Do reactive oxygen species or does oxygen itself confer obligate anaerobiosis? The case of Bacteroides thetaiotaomicron

Abstract

Bacteroides thetaiotaomicron was examined to determine whether its obligate anaerobiosis is imposed by endogenous reactive oxygen species or by molecular oxygen itself. Previous analyses established that aerated B. thetaiotaomicron loses some enzyme activities due to a high rate of endogenous superoxide formation. However, the present study establishes that another key step in central metabolism is poisoned by molecular oxygen itself. Pyruvate dissimilation was shown to depend upon two enzymes, pyruvate:formate lyase (PFL) and pyruvate:ferredoxin oxidoreductase (PFOR), that lose activity upon aeration. PFL is a glycyl-radical enzyme whose vulnerability to oxygen is already understood. The rate of PFOR damage was unaffected by the level of superoxide or peroxide, showing that molecular oxygen itself is the culprit. The cell cannot repair PFOR, which amplifies the impact of damage. The rates of PFOR and fumarase inactivation are similar, suggesting that superoxide dismutase is calibrated so the oxygen- and superoxide-sensitive enzymes are equally sensitive to aeration. The physiological purpose of PFL and PFOR is to degrade pyruvate without disrupting the redox balance, and they do so using catalytic mechanisms that are intrinsically vulnerable to oxygen. In this way, the anaerobic excellence and oxygen sensitivity of B. thetaiotaomicron are two sides of the same coin.

Keywords: iron-sulfur clusters; oxidative stress; pyruvate:ferredoxin oxidoreductase; pyruvate:formate lyase.

Figure 1.. Primary pathways of glucose fermentation in B. thetaiotaomicron.

PEP: phosphoenolpyruvate, OAA, oxaloacetate; Fdx: ferredoxin; PFL, pyruvate-formate lyase; PFOR, pyruvate:ferredoxin oxidoreductase; Rnf, ferrodoxin:NAD oxidoreductase, H₂ase: hydrogenase.

Figure 2.. B. thetaiotaomicron cells stop growing upon exposure to oxygen, but resume growth when anoxia is restored.

Wildtype B. thetaiotaomicron (BT5482) cells were grown in anoxic BHIS (no cysteine) media. They were then diluted and aerated for 2.5 hours (white markers). Cells were then returned to the anaerobic chamber, washed and resuspended in anoxic BHIS (no cysteine) (black markers). Error bars represent the standard error of the mean of three biological replicates. In this and some subsequent figures, error bars might be obscured by the markers.

Figure 3.. Fumarase, plus either PFL or PFOR, are essential for growth on glucose.

(A) Exponentially growing cells were subcultured at time zero into anoxic glucose medium. Where indicated, the Δfum mutant was supplemented with fumarate. (B) Exponentially growing cells in BHIS medium were washed and suspended at time zero into anoxic glucose medium. Strains: parent (BT5482 Δtdk), Δpfor (MK508), Δpfl4738 (MK550), Δpfl2955 (MK494), Δfdx (MK532), Δrnf (MK520), Δhyd1834 (MK540), Δhyd3472 (MK500), Δpfl4738 Δpfl2955 (MK644), Δpfl4738 Δpfor (MK635), Δpfor Δpfl2955 (MK630), and Δfum (LZ62). Error bars represent the standard error of the mean of three biological replicates.

Figure 4.. Pyruvate dissimilation is split between PFL4738 and PFOR.

Cells were cultured overnight in defined glucose media, and the concentrations of accumulated fermentation products were determined. (A) Formate was determined by enzyme-linked assay. The data identify PFL4738 as the functional pyruvate:formate lyase. (B) The mean values of integrated peak areas from (B) for formate, succinate, pyruvate and acetate, derived from three experiments. High values of each column are shaded. (C) End products were determined by NMR analysis. The Δpfl4738 Δpfor and Δfum mutants are not included because they were unable to grow in this medium. The formate signal is amplified 13-fold here for better representation. Note that Δpfor and Δfdx strains produce more formate. Form., formate; succ., succinate; pyr., pyruvate; acet., acetate; std, DSS standard. Strains: parent (BT5482 Δtdk), Δpfor (MK508), Δpfl4738 (MK550), Δpfl2955 (MK494), Δfdx (MK532), Δrnf (MK520), Δhyd1834 (MK540), Δhyd3472 (MK500), Δpfl4738 Δpfl2955 (MK644), and Δpfor Δpfl2955 (MK630). Error bars represent the standard error of the mean of three biological replicates.

Figure 5.. The rate of PFOR inactivation in aerated cells is independent of the intracellular concentrations of H₂O₂ and superoxide.

Cells growing exponentially in anoxic BHIS media were washed, resuspended in glucose buffer containing chloramphenicol, and aerated. At intervals aliquots were returned to the anaerobic chamber and PFOR activity was assayed. Strains: WT (BT5482), Hpx⁻ (SM135), SOD⁻ (LZ01), and pSOD (LZ200). Error bars and values after ± represent the standard error of the mean of three biological replicates.

Figure 6.. Physiological (<10 μM) concentrations of H₂O₂ cannot inactivate purified PFOR.

All cell growth, protein purification and enzyme assays were performed in the anaerobic chamber; see Materials & Methods for details. (A) Exponentially growing Hpx⁻ (SM135) cells were washed with and resuspended in Tris buffer and incubated with H₂O₂ for 15 minutes at 4 oC.. PFOR activity was then assayed. (B) In vitro: Cell lysates were prepared from Hpx⁻ cells. Lysates were incubated with H₂O₂ for 15 minutes at 4 °C, and PFOR activity was assayed. (C) The purified B. thetaiotaomicron PFOR was incubated with H₂O₂ for 15 minutes at 4 °C and then assayed. Error bars represent the standard error of the mean of three biological replicates.

Figure 7.. Unlike fumarase, oxidized PFOR cannot be reactivated in vitro or in vivo.

WT (BT5482) cells were grown in anoxic BHIS (no cysteine). Cells were centrifuged, suspended in Tris buffer containing glucose and chloramphenicol, and divided among 3 flasks. These were aerated for 0, 30 min or 60 min. Samples were removed to the anaerobic chamber, and the activities of fumarase (A) and PFOR (B) were measured in the same lysate. To test in vivo reactivation, the aerated cells were washed and resuspended in anoxic BHIS containing chloramphenicol, and at different time points cells were lysed and the activities of PFOR and fumarase were measured. For in vitro reactivation, the anoxic lysates of aerated cells were incubated with 0.5 mM ferrous ammonium sulfate and 5 mM DTT for 30 or 90 minutes, and the enzyme activities were measured. This experiment is representative of multiple experiments with different aeration and reactivation times, all of which yielded the same results. Error bars represent standard error of two technical replicates.

Figure 8.. PFOR inactivation depends upon the concentration of oxygen.

Exponentially growing WT cells (BT5482) in anoxic BHIS were washed, suspended in Tris buffer containing glucose and chloramphenicol, and incubated with the indicated percent of air saturation for 1 hr at 37 °C in tightly closed bottles. Cells were then harvested and their PFOR activities determined. The dotted line represents prior data (Lu et al., 2018) showing that the fumarase inactivation by superoxide parallels PFOR inactivation by oxygen. Error bars represent the standard error of the mean of six biological replicates.

Figure 9.. None of the tested enzymes are the primary source of H₂O₂ production in B. thetaiotaomicron.

The Hpx⁻ strain (SM135) and its mutant derivatives were grown in BHIS media, washed, resuspended in Tris buffer containing glucose and chloramphenicol, and then aerated at 37 °C. The rate of H₂O₂ production was measured as described. Strains: parent (SM135 aka Hpx⁻), Hpx⁻ Δpfor (MK592), Hpx⁻ Δpfl4738 (MK556), Hpx⁻ Δpfl2955 (MK562), Hpx⁻ Δfdx (MK654), Hpx⁻ Δrnf (MK584), Hpx⁻ Δhyd1834 (MK570), Hpx⁻ Δhyd3472 (MK580). Error bars represent the standard error of the mean of three biological replicates.