dependent with the introduction of a non-consecutive disulfide bond. These results s...

Quality control of disulfide bond formation in pilus subunits by the chaperone FimC

“…Only when the entire type 1 pilus gene cluster was overexpressed via a plasmid in a dsbA deletion strain could type 1 pilus biogenesis still be observed, but at reduced levels compared to wild-type cells 23,24 . The latter finding does not contradict the dependence on DsbA of type 1 pilus biogenesis in wild-type E. coli strains and may be explained by partial oxidation of overexpressed pilus subunits by air oxygen, which is possibly catalyzed by traces of transition metals such as copper 45 . The periplasmic disulfide isomerase DsbC might also act as an oxidant under certain growth conditions 19,20,46 .…”

Periplasmic proteins encoded by VCA0261–0260 and VC2216 genes together with copA and cueR products are required for copper tolerance but not for virulence in Vibrio cholerae

“…Since there is increasing evidence to indicate that the periplasm is an important cellular compartment for copper homeostasis (Bagai et al, 2008;Egler et al, 2005;Hiniker et al, 2005;Macomber et al, 2007), elucidating the involvement of VcCot (VCA0261-0260) and Vc-CopG (VC2216) homologues in V. cholerae copper tolerance represented an interesting topic of study. The evaluation of the role of VC2215 and VC0974 genes in copper resistance was also interesting, since their original annotations were only based on sequence similarity.…”

Copper-responsive gene regulation in bacteria

“…When in excess, however, copper is highly toxic to living cells, as it interacts with free proteinogenic thiol groups, destabilizes iron-sulfur cofactors, competes with other metals for protein binding sites, and possibly leads to formation of reactive oxygen species (Chillappagari et al, 2010;Hiniker et al, 2005;Macomber & Imlay, 2009). Remarkably, disturbance of copper homeostasis is thought to lead to human diseases such as Alzheimer's disease, Parkinson's disease, Wilson's disease and Menkes syndrome (Barnham & Bush, 2008;Lenartowicz et al, 2011;Lutsenko, 2010).…”