The Disulfide Bond Formation Pathway Is Essential for Anaerobic Growth of Escherichia coli

Abstract

Disulfide bonds are critical to the stability and function of many bacterial proteins. In the periplasm of Escherichia coli, intramolecular disulfide bond formation is catalyzed by the two-component disulfide bond forming (DSB) system. Inactivation of the DSB pathway has been shown to lead to a number of pleotropic effects, although cells remain viable under standard laboratory conditions. However, we show here that dsb strains of E. coli reversibly filament under aerobic conditions and fail to grow anaerobically unless a strong oxidant is provided in the growth medium. These findings demonstrate that the background disulfide bond formation necessary to maintain the viability of dsb strains is oxygen dependent. LptD, a key component of the lipopolysaccharide transport system, fails to fold properly in dsb strains exposed to anaerobic conditions, suggesting that these mutants may have defects in outer membrane assembly. We also show that anaerobic growth of dsb mutants can be restored by suppressor mutations in the disulfide bond isomerization system. Overall, our results underscore the importance of proper disulfide bond formation to pathways critical to E. coli viability under conditions where oxygen is limited.IMPORTANCE While the disulfide bond formation (DSB) system of E. coli has been studied for decades and has been shown to play an important role in the proper folding of many proteins, including some associated with virulence, it was considered dispensable for growth under most laboratory conditions. This work represents the first attempt to study the effects of the DSB system under strictly anaerobic conditions, simulating the environment encountered by pathogenic E. coli strains in the human intestinal tract. By demonstrating that the DSB system is essential for growth under such conditions, this work suggests that compounds inhibiting Dsb enzymes might act not only as antivirulents but also as true antibiotics.KEYWORDS disulfide bonds, anaerobiosis, LptD, dsbC, dsbA, dsbB, disulfide bond, lptD I ntramolecular disulfide bonds are critical to the structural integrity of many proteins found in the bacterial cell envelope. Among these disulfide-stabilized proteins are phosphatases (1), toxins (2), components of the flagellar motor (3), and enzymes involved in the assembly of the outer membrane (4). While it was long thought that disulfide bonds formed spontaneously in aerobic environments due to background oxidation (5), it has been suggested that this spontaneous rate of disulfide formation is too low for many physiological processes. Enzymatic systems dedicated to catalyzing disulfide bond formation were identified first in prokaryotes and subsequently in eukaryotes (6, 7). Many Gram-negative bacteria, like Escherichia coli, utilize the disulfide bond forming (DSB) pathway for introducing disulfides into newly secreted proteins. In this pathway, two electrons are passed from substrate proteins to the periplasmic thioredoxin-like protein DsbA via a critical cysteine residue. This process gives ri...

Inhibition of Pseudomonas aeruginosa and Mycobacterium tuberculosis disulfide bond forming enzymes

“…Animal experiments with the dsbA1 mutant showed slightly larger defects in the lungs than the dsbB1B2 double mutant, while in the eye model both mutant strains were similarly affected. This is not surprising as it is well known that DsbA mutants confer greater defects in disulfide bond formation than DsbB mutants, presumably because some degree of oxidation of DsbA takes place in the presence of oxygen (Meehan et al , 2017a). These animal studies, along with the availability of a sensitive assay for screens, provide additional reason to consider that the search for inhibitors of bacterial DsbBs may yet yield candidates for antibacterial development.…”

“…In contrast, the Dsb pathway in E. coli is only essential under anaerobic growth (Meehan et al , 2017a).…”

“…Additionally, VKOR has been shown to be essential for growth of M. tuberculosis and M. smegmatis (Sassetti and Rubin, ; Dutton et al, ), likely because one or more essential proteins require disulfide bonds to be active. In contrast, the Dsb pathway in E. coli is only essential under anaerobic growth (Meehan et al , ).…”

Producing Gene Deletions in <em>Escherichia coli</em> by P1 Transduction with Excisable Antibiotic Resistance Cassettes

“…One such application is the production of deletion mutants in other E. coli strains. The Kan cassette from a given deletion clone can be mobilized by generally transducing bacteriophages, such as P1 10,11,12,13,14 . A phage stock prepared from such a strain can then be used to infect a recipient E. coli strain of interest, where at a low but reliable frequency the Kan cassette-containing region can be incorporated into the recipient genome by homologous recombination (Figure 2).…”

Disulfide Bond Formation in the Periplasm of Escherichia coli

“…Neither DsbA nor its partner, DsbB (see below), are essential for aerobic growth of E. coli (20), despite the fact that two proteins which are essential have disulfide bonds that are required for either their stability, FtsN (78), or folding, LptD (79). The pathway is essential for anaerobic growth, which also requires these two proteins, suggesting that aerobic growth may be dependent on back-ground oxidation sufficient for the activity of at least those two proteins (79). An explanation involving an alternative pathway is possible.…”