ABSTRACT
The non-native oxidation of horse heart myoglobin with hydrogen peroxide produces compound II which autoreduces by utilizing an internal oxidation site. Here, we utilize full UV-visible time-dependent kinetics with global kinetic singular value decomposition analysis to explore the mechanism and uncover more detail about the high-valent heme spectral features. By varying the hydrogen peroxide and myoglobin concentration, we were able to uncover more detailed spectra of myoglobin compound II and the autoreduction rate under several different pH conditions. The compound II spectra demonstrate pH-dependent features with an inflection point around pH 5.7 ± 0.1. The rate of autoreduction of compound II, k 2, increases with lower pH with a half-power proton dependence and no indication of a pK a > 3.9 ± 0.2, indicating that the autoreduction is still dependent on the protonation of the ferryl oxo species. The k 2 also demonstrates both hydrogen peroxide and myoglobin dependency. At myoglobin concentrations greater than 6.6 µM, the k 2 is myoglobin-independent, but for lower concentrations, a pH-sensitive concentration dependence is seen.
ABSTRACT
We have constructed and structurally characterized a Pseudomonas aeruginosa azurin mutant Re126WWCuI , where two adjacent tryptophan residues (W124 and W122, indole separation 3.6-4.1 Å) are inserted between the CuI center and a Re photosensitizer coordinated to the imidazole of H126 (ReI(H126)(CO)3(4,7-dimethyl-1,10-phenanthroline)+). CuI oxidation by the photoexcited Re label (*Re) 22.9 Å away proceeds with a â¼70 ns time constant, similar to that of a single-tryptophan mutant (â¼40 ns) with a 19.4 Å Re-Cu distance. Time-resolved spectroscopy (luminescence, visible and IR absorption) revealed two rapid reversible electron transfer steps, W124 â *Re (400-475 ps, K 1 â 3.5-4) and W122 â W124â¢+ (7-9 ns, K 2 â 0.55-0.75), followed by a rate-determining (70-90 ns) CuI oxidation by W122â¢+ ca. 11 Å away. The photocycle is completed by 120 µs recombination. No photochemical CuI oxidation was observed in Re126FWCuI , whereas in Re126WFCuI , the photocycle is restricted to the ReH126W124 unit and CuI remains isolated. QM/MM/MD simulations of Re126WWCuI indicate that indole solvation changes through the hopping process and W124 â *Re electron transfer is accompanied by water fluctuations that tighten W124 solvation. Our finding that multistep tunneling (hopping) confers a â¼9000-fold advantage over single-step tunneling in the double-tryptophan protein supports the proposal that hole-hopping through tryptophan/tyrosine chains protects enzymes from oxidative damage.
ABSTRACT
The first posttranslational modification step in the biosynthesis of the tryptophan-derived quinone cofactors is the autocatalytic hydroxylation of a specific Trp residue at position C-7 on the indole side chain. Subsequent modifications are catalyzed by modifying enzymes, but the mechanism by which this first step occurs is unknown. LodA possesses a cysteine tryptophylquinone (CTQ) cofactor. Metal analysis as well as spectroscopic and kinetic studies of the mature and precursor forms of a D512A LodA variant provides evidence that copper is required for the initial hydroxylation of the precursor protein and that if alternative metals are bound, the modification does not occur and the precursor is unstable. It is shown that the mature native LodA also contains loosely bound copper, which affects the visible absorbance spectrum and quenches the fluorescence spectrum that is attributed to the mature CTQ cofactor. When copper is removed, the fluorescence appears, and when it is added back to the protein, the fluorescence is quenched, indicating that copper reversibly binds in the proximity of CTQ. Removal of copper does not diminish the enzymatic activity of LodA. This distinguishes LodA from enzymes with protein-derived tyrosylquinone cofactors in which copper is present near the cofactor and is absolutely required for activity. Mechanisms are proposed for the role of copper in the hydroxylation of the unactivated Trp side chain. These results demonstrate that the reason that the highly conserved Asp512 is critical for LodA, and possibly all tryptophylquinone enzymes, is not because it is required for catalysis but because it is necessary for CTQ biosynthesis, more specifically to facilitate the initial copper-dependent hydroxylation of a specific Trp residue.
Subject(s)
Amino Acid Oxidoreductases/chemistry , Aspartic Acid/metabolism , Copper/metabolism , Dipeptides/metabolism , Indolequinones/metabolism , Tryptophan/metabolism , Amino Acid Oxidoreductases/metabolism , Aspartic Acid/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Copper/chemistry , Dipeptides/chemistry , Hydroxylation , Indolequinones/chemistry , Marinomonas/enzymology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrometry, Fluorescence , Tryptophan/chemistryABSTRACT
In the absence of its substrate, the autoreduction of the high-valent bis-FeIV state of the hemes of MauG to the diferric state proceeds via a Compound I-like and then a Compound II-like intermediate. This process is coupled to oxidative damage to specific methionine residues and inactivation of MauG. The autoreduction of a P107V MauG variant, which is more prone to oxidative damage, proceeds directly from the bis-FeIV to the Compound II-like state with no detectable Compound I intermediate. Comparison of the crystal structures of native and P107V MauG reveals that this mutation alters the positions of amino acid residues in the heme site as well as the water network that delivers protons from the solvent to the hemes during their reduction. Kinetic, spectroscopic, and solvent kinetic isotope effect studies demonstrate that these changes in the heme site affect the protonation state of the ferryl heme and the relative efficiencies of two alternative pathways for the transfer of protons from solvent to the hemes. These changes enhance the rate of autoreduction of P107V MauG such that it competes with the catalytic reaction with substrate and causes the enzyme to inactivate itself during the steady-state reaction with H2O2 and its substrate. Thus, while this mutation has negligible effects on the initial steady-state kinetic parameters of MauG, it is a fatal mutation as it causes inactivation during catalysis.
Subject(s)
Heme/chemistry , Mutation , Catalysis , Kinetics , Protons , Spectrum Analysis/methods , ThermodynamicsABSTRACT
GoxA is a glycine oxidase that possesses a cysteine tryptophylquinone (CTQ) cofactor that is formed by posttranslational modifications that are catalyzed by a modifying enzyme GoxB. It is the second known tryptophylquinone enzyme to function as an oxidase, the other being the lysine ϵ-oxidase, LodA. All other enzymes containing CTQ or tryptophan tryptophylquinone (TTQ) cofactors are dehydrogenases. Kinetic analysis of GoxA revealed allosteric cooperativity for its glycine substrate, but not O2 This is the first CTQ- or TTQ-dependent enzyme to exhibit cooperativity. Here, we show that cooperativity and homodimer stabilization are strongly dependent on the presence of Phe-237. Conversion of this residue, which is a Tyr in LodA, to Tyr or Ala eliminates the cooperativity and destabilizes the dimer. These mutations also significantly affect the kcat and Km values for the substrates. On the basis of structural and modeling studies, a mechanism by which Phe-237 exerts this influence is presented. Two active site residues, Asp-547 and His-466, were also examined and shown by site-directed mutagenesis to be critical for CTQ biogenesis. This result is compared with the results of similar studies of mutagenesis of structurally conserved residues of other tryptophylquinone enzymes. These results provide insight into the roles of specific active-site residues in catalysis and CTQ biogenesis, as well as describing an interesting mechanism by which a single residue can dictate whether or not an enzyme exhibits cooperative allosteric behavior toward a substrate.
Subject(s)
Amino Acid Oxidoreductases/chemistry , Amino Acid Oxidoreductases/metabolism , Dipeptides/biosynthesis , Gammaproteobacteria/enzymology , Indolequinones/biosynthesis , Amino Acid Oxidoreductases/genetics , Amino Acid Sequence , Catalysis , Conserved Sequence , Dimerization , Dipeptides/chemistry , Gammaproteobacteria/chemistry , Gammaproteobacteria/genetics , Gammaproteobacteria/metabolism , Indolequinones/chemistry , Kinetics , Molecular Sequence Data , Sequence AlignmentABSTRACT
GoxA is a glycine oxidase bearing a protein-derived cysteine tryptophylquinone (CTQ) cofactor that is formed by posttranslational modifications catalyzed by a flavoprotein, GoxB. Two forms of GoxA were isolated: an active form with mature CTQ and an inactive precursor protein that lacked CTQ. The active GoxA was present as a homodimer with no detectable affinity for GoxB, whereas the precursor was isolated as a monomer in a tight complex with one GoxB. Thus, the interaction of GoxA with GoxB and subunit assembly of mature GoxA are each dependent on the extent of CTQ biosynthesis.
Subject(s)
Amino Acid Oxidoreductases/metabolism , Bacterial Proteins/metabolism , Dipeptides/metabolism , Indolequinones/metabolism , Marinomonas/metabolism , Amino Acid Oxidoreductases/chemistry , Amino Acid Oxidoreductases/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Dipeptides/chemistry , Escherichia coli/genetics , Indolequinones/chemistry , Marinomonas/chemistry , Marinomonas/genetics , Protein Interaction Maps , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
In the absence of its substrate, the auto-reduction of the high-valent bis-Fe(IV) state of the dihaem enzyme MauG is coupled to oxidative damage of a methionine residue. Transient kinetic and solvent isotope effect studies reveal that this process occurs via two sequential long-range electron transfer (ET) reactions from methionine to the haems. The first ET is coupled to proton transfer (PT) to the haems from solvent via an ordered water network. The second ET is coupled to PT at the methionine site and occurs during the oxidation of the methionine to a sulfoxide. This process proceeds via Compound I- and Compound II-like haem intermediates. It is proposed that the methionine radical is stabilized by a two-centre three-electron (2c3e) bond. This provides insight into how oxidative damage to proteins may occur without direct contact with a reactive oxygen species, and how that damage can be propagated through the protein.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Electron Transport/physiology , Heme/metabolism , Paracoccus denitrificans/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Bacterial Proteins/genetics , Electron Transport/genetics , Heme/chemistry , Models, Molecular , Oxidation-Reduction , Recombinant Proteins/genetics , Solvents/chemistryABSTRACT
The high-valent state of the diheme enzyme MauG exhibits charge-resonance (CR) stabilization in which the major species is a bis-Fe(IV) state with one heme present as Fe(IV)=O and the other as Fe(IV) with axial heme ligands provided by His and Tyr side chains. In the absence of its substrate, the high-valent state is relatively stable and returns to the diferric state over several minutes. It is shown that this process occurs in two phases. The first phase is redistribution of the resonance species that support the CR. The second phase is the loss of CR and reduction to the diferric state. Thermodynamic analysis revealed that the rates of the two phases exhibited different temperature dependencies and activation energies of 8.9 and 19.6 kcal/mol. The two phases exhibited kinetic solvent isotope effects of 2.5 and 2.3. Proton inventory plots of each reaction phase exhibited extreme curvature that could not be fit to models for one- or multiple-proton transfers in the transition state. Each did fit well to a model for two alternative pathways for proton transfer, each involving multiple protons. In each case the experimentally determined fractionation factors were consistent with one of the pathways involving tunneling. The percent of the reaction that involved the tunneling pathway differed for the two reaction phases. Using the crystal structure of MauG it was possible to propose proton-transfer pathways consistent with the experimental data using water molecules and amino acid side chains in the distal pocket of the high-spin heme.
Subject(s)
Electron Transport , Enzymes/metabolism , Ferric Compounds/chemistry , Protons , Enzymes/chemistry , Ion Transport , Kinetics , ThermodynamicsABSTRACT
The diheme enzyme MauG catalyzes a six-electron oxidation required for posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. One heme is low-spin with ligands provided by His205 and Tyr294, and the other is high-spin with a ligand provided by His35. The side chain methyl groups of Thr67 and Leu70 are positioned at a distance of 3.4Å on either side of His35, maintaining a hydrophobic environment in the proximal pocket of the high-spin heme and restricting the movement of this ligand. Mutation of Thr67 to Ala in the proximal pocket of the high-spin heme prevented reduction of the low-spin heme by dithionite, yielding a mixed-valent state. The mutation also enhanced the stabilization of the charge-resonance-transition of the high-valent bis-FeIV state that is generated by addition of H2O2. The rates of electron transfer from TTQ biosynthetic intermediates to the high-valent form of T67A MauG were similar to that of wild-type MauG. These results are compared to those previously reported for mutation of residues in the distal pocket of the high-spin heme that also affected the redox properties and charge resonance transition stabilization of the high-valent state of the hemes. However, given the position of residue 67, the structure of the variant protein and the physical nature of the T67A mutation, the basis for the effects of the T67A mutation must be different from those of the mutations of the residues in the distal heme pocket.
Subject(s)
Bacterial Proteins/chemistry , Ferric Compounds/chemistry , Ferrous Compounds/chemistry , Heme/chemistry , Hemeproteins/chemistry , Mutation/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Crystallography, X-Ray , Electron Transport , Ferric Compounds/metabolism , Ferrous Compounds/metabolism , Heme/genetics , Heme/metabolism , Hemeproteins/genetics , Hemeproteins/metabolism , Indolequinones/metabolism , Models, Molecular , Oxidation-Reduction , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Paracoccus denitrificans/genetics , Paracoccus denitrificans/growth & development , Paracoccus denitrificans/metabolism , Protein Processing, Post-Translational , Rhodobacter sphaeroides/genetics , Rhodobacter sphaeroides/growth & development , Rhodobacter sphaeroides/metabolism , Spectrum Analysis, Raman , Tryptophan/analogs & derivatives , Tryptophan/metabolismABSTRACT
Electron transfer (ET) through and between proteins is a fundamental biological process. The rates and mechanisms of these ET reactions are controlled by the proteins in which the redox centers that donate and accept electrons reside. The protein influences the magnitudes of the ET parameters, the electronic coupling and reorganization energy that are associated with the ET reaction. The protein can regulate the rates of the ET reaction by requiring reaction steps to optimize the system for ET, leading to kinetic mechanisms of gated or coupled ET. Amino acid residues in the segment of the protein through which long range ET occurs can also modulate the ET rate by serving as staging points for hopping mechanisms of ET. Specific examples are presented to illustrate these mechanisms by which proteins control rates of ET reactions.
Subject(s)
Electron Transport , Proteins/metabolism , Animals , Electrons , Humans , Kinetics , Models, Molecular , Proteins/chemistryABSTRACT
The 6×-Histidine tag which is commonly used for purification of recombinant proteins was converted to a catalytic redox-active center by incorporation of Co(2+). Two examples of the biological activity of this engineered protein-derived cofactor are presented. After inactivation of the natural diheme cofactor of MauG, it was shown that the Co(2+)-loaded 6×His-tag could substitute for the hemes in the H2O2-driven catalysis of tryptophan tryptophylquinone biosynthesis. To further demonstrate that the Co(2+)-loaded 6×His-tag could mediate long range electron transfer, it was shown that addition of H2O2 to the Co(2+)-loaded 6×His-tagged Cu(1+) amicyanin oxidizes the copper site which is 20Å away. These results provide proof of principle for this simple method by which to introduce a catalytic redox-active site into proteins for potential applications in research and biotechnology.
Subject(s)
Protein Engineering , Calcium/chemistry , Catalysis , Cobalt/chemistry , Models, Molecular , Oxidation-Reduction , Recombinant Proteins/chemistry , Tryptophan/biosynthesisABSTRACT
Transmission of M. ulcerans, the etiological agent of Buruli ulcer, from the environment to humans remains an enigma despite decades of research. Major transmission hypotheses propose 1) that M. ulcerans is acquired through an insect bite or 2) that bacteria enter an existing wound through exposure to a contaminated environment. In studies reported here, a guinea pig infection model was developed to determine whether Buruli ulcer could be produced through passive inoculation of M. ulcerans onto a superficial abrasion. The choice of an abrasion model was based on the fact that most bacterial pathogens infecting the skin are able to infect an open lesion, and that abrasions are extremely common in children. Our studies show that after a 90d infection period, an ulcer was present at intra-dermal injection sites of all seven animals infected, whereas topical application of M. ulcerans failed to establish an infection. Mycobacterium ulcerans was cultured from all injection sites whereas infected abrasion sites healed and were culture negative. A 14d experiment was conducted to determine how long organisms persisted after inoculation. Mycobacterium ulcerans was isolated from abrasions at one hour and 24 hours post infection, but cultures from later time points were negative. Abrasion sites were qPCR positive up to seven days post infection, but negative at later timepoints. In contrast, M. ulcerans DNA was detected at intra-dermal injection sites throughout the study. M. ulcerans was cultured from injection sites at each time point. These results suggest that injection of M. ulcerans into the skin greatly facilitates infection and lends support for the role of an invertebrate vector or other route of entry such as a puncture wound or deep laceration where bacteria would be contained within the lesion. Infection through passive inoculation into an existing abrasion appears a less likely route of entry.
Subject(s)
Buruli Ulcer/microbiology , Buruli Ulcer/transmission , Insect Bites and Stings/complications , Mycobacterium ulcerans/physiology , Skin/injuries , Skin/microbiology , Wound Infection/microbiology , Animals , Disease Models, Animal , Female , Guinea Pigs , Injections, Intradermal , Male , Mycobacterium ulcerans/growth & developmentABSTRACT
The di-heme enzyme MauG catalyzes the oxidative biosynthesis of a tryptophan tryptophylquinone cofactor on a precursor of the enzyme methylamine dehydrogenase (preMADH). Reaction of H2O2 with the diferric form of MauG, or reaction of O2 with diferrous MauG, forms the catalytic intermediate known as bis-Fe(IV), which acts as the key oxidant during turnover. The site of substrate oxidation is more than 40 Å from the high-spin heme iron where H2O2 initially reacts, and catalysis relies on radical hopping through an interfacial residue, Trp199 of MauG. In the absence of preMADH, the bis-Fe(IV) intermediate is remarkably stable, but repeated exposure to H2O2 results in suicide inactivation. Using mass spectrometry, we show that this process involves the oxidation of three Met residues (108, 114, and 116) near the high-spin heme through ancillary electron transfer pathways engaged in the absence of substrate. The mutation of a conserved Pro107 in the distal pocket of the high-spin heme results in a dramatic increase in the level of oxidation of these Met residues. These results illustrate structural mechanisms by which MauG controls reaction with its high-valent heme cofactor and limits uncontrolled oxidation of protein residues and loss of catalytic activity. The conservation of Met residues near the high-spin heme among MauG homologues from different organisms suggests that eventual deactivation of MauG may function in a biological context. That is, methionine oxidation may represent a protective mechanism that prevents the generation of reactive oxygen species by MauG in the absence of preMADH.
Subject(s)
Bacterial Proteins/metabolism , Heme/metabolism , Iron/metabolism , Paracoccus denitrificans/enzymology , Amino Acid Motifs , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Heme/chemistry , Iron/chemistry , Kinetics , Methionine/metabolism , Models, Molecular , Oxidation-Reduction , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Paracoccus denitrificans/chemistry , Paracoccus denitrificans/genetics , Paracoccus denitrificans/metabolismABSTRACT
BACKGROUND: Buruli ulcer is a skin disease often associated with proximity to certain water bodies in Africa. Much remains unknown about the reservoir and transmission of this disease. Previous studies have suggested that fish may concentrate Mycobacterium ulcerans, the etiological agent of the disease, in their gills and intestines and serve as passive reservoirs of the bacterium. The objective of this study was to test the hypothesis that fish and amphibians serve as natural reservoirs of M. ulcerans or other closely related mycolactone-producing mycobacteria. METHODS: Polymerase chain reaction targeting the enoyl reductase (ER) domain present in mlsA, which is required for mycolactone production, was used to screen water, fish, and amphibians from water bodies in Ghana for the presence of mycolactone-producing mycobacteria, and positive specimens were subjected to variable number tandem repeat (VNTR) typing. RESULTS: The use of VNTR typing revealed the presence of Mycobacterium liflandii in a tadpole and a fish, and M. ulcerans in an adult frog. Similarity percentage analysis (SIMPER) showed that the predatory cichlid Hemichromis bimaculatus was associated with ER-positive water bodies. No amphibian species or fish-feeding guild served as a reliable indicator of the presence of mycolactone-producing mycobacteria in a water body, and there was no significant difference between fish and amphibian positivity rates (P-value=0.106). There was a significant difference between water bodies in the total number of ER-positive specimens (P-value=0.0164). CONCLUSIONS: Although IS2404-positive tadpoles and fish have been reported, this is the first VNTR confirmation of M. ulcerans or M. liflandii in wild amphibian and fish populations in West Africa. Results from this study suggest that amphibians should be carefully examined as potential reservoirs for M. ulcerans in West Africa, and that H. bimaculatus may be useful as an indicator of habitats likely to support mycolactone-producing mycobacteria.
ABSTRACT
BACKGROUND: Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU). In West Africa there is an association between BU and residence in low-lying rural villages where aquatic sources are plentiful. Infection occurs through unknown environmental exposure; human-to-human infection is rare. Molecular evidence for M. ulcerans in environmental samples is well documented, but the association of M. ulcerans in the environment with Buruli ulcer has not been studied in West Africa in an area with accurate case data. METHODOLOGY/PRINCIPAL FINDING: Environmental samples were collected from twenty-five villages in three communes of Benin. Sites sampled included 12 BU endemic villages within the Ouheme and Couffo River drainages and 13 villages near the Mono River and along the coast or ridge where BU has never been identified. Triplicate water filtrand samples from major water sources and samples from three dominant aquatic plant species were collected. Detection of M. ulcerans was based on quantitative polymerase chain reaction. Results show a significant association between M. ulcerans in environmental samples and Buruli ulcer cases in a village (pâ=â0.0001). A "dose response" was observed in that increasing numbers of M. ulceran- positive environmental samples were associated with increasing prevalence of BU cases (R(2)â=â0.586). CONCLUSIONS/SIGNIFICANCE: This study provides the first spatial data on the overlap of M. ulcerans in the environment and BU cases in Benin where case data are based on active surveillance. The study also provides the first evidence on M. ulcerans in well-defined non-endemic sites. Most environmental pathogens are more broadly distributed in the environment than in human populations. The congruence of M. ulcerans in the environment and human infection raises the possibility that humans play a role in the ecology of M. ulcerans. Methods developed could be useful for identifying new areas where humans may be at high risk for BU.
Subject(s)
Buruli Ulcer/epidemiology , Environmental Microbiology , Mycobacterium ulcerans/isolation & purification , Animals , Benin/epidemiology , DNA, Bacterial/genetics , Geography , Humans , Prevalence , Real-Time Polymerase Chain Reaction , Rural PopulationABSTRACT
A novel mycolactone has been identified from Mycobacterium marinum infecting freshwater fish.
Subject(s)
Bacterial Toxins/chemistry , Mycobacterium marinum/chemistry , Animals , Bacterial Toxins/biosynthesis , Bacterial Toxins/isolation & purification , Fishes , Fresh Water , Macrolides , Molecular Structure , Mycobacterium marinum/metabolism , Seawater , StereoisomerismABSTRACT
Mycobacterium ulcerans, the causative agent of Buruli ulcer, is an emerging environmental bacterium in Australia and West Africa. The primary risk factor associated with Buruli ulcer is proximity to slow moving water. Environmental constraints for disease are shown by the absence of infection in arid regions of infected countries. A particularly mysterious aspect of Buruli ulcer is the fact that endemic and non-endemic villages may be only a few kilometers apart within the same watershed. Recent studies suggest that aquatic invertebrate species may serve as reservoirs for M. ulcerans, although transmission pathways remain unknown. Systematic studies of the distribution of M. ulcerans in the environment using standard ecological methods have not been reported. Here we present results from the first study based on random sampling of endemic and non-endemic sites. In this study PCR-based methods, along with biofilm collections, have been used to map the presence of M. ulcerans within 26 aquatic sites in Ghana. Results suggest that M. ulcerans is present in both endemic and non-endemic sites and that variable number tandem repeat (VNTR) profiling can be used to follow chains of transmission from the environment to humans. Our results suggesting that the distribution of M. ulcerans is far broader than the distribution of human disease is characteristic of environmental pathogens. These findings imply that focal demography, along with patterns of human water contact, may play a major role in transmission of Buruli ulcer.