Inactivation of a single gene enables microaerobic growth of the obligate anaerobe Bacteroides fragilis Brian M. Meehana,1, Anthony D. Baughna,b,1, Rene Gallegosa, and Michael H. Malamya,2 aDepartment of Molecular Biology and Microbiology, Tufts University School of Medicine and Sackler School of Biomedical Sciences, Boston, MA 02111; and bDepartment of Microbiology, University of Minnesota, Minneapolis, MN 55455 Edited by Caroline S. Harwood, University of Washington, Seattle, WA, and approved June 14, 2012 (received for review March 5, 2012) Bacteroides fragilis can replicate in atmospheres containing anaerobe,” this mammalian commensal has since been found to ≤0.05% oxygen, but higher concentrations arrest growth by an grow in and benefit from nanomolar concentrations of oxygen unknown mechanism. Here we show that inactivation of a single (21). In addition, even though it cannot replicate in room air, gene, oxe (i.e., oxygen enabled) in B. fragilis allows for growth in this organism is capable of mounting a strong response to concentrations as high as 2% oxygen while increasing the toler- aeration (22, 23), including the production of numerous ROS- ance of this organism to room air. Known components of the oxi- scavenging enzymes (24–29) and other factors (30) that con- dative stress response including the ahpC, kat, batA-E,andtpx tribute to its impressive aerotolerance. The unique position of genes were not individually important for microaerobic growth. B. fragilis in the oxygen tolerance spectrum has allowed us to Δ However, a oxe strain scavenged H2O2 at a faster rate than WT, gain valuable insights into the nature of anaerobiosis. Herein indicating that reactive oxygen species may play a critical role in we characterize derivatives of this bacterium that, as a result limiting growth of this organism to low-oxygen environments. Clin- ofmutationofasinglegene(oxe), are capable of growth in ical isolates of B. fragilis displayed a greater capacity for growth as much as 2% oxygen, representing a 40-fold increase vs. WT under microaerobic conditions than fecal isolates, with some encod- B. fragilis. Additionally, an oxe mutant is even more tolerant of ing polymorphisms in oxe. Additionally, isolation of oxygen-en- room air than its WT counterpart, thus providing an intriguing abled mutants of Bacteroides thetaiotaomicron suggests that Oxe link between those bacteria that thrive in aerobic environments may mediate growth arrest of other anaerobes in oxygenated and those that do not. environments. MICROBIOLOGY Results superoxide | peroxidase | anaerobiosis Isolation of Oxygen-Enabled Mutants. Recent work has shown that B. fragilis can grow under atmospheres containing as much as hen cyanobacteria developed the ability to strip electrons 0.05% oxygen (500 nM dissolved O2) (21). Higher concen- ∼ Wfrom water 2.5 billion years ago, they set in motion trations of O2, however, inhibit growth in a manner independent a series of environmental changes that would have profound of increases in environmental redox potential (31). With these effects on the evolution of life on this planet. The release of observations in mind, we sought to isolate mutants of B. fragilis molecular oxygen (i.e., O2) as a byproduct of photosynthesis left capable of growth under normally restrictive oxygen concen- many microbes vulnerable to oxidative damage, caused in part by trations. When WT B. fragilis was plated under microaerobic the reactivity of key components of the metabolic networks used conditions (0.25–2% oxygen by volume), colonies were found to − by these organisms. Free iron and flavin-containing proteins (1– form at a frequency of ∼10 6 relative to an anaerobic control 5) can adventitiously donate electrons to oxygen to produce fi − (Table 1). Puri cation of these colonies revealed that they were reactive oxygen species (ROS) like superoxide (i.e., O2 ) or hy- capable of robust growth under microaerobic conditions, sug- drogen peroxide. These endogenously generated ROSs can gesting that a spontaneous mutation had somehow given rise to destroy important iron–sulfur clusters, inactivate key metabolic “oxygen-enabled” variants of B. fragilis. To gain insights into the enzymes, and cause DNA lesions that may culminate in death of molecular nature of this microaerobic growth, we tested whether the organism (6–13). Primordial microbes facing extinction in various mutations on the B. fragilis genome would alter the fre- this newly oxygenated environment were forced to retreat to quencies at which oxygen-enabled colonies arose. protected niches in reducing sediments or to evolve mechanisms B. fragilis encodes numerous oxidative stress defense enzymes of protection from oxidative stress. These defenses included the that play a crucial role in protecting the organism from the elaboration of more robust ROS detoxifying enzymes like su- damaging effects of ROS, and the expression of some of these peroxide dismutase (14), rubrerythrins, catalases, and perox- proteins is activated at the transcriptional level by OxyR (23). A idases (15), as well as metabolic enzymes fortified against ROS strain encoding a constitutively active allele of oxyR (32), as well damage (16–18). With such protective measures in place, these as a ΔoxyR strain, were found to give rise to colonies at the same bacteria could not only grow despite the presence of oxygen, but frequency as WT under microaerobic conditions (Table 1), in- could further evolve to harness the enormous energy-generating dicating that the OxyR-mediated oxidative stress response potential of this powerful electron acceptor. (OSR) is neither sufficient nor essential for the oxygen-enabled Obligately anaerobic bacteria cannot replicate in the presence phenotype. However, we reasoned that we might still be able to of oxygen, and present-day species may have evolved from the sediment-dwelling ancient microbes. For this reason, obligate anaerobiosis was once thought to be rooted in the lack of ROS- Author contributions: B.M.M., A.D.B., and M.H.M. designed research; B.M.M., A.D.B., R.G., detoxifying enzymes (19). Extensive evidence to the contrary has and M.H.M. performed research; B.M.M., A.D.B., R.G., and M.H.M. analyzed data; and B. since negated this hypothesis, but the molecular mechanisms M.M., A.D.B., and M.H.M. wrote the paper. underlying anaerobiosis have remained elusive. Although the The authors declare no conflict of interest. possibility remains that molecular oxygen itself can cause growth This article is a PNAS Direct Submission. 1 cessation in this class of organisms (20), a complete picture of O2 B.M.M. and A.D.B. contributed equally to this work. sensitivity has not yet taken shape. 2To whom correspondence should be addressed. E-mail: [email protected]. Bacteroides fragilis provides a convenient model for testing This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. theories of oxygen sensitivity. Previously classified as a “strict 1073/pnas.1203796109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1203796109 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 Table 1. Log10 efficiencies of plating for various strains of B. fragilis under microaerobic conditions Relevant Strain characteristics Log10 (efficiency of plating) ADB77 TM4000ΔthyA −5.9 ADB267 ADB77Δoxe −0.03 ADB266 ADB77ΔcydAB −6.3 BM37 ADB77Δkat −5.1 BM28 ADB77ΔahpC −5.0 IB263 638RoxyRC −6.3 MBD616 TM4000 thyA2ΔsodA −6.2 YT135 TM4000 batD::Tn4400′ −5.9 BM50 ADB77ΔahpCΔkat −5.7 BM118 ADB77ΔoxyR −5.9 BM95 ADB77Δtpx −4.9 BM131 ADB77 ΔahpCΔtpx −5.2 Fig. 1. Map of the oxe region of B. fragilis.(A) Diagram of the oxe gene BM105 ADB77ΔahpCΔkatΔtpx ≤8.7 (blue box). Predicted protein domains are shown below. Red arrows depict BM134 ADB77Δrubrerythrins −5.4 IS4400′ insertions. Shown in the green boxes are the mutations found in Δ − BM2 ADB77 rub 4.9 spontaneous oxygen-enabled mutants of ADB77, with nucleotide positions in parentheses. Orange boxes denote mutations found in oxygen-enabled Strains were grown anaerobically, serially diluted, and plated on BHIS. mutants from transposon-insertion pool. Purple boxes denote poly- Test plates were transferred to microaerobic conditions, and control plates morphisms in Oxe sequence from clinical strains. (B) Genomic region sur- were transferred to anaerobic conditions. After several days, counts (in cfu) rounding the oxe gene. nagB, glucosamine 6-P deaminase. were enumerated. Log10 (efficiency of plating) is defined as the log10 (num- ber of colonies arising in the presence of oxygen/number of colonies arising anaerobically). insertion (Fig. 1). Importantly, introduction of a plasmid encoding WT oxe into these strains prevented growth under suppress the formation of oxygen-enabled colonies by deleting microaerobic conditions, indicating that the O2-enabled pheno- enzymes crucial to the reduction of oxygen or its reactive inter- type was a result of the mutations in oxe. Additionally, four mediates. As cytochrome oxidase (cydAB) is used during isolates that arose from microaerobic plating of the WT B. fragilis nanaerobic growth of B. fragilis and appears to be the major strain were sequenced across the oxe locus. All were found to reducer of O2 under low-oxygen conditions (21), we hypothe- contain mutations in oxe (Fig. 1). sized that it would also play a crucial role in microaerobic growth. However, a ΔcydAB strain plated with the same low Δoxe Strain Plates at Unity in as Much as 2% Oxygen. Although both efficiency as WT under microaerobic conditions, as did strains WT and Δoxe strains plated with ∼100% efficiency in envi- missing catalase (kat), superoxide dismutase (sod), alkylhy- ronments containing ≤0.05% oxygen compared with their re- droperoxide reductase (ahpC), thioredoxin-dependent peroxi- spective anaerobic controls, they differed significantly in plating dase (tpx), or the Bacteroides aerotolerance BAT operon (30) efficiencies at higher O2 concentrations (Fig. 2). WT B. fragilis − (Table 1), suggesting that a fully functional OSR was not nec- gave rise to colonies at a frequency of ∼10 6 under 0.2% to 2% essary for the oxygen-enabled phenotype.