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Endogenous Superoxide Is a Key Effector of the Oxygen Sensitivity of a Model Obligate Anaerobe

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Endogenous Superoxide Is a Key Effector of the Oxygen Sensitivity of a Model Obligate Anaerobe Endogenous superoxide is a key effector of the oxygen sensitivity of a model obligate anaerobe Zheng Lua,1, Ramakrishnan Sethua,1, and James A. Imlaya,2 aDepartment of Microbiology, University of Illinois, Urbana, IL 61801 Edited by Irwin Fridovich, Duke University Medical Center, Durham, NC, and approved March 1, 2018 (received for review January 3, 2018) It has been unclear whether superoxide and/or hydrogen peroxide fects (3, 4). Thus, these phenotypes confirmed the potential tox- play important roles in the phenomenon of obligate anaerobiosis. icity of reactive oxygen species (ROS), and they broadly supported This question was explored using Bacteroides thetaiotaomicron,a the idea that anaerobes might be poisoned by endogenous major fermentative bacterium in the human gastrointestinal tract. oxidants. Aeration inactivated two enzyme families—[4Fe-4S] dehydratases The metabolic defects of the mutant E. coli strains were sub- and nonredox mononuclear iron enzymes—whose homologs, in sequently traced to damage to two types of enzymes: dehy- contrast, remain active in aerobic Escherichia coli. Inactivation- dratases that depend upon iron-sulfur clusters and nonredox rate measurements of one such enzyme, B. thetaiotaomicron fu- enzymes that employ a single atom of ferrous iron (5–9). In both marase, showed that it is no more intrinsically sensitive to oxi- enzyme families, the metal centers are solvent exposed so that dants than is an E. coli fumarase. Indeed, when the E. coli they can directly bind and activate their substrates. Superoxide B. thetaiotaomicron enzymes were expressed in , they no longer and H2O2 are tiny molecules that cannot easily be excluded from could tolerate aeration; conversely, the B. thetaiotaomicron en- active sites, and they have high affinity for iron. The upshot is zymes maintained full activity when expressed in aerobic E. coli. that they directly ligand and oxidize the enzyme metal centers. Thus, the aerobic inactivation of the B. thetaiotaomicron enzymes The oxidized iron atoms dissociate, activity is lost, and the is a feature of their intracellular environment rather than of the pathways fail. B. thetaiotaomicron enzymes themselves. possesses superoxide Superoxide and H2O2 are continuously formed in aerobic cells dismutase and peroxidases, and it can repair damaged enzymes. because molecular oxygen adventitiously oxidizes redox enzymes However, measurements confirmed that the rate of reactive oxy- (10–12). Due to its substantial titers of scavenging enzymes, WT gen species production inside aerated B. thetaiotaomicron is far E. coli can suppress this threat. The question remains as to higher than in E. coli. Analysis of the damaged enzymes recovered whether these ROS poison obligate anaerobes. Among the from aerated B. thetaiotaomicron suggested that they had been bacteria whose oxygen sensitivity has received particular atten- inactivated by superoxide rather than by hydrogen peroxide. Ac- tion are members of the Bacteroidetes (13–18). These carbohy- cordingly, overproduction of superoxide dismutase substantially drate fermenters are among the dominant bacteria in the protected the enzymes from aeration. We conclude that when this mammalian gut (19), where they grow alongside E. coli. How- anaerobe encounters oxygen, its internal superoxide levels rise ever, in contrast to E. coli, Bacteroides species quickly stop high enough to inactivate key catabolic and biosynthetic enzymes. growing upon aeration. Notably, they do so despite possessing a Superoxide thus comprises a major element of the oxygen sensi- substantial retinue of SOD, catalase, and peroxidases (16, 20– tivity of this anaerobe. The extent to which molecular oxygen 22). Product analysis of aerated Bacteroides thetaiotaomicron exerts additional direct effects remains to be determined. Significance oxidative stress | obligate anaerobiosis | Bacteroides | reactive oxygen species Microbes display profound differences in their tolerance for oxygen, and this trait organizes the structure of many micro- he phenomenon of obligate anaerobiosis is the most obvious bial communities. However, the molecular basis of oxygen Tnatural manifestation of oxidative stress. Many microorgan- sensitivity is not well understood. In this study we determined isms can only grow in anoxic places. This restriction is a domi- that Bacteroides thetaiotaomicron, an abundant member of nant factor in the organization of microbial ecosystems in soil the human intestinal flora, is incapacitated by superoxide and gut, where respiring organisms help to shield the majority of stress when it enters a fully oxic environment. The key differ- anaerobes from the encroachment of oxygen. In 1971, McCord ence from oxygen-tolerant bacteria lies not in its defensive et al. (1) published a survey of scavenging enzymes that implied a systems, nor in the nature of the affected enzymes, but in the possible cause of obligate anaerobiosis. In contrast to oxygen- rate of endogenous oxidant formation. Anaerobes thrive in tolerant microbes, the anaerobes that they examined contained oxygen-poor environments because they deploy low-potential little or no superoxide dismutase (SOD) or catalase—which electron-transfer pathways; these results suggest that an an- suggested that, upon aeration, these microbes would be poisoned cillary effect is the reactivity of these pathways with oxygen, − by superoxide (O2 ) or hydrogen peroxide (H2O2). The table thereby generating enough reactive oxygen species to pre- that was published has been widely circulated, and this correla- clude oxic growth. tion is still cited in textbooks as a likely explanation for obligate anaerobiosis. Author contributions: Z.L., R.S., and J.A.I. designed research, performed research, ana- In 1986, Carlioz and Touati (2) performed a key experimental lyzed data, and wrote the paper. test of the idea, by deleting the SOD genes from the facultative The authors declare no conflict of interest. bacterium Escherichia coli. The resultant mutant grew at normal This article is a PNAS Direct Submission. rates in the absence of oxygen, but upon aeration it exhibited a Published under the PNAS license. set of severe biosynthetic and catabolic defects. These included 1Z.L. and R.S. contributed equally to this work. deficiencies in the biosynthesis of eight amino acids plus an in- 2To whom correspondence should be addressed. Email: [email protected]. ability to use TCA-cycle substrates as carbon sources. Analogous This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mutants that lacked catalase and peroxidase were generated 1073/pnas.1800120115/-/DCSupplemental. much later, and these mutants exhibited many of the same de- Published online March 20, 2018. E3266–E3275 | PNAS | vol. 115 | no. 14 www.pnas.org/cgi/doi/10.1073/pnas.1800120115 Downloaded by guest on September 29, 2021 showed that stoppage of growth occurs concomitant with a loss Results PNAS PLUS of carbohydrate catabolism (15). Two enzymes in central me- When B. thetaiotaomicron cultures in rich medium were aerated, tabolism lose activity (Fig. 1): fumarase, a member of the iron- growth stopped after ∼40 min (Fig. 2). The static cells remained sulfur dehydratase family, and pyruvate:ferredoxin oxidoreduc- viable; when anoxia was restored hours later, growth resumed tase (PFOR), a key pyruvate-dissimilating enzyme that passes within minutes. We previously noted that the cessation of growth low-potential electrons toward hydrogen formation and/or NAD was accompanied by a diminution of glucose catabolism and the reduction. The fumarase bottleneck is marked by a cessation of parallel inactivation of fumarase and PFOR, key enzymes in succinate production and an unusual release of lactate. When central metabolism (15). Fumarase drew our attention because this injury was bypassed by the addition of exogenous fumarate, this enzyme belongs to the family of [4Fe-4S] dehydratases, some succinate production was restored, but the cell instead which are vulnerable to oxygen species that can oxidize their iron-sulfur clusters (5, 25–27). Assays revealed that two other excreted pyruvate, reflecting PFOR failure. Either block should members of this enzyme family, aconitase and isopropylmalate be enough to prohibit fermentative growth. − isomerase, also progressively lost activity when B. thetaiotaomicron In this study we tested whether O or H O might be in- 2 2 2 was aerated (Fig. 3). volved. Our immediate focus was drawn to fumarase, because its The other family known to be vulnerable to these oxidants vulnerability to ROS is well understood (5, 23, 24). We found comprises enzymes that use solvent-exposed ferrous iron atoms that aeration simultaneously inactivated other iron-sulfur dehy- to catalyze nonredox reactions (6, 7, 9). When E. coli is stripped dratases and mononuclear iron enzymes. These failures were not of its scavenging enzymes, both superoxide and H2O2 can oxidize due to any special sensitivity of the B. thetaiotaomicron enzymes, enzymic Fe(II) cofactors, triggering iron release, the loss of ac- which maintained activity when expressed in aerobic E. coli. tivity, and collapse of the processes to which these enzymes Instead, the cellular environment of aerated B. thetaiotaomicron contribute. We examined two such enzymes in B. thetaiotaomicron: is much more oxidizing than that of E. coli due to a much higher ribulose-5-phosphate 3-epimerase (Rpe) and peptide deformylase rate of endogenous ROS formation. Finally, analysis indicated (Pdf).
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