Disulfide Bond Formation and Cysteine Exclusion in Gram-positive Bacteria

Abstract

Most secretion pathways in bacteria and eukaryotic cells are challenged by the requirement for their substrate proteins to mature after they traverse a membrane barrier and enter a reactive oxidizing environment. For Gram-positive bacteria, the mechanisms that protect their exported proteins from misoxidation during their post-translocation maturation are poorly understood. To address this, we separated numerous bacterial species according to their tolerance for oxygen and divided their proteomes based on the predicted subcellular localization of their proteins. We then applied a previously established computational approach that utilizes cysteine incorporation patterns in proteins as an indicator of enzymatic systems that may exist in each species. The Sec-dependent exported proteins from aerobic Gram-positive Actinobacteria were found to encode cysteines in an even-biased pattern indicative of a functional disulfide bond formation system. In contrast, aerobic Gram-positive Firmicutes favor the exclusion of cysteines from both their cytoplasmic proteins and their substantially longer exported proteins. Supporting these findings, we show that Firmicutes, but not Actinobacteria, tolerate growth in reductant. We further demonstrate that the actinobacterium Corynebacterium glutamicum possesses disulfide-bonded proteins and two dimeric Dsb-like enzymes that can efficiently catalyze the formation of disulfide bonds. Our results suggest that cysteine exclusion is an important adaptive strategy against the challenges presented by oxidative environments.

Functional Analysis of Paralogous Thiol-disulfide Oxidoreductases in Streptococcus gordonii

“…Screening for potential SdbA substrates was carried out using a protocol modified from Daniels et al (8). The S. gordonii proteome was downloaded from the UniProtKB database.…”

“…In contrast, little is known about disulfide bond formation in Gram-positive bacteria, which are predicted to have a lower prevalence of disulfide-bonded proteins (4,8). Although Grampositive DsbA homologs have been identified, few have demonstrated functions, and those that do appear to perform specialized functions in the cell.…”

“…Novel oxidases identified in these species include Mycobacterium tuberculosis DsbE and DsbF (15,16), which show greater homology to E. coli reductases than to DsbA, and a TDOR from Corynebacterium glutamicum, CG.0026, which is predicted to be widespread among the Actinobacteria (8). In vitro analyses have confirmed that these enzymes function as oxidases, although no in vivo functions or phenotypes have been identified (8,15,16).…”

“…Bioinformatic screens for TDORs among diverse groups of bacteria have noted that many anaerobic Firmicutes, including streptococci and clostridia, lack homologs to E. coli DsbA and DsbB (1,4,8). It has been proposed that these bacteria do not engage in disulfide bond formation; however, there are important examples of disulfide-bonded proteins produced by these species, including secreted toxins that contribute to virulence (17,18).…”

“…Emerging evidence suggests that Gram-positive Actinobacteria may use different types of TDORs, with low homology to E. coli DsbA, to form disulfide bonds (8,15,16). Novel oxidases identified in these species include Mycobacterium tuberculosis DsbE and DsbF (15,16), which show greater homology to E. coli reductases than to DsbA, and a TDOR from Corynebacterium glutamicum, CG.0026, which is predicted to be widespread among the Actinobacteria (8).…”