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.

FIGURE 1.

Exported proteins from aerobic Firmicutes largely exclude cysteines, whereas those from Actinobacteria favor paired cysteines. A, the schematics depict the protein characteristics used to classify the subproteomes. The SPase type I and II signal peptides (SP) and TM regions were used to identify and sort each proteome into its respective topology or subproteome. B, the exported proteins (secreted proteins and lipoproteins) from Actinobacteria show a profile suggestive of disulfide bond formation, whereas those from Firmicutes preferentially exclude cysteines_. The subproteomes containing the exported proteins from Gram-positive bacteria were separated into two major cysteine incorporation patterns. All aerobic Gram-positive Actinobacteria possess an “even-numbered cysteine bias” (left panel) in their exported proteins, and the large majority of Gram-positive Firmicutes show a cysteine exclusion trend (right panel). Each line represents the cysteine distribution in the exported proteins from one bacterial proteome.

FIGURE 2.

Cysteine exclusion from the exported proteins, but not from the cytoplasmic proteins, of aerobic Firmicutes correlates with a substantial increase in protein length. A, the average lengths of the predicted cytoplasmic and exported proteins from the indicated class of bacteria are displayed. The exported proteins from aerobic and anaerobic Firmicutes have an average length that is 22 and 32% longer than their respective cytoplasmic proteins, whereas those from aerobic and anaerobic Actinobacteria have average lengths that are only 2 and 5% longer, respectively. B, aerobic Gram-positive bacteria have a unique distribution in their cytoplasmic proteins with respect to cysteine content. Each line represents the percentage of cytoplasmic proteins from the different classes of bacteria that possess the indicated number of cysteine residues. C, the majority of aerobic Gram-positive Firmicutes show a cysteine incorporation pattern in their cytoplasmic proteins that follows the cysteine exclusion trend (left panel) versus the semi-normal cysteine distribution (right panel) favored by Actinobacteria and anaerobic Firmicutes.

FIGURE 3.

Actinobacteria possess disulfide-bonded proteins. Actinobacteria (C. glutamicum) and Gram-negative Proteobacteria (E. coli) express proteins with both inter- and intramolecular disulfide bonds. Whole cell lysates from the indicated bacteria were separated by two-dimensional nonreducing/reducing SDS-PAGE and visualized by Coomassie Blue staining. Intramolecular disulfide-bonded proteins lie above the diagonal, and intermolecular disulfide-bonded proteins lie below the diagonal.

FIGURE 4.

Firmicutes have a unique tolerance for high levels of reductant. The reductant DTT severely impaired the growth of aerobic Gram-negative Proteobacteria and Actinobacteria, whereas aerobic Firmicutes were capable of growth even at high DTT concentrations (for additional species, see supplemental Fig. S9). Representative growth curves from three independent experiments are displayed as changes in OD_{600 nm}. The indicated bacterial strains were grown in brain-heart infusion medium in the presence of 0, 1, 5, 10, and 20 mm DTT (E. coli and C. glutamicum) or 0, 10, 20, 80, and 100 mm DTT (S. aureus).

FIGURE 5.

The Actinobacterium C. glutamicum possesses two secreted dimeric oxidoreductases. A, schematic representations and characteristics of the three putative oxidoreductases from C. glutamicum and DsbA from E. coli. The recombinant proteins were expressed with an N-terminal His tag replacing the signal peptide. The amino acid numbering refers to the unprocessed wild-type proteins, and the active-site Cys-X-X-Cys motifs are displayed. B, Coomassie Blue-stained gel of the recombinant proteins that were purified following expression in E. coli. C, size exclusion profiles of the purified proteins separated on a Superdex 75 column and monitored by the absorbance at 280 nm. The relative size of each standard is indicated with an arrow. mAbs, milli-absorbance. D, the actinobacterial enzyme CG26 from C. glutamicum efficiently reduces insulin in vitro, whereas CG2799 shows slight activity. Each enzyme was analyzed for the capacity to reduce insulin at 27 °C by monitoring precipitation of the reduced B chain observed by changes in OD_{600 nm}. E, CG26 and CG2799 can correctly oxidize reduced RNase A in vitro. The ability of each enzyme to properly oxidize reduced RNase A (rRnase) in a glutathione redox buffer was assayed at 27 °C by monitoring the change in the absorbance at 296 nm that results from cleavage of cCMP by RNase A. The activities of native RNase A and reduced RNase A were included as controls.