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The advent of the genomic era has opened up enormous possibilities for the discovery of new natural products. Also known as specialized metabolites, these compounds produced by bacteria, fungi, and plants have long been sought for their bioactive properties. Innovations in both DNA sequencing technologies and bioinformatics now allow the wealth of sequence data to be mined at both the genome and metagenome levels for new specialized metabolites. However, a key problem that remains is rapidly and efficiently linking these identified genes to their corresponding compounds. Within this review, we provide specific examples of studies that have used the power of genomic or metagenomic data to overcome these problems and identify new small molecules and their biosynthetic pathways.

Anaerobic bacteria have only recently been recognized as a source of antibiotics; yet, the metabolic potential of Negativicutes (Gram‐negative staining Firmicutes) such as the oak‐associated Dendrosporobacter quercicolus has remained unknown. Genome mining of D. quercicolus and phylogenetic analyses revealed a gene cluster for a type II polyketide synthase (PKS) complex that belongs to the most ancestral enzyme systems of this type. Metabolic profiling, NMR analyses, and stable‐isotope labeling led to the discovery of a new family of anthraquinone‐type polyphenols, the dendrubins, which are diversified by acylation, methylation, and dimerization. Dendrubin A and B were identified as strong antibiotics against a range of clinically relevant, human‐pathogenic mycobacteria., A path of polyketide ancestry: Genome mining revealed an ancestral type II polyketide synthase (PKS) system of the oak‐associated anaerobic bacterium Dendrosporobacter quercicolus, a member of the underexplored class of Negativicutes. Metabolic profiling led to the discovery of a new family of aromatic polyketides (dendrubins) that exhibit potent activities against a range of clinically relevant pathogenic mycobacteria (see figure).

Mosquito breeding sites are complex aquatic environments with wide microbial diversity and physicochemical parameters that can change over time during the development of immature insect stages. Changes in biotic and abiotic conditions in water can alter life-history traits of adult mosquitos but this area remains understudied. Here, using microbial genomic and metabolomics analyses, we explored the metabolites associated with Aedes aegypti breeding sites as well as the potential contribution of Klebsiella sp., symbiotic bacteria highly associated with mosquitoes. We sought to address whether breeding sites have a signature metabolic profile and understand the metabolite contribution of the bacteria in the aquatic niches where Ae. aegypti larvae develop. An analysis of 32 mosquito-associated bacterial genomes, including Klebsiella, allowed us to identify gene clusters involved in primary metabolic pathways. From them, we inferred metabolites that could impact larval development (e.g., spermidine), as well as influence the quality assessment of a breeding site by a gravid female (e.g., putrescine), if produced by bacteria in the water. We also detected significant variance in metabolite presence profiles between water samples representing a decoupled oviposition event (oviposition by single females and manually deposited eggs) versus a control where no mosquito interactions occurred (PERMANOVA: p < 0.05; R2 = 24.64% and R2 = 30.07%). Five Klebsiella metabolites were exclusively linked to water samples where oviposition and development occurred. These data suggest metabolomics can be applied to identify compounds potentially used by female Ae. aegypti to evaluate the quality of a breeding site. Elucidating the physiological mechanisms by which the females could integrate these sensory cues while ovipositing constitutes a growing field of interest, which could benefit from a more depurated list of candidate molecules.

Siderophores are key players in bacteria–host interactions, with the main function to provide soluble iron for their producers. Gramibactin from rhizosphere bacteria expands siderophore function and diversity as it delivers iron to the host plant and features an unusual diazeniumdiolate moiety for iron chelation. By mutational analysis of the grb gene cluster, we identified genes (grbD and grbE) necessary for diazeniumdiolate formation. Genome mining using a GrbD‐based network revealed a broad range of orthologous gene clusters in mainly plant‐associated Burkholderia/Paraburkholderia species. Two new types of diazeniumdiolate siderophores, megapolibactins and plantaribactin were fully characterized. In vitro assays and in vivo monitoring experiments revealed that the iron chelators also liberate nitric oxide (NO) in plant roots. This finding is important since NO donors are considered as biofertilizers that maintain iron homeostasis and increase overall plant fitness.

The bacterial endosymbiont (Burkholderia rhizoxinica) of the rice seedling blight fungus (Rhizopus microsporus) harbors a large number of cryptic biosynthesis gene clusters. Genome mining and sequence similarity networks based on an encoded nonribosomal peptide assembly line and the associated pyrrole-forming enzymes in the symbiont indicated that the encoded metabolites are unique among a large number of tentative pyrrole natural products in diverse and unrelated bacterial phyla. By performing comparative metabolic profiling using a mutant generated with an improved pheS Burkholderia counterselection marker, we found that the symbionts' biosynthetic pathway is mainly activated under salt stress and exclusively in symbiosis with the fungal host. The cryptic metabolites were fully characterized as novel pyrrole-substituted depsipeptides (endopyrroles). A broader survey showed that endopyrrole production is a hallmark of geographically distant endofungal bacteria, which produce the peptides solely under symbiotic conditions.

The nargenicin family of antibiotics are macrolides containing a rare ether-bridged cis-decalin motif. Several of these compounds are highly active against multi-drug resistant organisms. Despite the identification of the first members of this family almost 40 years ago, the genetic basis for the production of these molecules and the enzyme responsible for formation of the oxa bridge, remain unknown. Here, the 85 kb nargenicin biosynthetic gene cluster was identified from a human pathogenic Nocardia arthritidis isolate and this locus is solely responsible for nargenicin production. Further investigation of this locus revealed a putative iron-α-ketoglutarate-dependent dioxygenase, which was found to be responsible for the formation of the ether bridge from the newly identified deoxygenated precursor, 8,13-deoxynargenicin. Uncovering the nargenicin biosynthetic locus provides a molecular basis for the rational bioengineering of these interesting antibiotic macrolides.

2.AbstractIntroductionThe SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has necessitated the exploration of alternative diagnostic options to alleviate supply chain issues.AimTo establish and validate a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs.MethodologyWe used a commercial RT-LAMP mastermix from OptiGene Ltd in combination with a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A single-tube, single-step fluorescence assay was implemented whereby as little as 1 μL of universal transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing the requirement for RNA purification. Amplification and detection could be conducted in any thermocycler capable of holding 65°C for 30 minutes and measure fluorescence in the FAM channel at one-minute intervals.ResultsAssay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed assay sensitivity and specificity of 87% and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical samples of 14 minutes (SD ±7 minutes). Using dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 Tissue Culture Infectious Dose 50 per ml (TCID50 mL−1), with satisfactory assay sensitivity and specificity. A comparison of 20 clinical specimens between four laboratories showed excellent interlaboratory concordance; performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay.ConclusionWith a simplified workflow, N1-STOP-LAMP is a powerful, scalable option for specific and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium against COVID-19.3.Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

BackgroundMycobacterium ulcerans is the causative agent of a debilitating skin and soft tissue infection known as Buruli ulcer (BU). There is no vaccine against BU. The purpose of this study was to investigate the vaccine potential of two previously described immunogenic M. ulcerans proteins, MUL_3720 and Hsp18, using a mouse tail infection model of BU.MethodsRecombinant versions of the two proteins were each electrostatically coupled with a previously described lipopeptide adjuvant. Seven C57BL/6 and seven BALB/c mice were vaccinated and boosted with each of the formulations. Vaccinated mice were then challenged with M. ulcerans via subcutaneous tail inoculation. Vaccine performance was assessed by time-to-ulceration compared to unvaccinated mice.ResultsThe MUL_3720 and Hsp18 vaccines induced high titres of antigen-specific antibodies that were predominately subtype IgG1. However, all mice developed ulcers by day-40 post-M. ulcerans challenge. No significant difference was observed in the time-to-onset of ulceration between the experimental vaccine groups and unvaccinated animals.ConclusionsThese data align with previous vaccine experiments using Hsp18 and MUL_3720 that indicated these proteins may not be appropriate vaccine antigens. This work highlights the need to explore alternative vaccine targets and different approaches to understand the role antibodies might play in controlling BU.

A spider‐transmitted fungus (Rhizopus microsporus) that was isolated from necrotic human tissue was found to harbor endofungal bacteria (Burkholderia sp.). Metabolic profiling of the symbionts revealed a complex of cytotoxic agents (necroximes). Their structures were characterized as oxime‐substituted benzolactone enamides with a peptidic side chain. The potently cytotoxic necroximes are also formed in symbiosis with the fungal host and could have contributed to the necrosis. Genome sequencing and computational analyses revealed a novel modular PKS/NRPS assembly line equipped with several non‐canonical domains. Based on gene‐deletion mutants, we propose a biosynthetic model for bacterial benzolactones. We identified specific traits that serve as genetic handles to find related salicylate macrolide pathways (lobatamide, oximidine, apicularen) in various other bacterial genera. Knowledge of the biosynthetic pathway enables biosynthetic engineering and genome‐mining approaches., Spider sense: The fungus Rhizopus microsporus, isolated from necrotic tissue resulting from a spider bite, was found to contain bacteria (Burkholderia sp.) that produce a complex of highly cytotoxic compounds (necroximes). Analysis of the PKS/NRPS hybrid assembly line and genome mining revealed the molecular basis of the biosynthesis of V‐ATPase‐inhibiting antitumor agents from diverse biological origins.

Enzymes that cleave ATP to activate carboxylic acids play essential roles in primary and secondary metabolism in all domains of life. Class I adenylate-forming enzymes share a conserved structural fold but act on a wide range of substrates to catalyze reactions involved in bioluminescence, nonribosomal peptide biosynthesis, fatty acid activation, and β-lactone formation. Despite their metabolic importance, the substrates and catalytic functions of the vast majority of adenylate-forming enzymes are unknown without tools available to accurately predict them. Given the crucial roles of adenylate-forming enzymes in biosynthesis, this also severely limits our ability to predict natural product structures from biosynthetic gene clusters. Here we used machine learning to predict adenylate-forming enzyme function and substrate specificity from protein sequence. We built a web-based predictive tool and used it to comprehensively map the biochemical diversity of adenylate-forming enzymes across >50,000 candidate biosynthetic gene clusters in bacterial, fungal, and plant genomes. Ancestral enzyme reconstruction and sequence similarity networking revealed a ‘hub’ topology suggesting radial divergence of the adenylate-forming superfamily from a core enzyme scaffold most related to contemporary aryl-CoA ligases. Our classifier also predicted β-lactone synthetases in novel biosynthetic gene clusters conserved across >90 different strains ofNocardia. To test our computational predictions, we purified a candidate β-lactone synthetase fromNocardia brasiliensisand reconstituted the biosynthetic pathwayin vitroto link the gene cluster to the β-lactone natural product, nocardiolactone. We anticipate our machine learning approach will aid in functional classification of enzymes and advance natural product discovery.

The neglected tropical disease Buruli ulcer (BU) is an infection of subcutaneous tissue with Mycobacterium ulcerans. There is no effective BU vaccine. Here, we assessed an experimental prime-boost vaccine in a low-dose murine tail infection model. We used the enoyl-reductase (ER) domain of the M. ulcerans mycolactone polyketide synthases electrostatically coupled with a previously described TLR-2 agonist-based lipopeptide adjuvant, R4Pam2Cys. Mice were vaccinated and then challenged via tail inoculation with 14-20 colony forming units (CFU) of an engineered bioluminescent strain of M. ulcerans. Mice receiving either the experimental ER vaccine or Mycobacterium bovis Bacille Calmette-Guérin (BCG) were equally well protected, with both groups faring significantly better than non-vaccinated animals (p-γ produced in the spleen best predicted control of infection across all vaccine groups. Univariate logistic regression then revealed vaccine-specific profiles of protection. High titres of ER-specific IgG serum antibodies together with IL-2 and IL-4 in the draining lymph node (DLN) were associated with protection induced by the experimental ER vaccine. In contrast, high titres of IL-6, TNF-α, IFN-γ and IL-10 in the DLN and low IFNγ titres in the spleen were associated with protection following BCG vaccination. This study suggests an effective BU vaccine must induce localized, tissue-specific immune profiles with controlled inflammatory responses at the site of infection.

The nargenicin family of antibiotic macrolides comprise a group of bacterial natural products with a rare ether bridged cis-decalin moiety and a narrow spectrum of activity. Most family members were identified almost four decades ago and were placed on the shelf due to the numbers of broad-spectrum compounds available at the time. However, in light of rising rates of antimicrobial resistance, there has been a renewed interest in the use of narrow-spectrum antimicrobials. Here, we review the history of this family of compounds, including synthetic approaches, and highlight the recently uncovered genetic basis for nargenicin production. Given the renewed pharmaceutical interest in these compounds, we also investigate structure-activity relationships among these molecules, with a view to the future development of members of this unusual antibiotic family.

WalKR (YycFG) is the only essential two-component regulator in the human pathogen Staphylococcus aureus. WalKR regulates peptidoglycan synthesis, but this function alone does not explain its essentiality. Here, to further understand WalKR function, we investigate a suppressor mutant that arose when WalKR activity was impaired; a histidine to tyrosine substitution (H271Y) in the cytoplasmic Per-Arnt-Sim (PASCYT) domain of the histidine kinase WalK. Introducing the WalKH271Y mutation into wild-type S. aureus activates the WalKR regulon. Structural analyses of the WalK PASCYT domain reveal a metal-binding site, in which a zinc ion (Zn2+) is tetrahedrally-coordinated by four amino acids including H271. The WalKH271Y mutation abrogates metal binding, increasing WalK kinase activity and WalR phosphorylation. Thus, Zn2+-binding negatively regulates WalKR. Promoter-reporter experiments using S. aureus confirm Zn2+ sensing by this system. Identification of a metal ligand recognized by the WalKR system broadens our understanding of this critical S. aureus regulon., WalKR is an essential two-component regulator that controls peptidoglycan synthesis in the human pathogen Staphylococcus aureus. Here, the authors provide biochemical, structural, and functional evidence supporting that the binding of a zinc ion inhibits autophosphorylation and thus alters WalKR regulatory activity.

Metabolic profiling and genome mining revealed that anaerobic bacteria have the potential to produce acyloin natural products. In addition to sattazolin A and B, three new sattazolin congeners and a novel acyloin named clostrocyloin were isolated from three strains of Clostridium beijerinckii, a bacterium used for industrial solvent production. Bioactivity profiling showed that the sattazolin derivatives possess antimicrobial activities against mycobacteria and pseudomonads with only low cytotoxicity. Clostrocyloin was found to be mainly active against fungi. The thiamine diphosphate (ThDP)-dependent sattazolin-producing synthase was identified in silico and characterized both in vivo and in in vitro enzyme assays. A related acyloin synthase from the clostrocyloin producer was shown to be responsible for the production of the acyloin core of clostrocyloin. The biotransformation experiments provided first insights into the substrate scope of the clostrocyloin synthase and revealed biosynthetic intermediates.

Our inability to predict whether certain mutations will confer antibiotic resistance has made it difficult to rapidly detect the emergence of resistance, identify pre-existing resistant populations and manage our use of antibiotics to effective treat patients and prevent or slow the spread of resistance. Here we investigated the potential for resistance against the new antitubercular nitroimidazole prodrugs pretomanid and delamanid to emerge in Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). Deazaflavin-dependent nitroreductase (Ddn) is the only identified enzyme within M. tuberculosis that activates these prodrugs, via an F420H2-dependent reaction. We show that the native menaquinone-reductase activity of Ddn is important in aerobic respiration and essential for emergence from dormancy, which suggests that for resistance to spread and pose a threat to human health, the native activity of Ddn must be at least partially retained. We tested 75 unique mutations, including all known sequence polymorphisms identified among ~15,000 sequenced M. tuberculosis genomes. Several mutations abolished pretomanid activation in vitro, without causing complete loss of the native activity. We confirmed that a transmissible M. tuberculosis isolate from the hypervirulent Beijing family already possesses one such mutation and is resistant to pretomanid, even though it was never exposed to pretomanid. Notably, delamanid was still effective against this strain, which is consistent with structural analysis that indicates delamanid and pretomanid bind to Ddn differently. We suggest that the mutations identified in this work be monitored for informed use of delamanid and pretomanid treatment and to slow the emergence of resistance.

The bactericidal activity of isopropanol was determined against Enterococcus faecium ATCC 6057, ST 796 (isopropanol-tolerant strain) and Enterococcus hirae ATCC 10541 (EN 13727). Isopropanol at 60% and 70% were effective (≥5.38 log10-reduction) in 15 s against all strains but 23% isopropanol was not (

Atomic force microscopy measurements of capsule thickness revealed that that the wild-type Klebsiella pneumoniae AJ218 capsular polysaccharides were rearranged by exposure to colistin. The increase in capsule thickness measured near minimum inhibitory/bactericidal concentration (MIC/MBC) is consistent with the idea that colistin displaces the divalent cations that cross-bridge adjacent lipopolysaccharide (LPS) molecules through the capsule network. Cryo-electron microscopy demonstrated that the measured capsule thickness at near MIC/MBC of 1.2 μM was inflated by the disrupted outer membrane, through which the capsule is excreted and LPS is bound. Since wild-type and capsule-deficient strains of K. pneumoniae AJ218 have equivalent MICs and MBCs, the presence of the capsule appeared to confer no protection against colistin in AJ218. A spontaneously arising colistin mutant showed a tenfold increase in resistance to colistin; genetic analysis identified a single amino acid substitution (Q95P) in the PmrB sensor kinase in this colistin-resistant K. pneumoniae AJ218. Modification of the lipid A component of the LPS could result in a reduction of the net-negative charge of the outer membrane, which could hinder binding of colistin to the outer membrane and displacement of the divalent cations that bridge adjacent LPS molecules throughout the capsular polysaccharide network. Retention of the cross-linking divalent cations may explain why measurements of capsule thickness did not change significantly in the colistin-resistant strain after colistin exposure. These results contrast with those for other K. pneumoniae strains that suggest that the capsule confers colistin resistance.

BackgroundMycobacterium ulceransis the causative agent of a debilitating skin and soft tissue infection known as Buruli ulcer (BU). There is no vaccine against BU. The purpose of this study was to investigate the vaccine potential of two previously described immunogenicM. ulceransproteins, MUL_3720 and Hsp18, using a mouse tail infection model of BU.MethodsRecombinant versions of the two proteins were each electrostatically coupled with a previously described lipopeptide adjuvant. Seven C57BL/6 and seven BALB/c mice were vaccinated and boosted with each of the formulations. Vaccinated mice were then challenged withM. ulceransvia subcutaneous tail inoculation. Vaccine performance was assessed by time-to-ulceration compared to unvaccinated mice.ResultsThe MUL_3720 and Hsp18 vaccines induced high titres of antigen-specific antibodies that were predominately subtype IgG1. However, all mice developed ulcers by day-40 post-M. ulceranschallenge. No significant difference was observed in the time-to-onset of ulceration between the experimental vaccine groups and unvaccinated animals.ConclusionsThese data align with previous vaccine experiments using Hsp18 and MUL_3720 that indicated these proteins may not be appropriate vaccine antigens. This work highlights the need to explore alternative vaccine targets and different approaches to understand the role antibodies might play in controlling BU.

Our inability to predict which mutations could result in antibiotic resistance has made it difficult to rapidly identify the emergence of resistance, identify pre-existing resistant populations, and manage our use of antibiotics to effectively treat patients and prevent or slow the spread of resistance. Here we investigated the potential for resistance against the new antitubercular nitroimidazole prodrugs pretomanid and delamanid to emerge in Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). Deazaflavin-dependent nitroreductase (Ddn) is the only identified enzyme within M. tuberculosis that activates these prodrugs, via an F420H2-dependent reaction. We show that the native menaquinone-reductase activity of Ddn is essential for emergence from hypoxia, which suggests that for resistance to spread and pose a threat to human health, the native activity of Ddn must be at least partially retained. We tested 75 unique mutations, including all known sequence polymorphisms identified among ~15,000 sequenced M. tuberculosis genomes. Several mutations abolished pretomanid and delamanid activation in vitro, without causing complete loss of the native activity. We confirmed that a transmissible M. tuberculosis isolate from the hypervirulent Beijing family already possesses one such mutation and is resistant to pretomanid, before being exposed to the drug. Notably, delamanid was still effective against this strain, which is consistent with structural analysis that indicates delamanid and pretomanid bind to Ddn differently. We suggest that the mutations identified in this work be monitored for informed use of delamanid and pretomanid treatment and to slow the emergence of resistance., Author summary Bacterial pathogens often evolve resistance to antibiotics via mutations in the coding sequences of genes–frequently the target, an enzyme that metabolizes or transports the active drug, or an enzyme that activates a prodrug. In the case of tuberculosis, antibiotic resistance is a growing problem, with the rapid emergence of multi-drug resistant strains. New nitroimidazole-based antibiotic prodrugs, such as pretomanid and delamanid have the potential to help infected individuals, but we must guard against the evolution of resistance to these new compounds in Mycobacterium tuberculosis. In this report we use protein engineering to identify mutations that could potentially result in antibiotic resistance by knocking out the prodrug activating activity of the deazaflavin dependent nitroreductase (DDN), without completely abolishing its native menaquinone reductase activity. The retention of its native activity is important as DDN appears to be required for M. tuberculosis to emerge from hypoxia. Strikingly, when we analysed ~15,000 M. tuberculosis genomes from clinical strains, we identified several that harboured mutations that we identified as abolishing prodrug activation in vitro. A hypervirulent Beijing strain N0008 from Vietnam (which had not been exposed to pretomanid in the clinic) was predicted, and confirmed, to be resistant to pretomanid, revealing that resistance to this drug has arisen through genetic drift and not selective pressure in this instance. These data show that by testing potential resistant mutations in the laboratory before large-scale use of antibiotics, we should be able to use them more judiciously in order to slow the spread of resistance.

Bacterial outer membrane vesicles (OMVs) are extracellular sacs containing biologically active products, such as proteins, cell wall components and toxins. OMVs are reported to contain DNA, however, little is known about the nature of this DNA, nor whether it can be transported into host cells. Our work demonstrates that chromosomal DNA is packaged into OMVs shed by bacteria during exponential phase. Most of this DNA was present on the external surfaces of OMVs, with smaller amounts located internally. The DNA within the internal compartments of Pseudomonas aeruginosa OMVs were consistently enriched in specific regions of the bacterial chromosome, encoding proteins involved in virulence, stress response, antibiotic resistance and metabolism. Furthermore, we demonstrated that OMVs carry DNA into eukaryotic cells, and this DNA was detectable by PCR in the nuclear fraction of cells. These findings suggest a role for OMV-associated DNA in bacterial-host cell interactions and have implications for OMV-based vaccines.

WalKR (YycFG) is the only essential two-component regulator in the human pathogenStaphylococcus aureus.WalKR regulates peptidoglycan synthesis, but this function alone appears not to explain its essentiality. To understand WalKR function we investigated a suppressor mutant that arose when WalKR activity was impaired; a single histidine to tryptophan substitution (H271Y) in the cytoplasmic Per-Arnt-Sim (PASCYT) domain of the histidine kinase WalK. Introduction of the WalKH271Ymutation into wild-typeS.aureusactivated the WalKR regulon. Structural analyses of the WalK PASCYTdomain revealed a hitherto unknown metal binding site, in which a zinc ion (Zn2+) was tetrahedrally-coordinated by four amino acid residues including H271. The WallkH271Ymutation abrogated metal binding, increasing WalK kinase activity and WalR phosphorylation. Thus, Zn2+-binding negatively regulates WalKR activity. Identification of a metal ligand sensed by the WalKR system substantially expands our understanding of this criticalS.aureusregulon.

BackgroundBuruli ulcer is a disease of the skin and soft tissues caused by infection with a slow growing pathogen,Mycobacterium ulcerans. A vaccine for this disease is not available butM. ulceranspossesses a giant plasmid pMUM001 that harbours the polyketide synthase (PKS) genes encoding a multi-enzyme complex needed for the production of its unique lipid toxin called mycolactone, which is central to the pathogenesis of Buruli ulcer. We have studied the immunogenicity of enzymatic domains in humans withM. ulceransdisease, their contacts, as well as non-endemic areas controls.MethodsBetween March 2013 and August 2015, heparinized whole blood was obtained from patients confirmed with Buruli ulcer. The blood samples were diluted 1 in 10 in Roswell Park Memorial Institute (RPMI) medium and incubated for 5 days with recombinant mycolactone PKS domains and mycolyltransferase antigen 85A (Ag85A). Blood samples were obtained before and at completion of antibiotic treatment for 8 weeks and again 8 weeks after completion of treatment. Supernatants were assayed for interferon-γ (IFN-γ) and interleukin-5 (IL-5) by enzyme-linked immunosorbent assay. Responses were compared with those of contacts and non-endemic controls.ResultsMore than 80% of patients and contacts from endemic areas produced IFN-γ in response to all the antigens except acyl carrier protein type 3 (ACP3) to which only 47% of active Buruli ulcer cases and 71% of contacts responded. The highest proportion of responders in cases and contacts was to load module ketosynthase domain (Ksalt) (100%) and enoylreductase (100%). Lower IL-5 responses were induced in a smaller proportion of patients ranging from 54% after ketoreductase type B stimulation to only 21% with ketosynthase type C (KS C). Among endemic area contacts, the, highest proportion was 73% responding to KS C and the lowest was 40% responding to acyltransferase with acetate specificity type 2. Contacts of Buruli ulcer patients produced significantly higher IFN-γ and IL-5 responses compared with those of patients to PKS domain antigens and to mycolyltransferase Ag85A ofM. ulcerans. There was low or no response to all the antigens in non-endemic areas controls. IFN-γ and IL-5 responses of patients improved after treatment when compared to baseline results.DiscussionThe major response to PKS antigen stimulation was IFN-γ and the strongest responses were observed in healthy contacts of patients living in areas endemic for Buruli ulcer. Patients elicited lower responses than healthy contacts, possibly due to the immunosuppressive effect of mycolactone, but the responses were enhanced after antibiotic treatment. A vaccine made up of the most immunogenic PKS domains combined with the mycolyltransferase Ag85A warrants further investigation.

Plasmid vectors based on bacteriophage integrases are important tools in molecular microbiology for the introduction of foreign DNA, especially into bacterial species where other systems for genetic manipulation are limited. Site specific integrases catalyze recombination between phage and bacterial attachment sites (attP and attB, respectively) and the best studied integrases in the actinomycetes are the serine integrases from the Streptomyces bacteriophages ΦC31 and ΦBT1. As this reaction is unidirectional and highly stable, vectors containing phage integrase systems have been used in a number of genetic engineering applications. Plasmids bearing the ΦBT1 integrase have been used to introduce DNA into Streptomyces and Amycolatopsis strains; however, they have not been widely studied in other actinobacterial genera. Here, we show that vectors based on ΦBT1 integrase can stably integrate into the chromosomes of a range of Nocardia species, and that this integration occurs despite the absence of canonical attB sites in these genomes. Furthermore, we show that a ΦBT1 integrase-based vector can insert at multiple pseudo-attB sites within a single strain and we determine the sequence of a pseudo-attB motif. These data suggest that ΦBT1 integrase-based vectors can be used to readily and semi-randomly introduce foreign DNA into the genomes of a range of Nocardia species. However, the precise site of insertion will likely require empirical determination in each species to avoid unexpected off-target effects.

Addressing the transmission enigma of the neglected disease Buruli ulcer (BU) is a World Health Organization priority. In Australia, we have observed an association between mosquitoes harboring the causative agent, Mycobacterium ulcerans, and BU. Here we tested a contaminated skin model of BU transmission by dipping the tails from healthy mice in cultures of the causative agent, Mycobacterium ulcerans. Tails were exposed to mosquito (Aedes notoscriptus and Aedes aegypti) blood feeding or punctured with sterile needles. Two of 12 of mice with M. ulcerans contaminated tails exposed to feeding A. notoscriptus mosquitoes developed BU. There were no mice exposed to A. aegypti that developed BU. Eighty-eight percent of mice (21/24) subjected to contaminated tail needle puncture developed BU. Mouse tails coated only in bacteria did not develop disease. A median incubation time of 12 weeks, consistent with data from human infections, was noted. We then specifically tested the M. ulcerans infectious dose-50 (ID50) in this contaminated skin surface infection model with needle puncture and observed an ID50 of 2.6 colony-forming units. We have uncovered a biologically plausible mechanical transmission mode of BU via natural or anthropogenic skin punctures., Author summary Buruli ulcer is a neglected tropical disease caused by infection with Mycobacterium ulcerans. Unfortunately, how people contract this disease is not well understood. Here we show for the first time using experimental infections in mice that a very low dose of M. ulcerans delivered beneath the skin by a minor injury caused by a blood-feeding insect (mosquito) or an experimental needle puncture is sufficient to cause Buruli ulcer. This research provides important laboratory evidence to advance our understanding of Buruli ulcer disease transmission.

The natural products isatropolone A-C (1-3) were reisolated from Streptomyces Go66, with 1 and 3 showing potent activity against Leishmania donovani. They contain a rare tropolone ring derived from a type II polyketide biosynthesis pathway. Their biosynthesis was elucidated by labeling experiments, analysis of the biosynthesis gene cluster, its partial heterologous expression, and structural characterization of various intermediates. Owing to their 1,5-diketone moiety, they can react with ammonia, amines, lysine, and lysine-containing peptides and proteins, which results in the formation of a covalent bond and subsequent pyridine ring formation. Their fluorescence properties change upon amine binding, enabling the simple visualization of reacted amines including proteins.

Mycobacterium ulcerans is the causative agent of the debilitating skin disease Buruli ulcer, which is most prevalent in Western and Central Africa. M. ulcerans shares >98% DNA sequence identity with Mycobacterium marinum, however, M. marinum produces granulomatous, but not ulcerative, lesions in humans and animals. Here we report the differential expression of a small heat shock protein (Hsp18) between strains of M. ulcerans (Hsp18(+) ) and M. marinum (Hsp18(-) ) and describe the molecular basis for this difference. We show by gene deletion and GFP reporter assays in M. marinum that a divergently transcribed gene called hspR_2, immediately upstream of hsp18, encodes a MerR-like regulatory protein that represses hsp18 transcription while promoting its own expression. Naturally occurring mutations within a 70 bp segment of the 144 bp hspR_2-hsp18 intergenic region among M. ulcerans strains inhibit hspR_2 transcription and explain the Hsp18(+) phenotype. We also propose a biological role for Hsp18, as we show that this protein significantly enhances bacterial attachment or aggregation during biofilm formation. This study has uncovered a new member of the MerR family of transcriptional regulators and suggests that upregulation of hsp18 expression was an important pathoadaptive response in the evolution of M. ulcerans from a M. marinum-like ancestor.

Genome mining of the strictly anaerobic bacterium Clostridium beijerinckii, an industrial producer of solvents, revealed the presence of several cryptic gene clusters for secondary metabolite biosynthesis. To unearth its metabolic potential, a C. beijerinckii strain was cultured under various conditions, which led to the discovery of a deep purple pigment. This novel metabolite, named clostrubin (1), was isolated and its structure was fully elucidated. The pentacyclic polyphenol features a benzo(a)tetraphene ring topology that is unprece- dented for natural products. Stable-isotope labeling experi- ments showed that 1 is an aromatic polyketide that folds in a noncanonical manner to form the unusual perifused ring system. In addition to being the first reported polyketide from an anaerobic bacterium, 1 is a potent antibiotic with pro- nounced activity against various pathogenic bacteria, such as MRSA, VRE, and mycobacteria, with minimum inhibitory concentrations (MIC) of 0.12-0.97 mm. Bacterial infectious diseases are a persistent global concern and causing millions of deaths annually. According to the WHO, the rapid emergence of resistant pathogens and the lack of new therapeutics portend a return to the pre-antibiotic era. (1) Increased infections caused by the hospital-acquired pathogens methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterocci (VRE), com- bined with the emergence of multidrug-resistant tuberculosis (MDR-TB), demand the urgent development of new anti- biotic agents. (2, 3) Natural products, in particular microbial secondary metabolites, are the most important source of pharmacologically active compounds. (4) Traditionally, actino

Genome mining of the strictly anaerobic bacterium Clostridium beijerinckii, an industrial producer of solvents, revealed the presence of several cryptic gene clusters for secondary metabolite biosynthesis. To unearth its metabolic potential, a C. beijerinckii strain was cultured under various conditions, which led to the discovery of a deep purple pigment. This novel metabolite, named clostrubin (1), was isolated and its structure was fully elucidated. The pentacyclic polyphenol features a benzo(a)tetraphene ring topology that is unprece- dented for natural products. Stable-isotope labeling experi- ments showed that 1 is an aromatic polyketide that folds in a noncanonical manner to form the unusual perifused ring system. In addition to being the first reported polyketide from an anaerobic bacterium, 1 is a potent antibiotic with pro- nounced activity against various pathogenic bacteria, such as MRSA, VRE, and mycobacteria, with minimum inhibitory concentrations (MIC) of 0.12-0.97 mm. Bacterial infectious diseases are a persistent global concern and causing millions of deaths annually. According to the WHO, the rapid emergence of resistant pathogens and the lack of new therapeutics portend a return to the pre-antibiotic era. (1) Increased infections caused by the hospital-acquired pathogens methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterocci (VRE), com- bined with the emergence of multidrug-resistant tuberculosis (MDR-TB), demand the urgent development of new anti- biotic agents. (2, 3) Natural products, in particular microbial secondary metabolites, are the most important source of pharmacologically active compounds. (4) Traditionally, actino

There is no effective vaccine against Buruli ulcer. In experimental footpad infection of C57BL/6 mice with M. ulcerans, a prime-boost vaccination protocol using plasmid DNA encoding mycolyltransferase Ag85A of M. ulcerans and a homologous protein boost has shown significant, albeit transient protection, comparable to the one induced by M. bovis BCG. The mycolactone toxin is an obvious candidate for a vaccine, but by virtue of its chemical structure, this toxin is not immunogenic in itself. However, antibodies against some of the polyketide synthase domains involved in mycolactone synthesis, were found in Buruli ulcer patients and healthy controls from the same endemic region, suggesting that these domains are indeed immunogenic. Here we have analyzed the vaccine potential of nine polyketide synthase domains using a DNA prime/protein boost strategy. C57BL/6 mice were vaccinated against the following domains: acyl carrier protein 1, 2, and 3, acyltransferase (acetate) 1 and 2, acyltransferase (propionate), enoylreductase, ketoreductase A, and ketosynthase load module. As positive controls, mice were vaccinated with DNA encoding Ag85A or with M. bovis BCG. Strongest antigen specific antibodies could be detected in response to acyltransferase (propionate) and enoylreductase. Antigen-specific Th1 type cytokine responses (IL-2 or IFN-γ) were induced by vaccination against all antigens, and were strongest against acyltransferase (propionate). Finally, vaccination against acyltransferase (propionate) and enoylreductase conferred some protection against challenge with virulent M. ulcerans 1615. However, protection was weaker than the one conferred by vaccination with Ag85A or M. bovis BCG. Combinations of these polyketide synthase domains with the vaccine targeting Ag85A, of which the latter is involved in the integrity of the cell wall of the pathogen, and/or with live attenuated M. bovis BCG or mycolactone negative M. ulcerans may eventually lead to the development of an efficacious BU vaccine., Author Summary Buruli ulcer (BU) is an infectious disease, characterized by deep, ulcerating skin lesions, particularly on arms and legs, which are provoked by a toxin. BU is caused by a microbe of the genus that also cause tuberculosis and leprosy. The 33 countries where Buruli ulcer has been detected, especially in West Africa, have mainly tropical and subtropical climates, although the disease is also present in temperate areas of Australia and Japan. There is no effective vaccine against BU and it is still not fully understood which immune defence mechanisms (antibodies and/or T cells) are needed to control the infection. The identification of microbial components that are involved in immune control is an essential step in the development of an effective vaccine. In this paper, we used an experimental mouse model to demonstrate the immunogenicity and the vaccine potential of enzymes involved in the toxin synthesis. Combinations with other vaccine candidates, such as a subunit vaccine against Ag85A targeting cell wall synthesis or with live, attenuated M. bovis BCG or mycolactone negative Mycobacterium ulcerans remain to be tested.

Strictly anaerobic microorganisms, which represent the oldestliving beings on earth, are ubiquitous in the environment andin the intestines of higher organisms. Despite their hugeimpact on human health, ecology, and industrial biotech-nology, surprisingly little is known about the natural productchemistry of the oxygen-free world.

Mycolactones are polyketide-derived lipid virulence factors made by the slow-growing human pathogen, Mycobacterium ulcerans. Three unusually large and homologous plasmid-borne genes (mlsA1: 51 kb, mlsB: 42 kb and mlsA2: 7 kb) encode the mycolactone type I polyketide synthases (PKS). The extreme size and low sequence diversity of these genes has posed significant barriers for exploration of the genetic and biochemical basis of mycolactone synthesis. Here, we have developed a truncated, more tractable 3-module version of the 18-module mycolactone PKS and we show that this engineered PKS functions as expected in the natural host M. ulcerans to produce an additional polyketide; a triketide lactone (TKL). Cell fractionation experiments indicated that this 3-module PKS and the putative accessory enzymes encoded by mup045 and mup038 associated with the mycobacterial cell wall, a finding supported by confocal microscopy. We then assessed the capacity of the faster growing, Mycobacterium marinum to harbor and express the 3-module Mls PKS and accessory enzymes encoded by mup045 and mup038. RT-PCR, immunoblotting, and cell fractionation experiments confirmed that the truncated Mls PKS multienzymes were expressed and also partitioned with the cell wall material in M. marinum. However, this heterologous host failed to produce TKL. The systematic deconstruction of the mycolactone PKS presented here suggests that the Mls multienzymes are necessary but not sufficient for mycolactone synthesis and that synthesis is likely to occur (at least in part) within the mycobacterial cell wall. This research is also the first proof-of-principle demonstration of the potential of this enzyme complex to produce tailored small molecules through genetically engineered rearrangements of the Mls modules.

A specific and sensitive serodiagnostic test for Mycobacterium ulcerans infection would greatly assist the diagnosis of Buruli ulcer and would also facilitate seroepidemiological surveys. By comparative genomics, we identified 45 potential M. ulcerans specific proteins, of which we were able to express and purify 33 in E. coli. Sera from 30 confirmed Buruli ulcer patients, 24 healthy controls from the same endemic region and 30 healthy controls from a non-endemic region in Benin were screened for antibody responses to these specific proteins by ELISA. Serum IgG responses of Buruli ulcer patients were highly variable, however, seven proteins (MUP045, MUP057, MUL_0513, Hsp65, and the polyketide synthase domains ER, AT propionate, and KR A) showed a significant difference between patient and non-endemic control antibody responses. However, when sera from the healthy control subjects living in the same Buruli ulcer endemic area as the patients were examined, none of the proteins were able to discriminate between these two groups. Nevertheless, six of the seven proteins showed an ability to distinguish people living in an endemic area from those in a non-endemic area with an average sensitivity of 69% and specificity of 88%, suggesting exposure to M. ulcerans. Further validation of these six proteins is now underway to assess their suitability for use in Buruli ulcer seroepidemiological studies. Such studies are urgently needed to assist efforts to uncover environmental reservoirs and understand transmission pathways of the M. ulcerans., Author Summary Buruli ulcer is a slowly progressive but potentially devastating disease of skin and subcutaneous tissue caused by the bacterium Mycobacterium ulcerans. The disease is widespread throughout West and Central Africa, and some countries in the region have established Buruli ulcer control programs. Buruli ulcer is difficult to distinguish from other chronic skin conditions that require different treatments, and there is an urgent need for an accurate point-of-care diagnostic test. In this study, we have used genomic techniques to identify 45 potential M. ulcerans–specific antigens, 33 of which we have been able to produce and purify. We tested these proteins against sera from patients, healthy people living in the same region as the patients and from individuals living in a region with no cases of Buruli ulcer. We found that seven proteins were able to elicit antibody responses that were significantly different between patients and the control subjects from the non-endemic region but not from the healthy individuals in the same Buruli ulcer endemic region. Further analysis showed that six of these M. ulcerans proteins might be useful as markers of exposure to M. ulcerans and could be developed into tools to uncover environmental reservoirs and understand transmission pathways of the bacterium.

Mycobacterium ulcerans is the third most common mycobacterial infection of humans and causes a destructive disease of subcutaneous tissue known as Buruli ulcer. A cytotoxic lipid known as mycolactone mediates the characteristic necrosis seen in Buruli ulcers. A family of highly related mycolactone structural variants have been discovered, all of which are produced by large polyketide synthases (PKSs), encoded by genes on large plasmids harbored by M. ulcerans strains. The prototype mycolactone plasmid is pMUM001, a 174-kb circular molecule from a West African epidemic strain of M. ulcerans. A striking feature of pMUM001 is that it has one-third of its DNA devoted to three very large genes that encode the mycol-actone biosynthetic machinery. The plasmid (pMUM002) from M. ulcerans subsp. liflandii has recently been completely sequenced, and partial sequence has been obtained for a further pMUM plasmid, pMUM003, from M. ulcerans DL240490. Interstrain comparisons of pMUM sequences has revealed the highly mutable nature of the mycolactone PKS genes and given us greater insight into the origins of these large replicons.

Mycolactone A/B is a lipophilic macrocyclic polyketide that is the primary virulence factor produced by Mycobacterium ulcerans, a human pathogen and the causative agent of Buruli ulcer. In M. ulcerans strain Agy99 the mycolactone polyketide synthase (PKS) locus spans a 120 kb region of a 174 kb megaplasmid. Here we have identified promoter regions of this PKS locus using GFP reporter assays, in silico analysis, primer extension, and site-directed mutagenesis. Transcription of the large PKS genes mlsA1 (51 kb), mlsA2 (7 kb) and mlsB (42 kb) is driven by a novel and powerful SigA-like promoter sequence situated 533 bp upstream of both the mlsA1 and mlsB initiation codons, which is also functional in Escherichia coli, Mycobacterium smegmatis and Mycobacterium marinum. Promoter regions were also identified upstream of the putative mycolactone accessory genes mup045 and mup053. We transformed M. ulcerans with a GFP-reporter plasmid under the control of the mls promoter to produce a highly green-fluorescent bacterium. The strain remained virulent, producing both GFP and mycolactone and causing ulcerative disease in mice. Mosquitoes have been proposed as a potential vector of M. ulcerans so we utilized M. ulcerans-GFP in microcosm feeding experiments with captured mosquito larvae. M. ulcerans-GFP accumulated within the mouth and midgut of the insect over four instars, whereas the closely related, non-mycolactone-producing species M. marinum harbouring the same GFP reporter system did not. This is the first report to identify M. ulcerans toxin gene promoters, and we have used our findings to develop M. ulcerans-GFP, a strain in which fluorescence and toxin gene expression are linked, thus providing a tool for studying Buruli ulcer pathogenesis and potential transmission to humans., Author Summary Buruli ulcer (BU) is a serious skin infection of humans predominantly occurring in West and Central Africa. The disease is caused by infection with Mycobacterium ulcerans, a bacterium that produces an unusual toxin called mycolactone. There are many unanswered questions surrounding BU, particularly regarding the role of mycolactone in disease and how M. ulcerans is transmitted to humans. Here, we have partly addressed these questions by identifying genetic factors controlling the transcription of the mycolactone genes. Using a variety of experimental approaches, including green fluorescent protein (GFP) as a reporter of gene expression, we have identified strong promoters that drive transcription of the mycolactone genes in M. ulcerans. We then used our GFP reporters to produce highly fluorescent M. ulcerans-GFP that were readily visualized by microscopy. Mosquitoes have been proposed as a potential vector of M. ulcerans so we used M. ulcerans-GFP in feeding experiments with mosquito larvae. M. ulcerans-GFP accumulated within the insects, whereas other mycobacteria did not. This is the first report of the mycolactone gene promoters, and we have used our findings to develop M. ulcerans-GFP, a strain in which fluorescence and toxin gene expression are linked, thus providing a powerful tool for studying Buruli ulcer.

Background Mycolactones are immunosuppressive and cytotoxic polyketides, comprising five naturally occurring structural variants (named A/B, C, D, E and F), produced by different species of very closely related mycobacteria including the human pathogen, Mycobacterium ulcerans. In M. ulcerans strain Agy99, mycolactone A/B is produced by three highly homologous type I polyketide megasynthases (PKS), whose genes (mlsA1: 51 kb, mlsA2: 7.2 kb and mlsB: 42 kb) are found on a 174 kb plasmid, known as pMUM001. Results We report here comparative genomic analysis of pMUM001, the complete DNA sequence of a 190 kb megaplasmid (pMUM002) from Mycobacterium liflandii 128FXT and partial sequence of two additional pMUM replicons, combined with liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. These data reveal how PKS module and domain differences affecting MlsB correlate with the production of mycolactones E and F. For mycolactone E these differences from MlsB in M. ulcerans Agy99 include replacement of the AT domain of the loading module (acetate to propionate) and the absence of an entire extension module. For mycolactone F there is also a reduction of one extension module but also a swap of ketoreductase domains that explains the characteristic stereochemistry of the two terminal side-chain hydroxyls, an arrangement unique to mycolactone F Conclusion The mycolactone PKS locus on pMUM002 revealed the same large, three-gene structure and extraordinary pattern of near-identical PKS domain sequence repetition as observed in pMUM001 with greater than 98.5% nucleotide identity among domains of the same function. Intra- and inter-strain comparisons suggest that the extreme sequence homogeneity seen among the mls PKS genes is caused by frequent recombination-mediated domain replacement. This work has shed light on the evolution of mycolactone biosynthesis among an unusual group of mycobacteria and highlights the potential of the mls locus to become a toolbox for combinatorial PKS biochemistry.

Mycolactones are a family of highly related macrocyclic polyketides that exhibit immunosuppressive and cytotoxic properties. First discovered in 1999, they are the primary virulence factors produced by the environmental human pathogen Mycobacterium ulcerans, the causative agent of Buruli ulcer, and by some closely-related aquatic mycobacteria that cause disease in fish and frogs. Mycolactones are characterized by a common 12-membered lactone core to which is appended an unsaturated fatty acyl side-chain of variable length and oxidation state. This Highlight summarizes recent progress in understanding the structural diversity of the mycolactones, their biological activity and mode of action in mammalian cells, and the genetics, evolution, and enzymology of their biosynthesis.

Mycolactone structural variants, produced by Mycobacterium ulcerans and related mycobacteria, are caused by rearrangements among the highly homologous domains and modules within the MlsB polyketide megasynthase, that in turn alter the immunosuppressive and cytotoxic potency of these natural products., Covering: up to January 2008, Mycolactones are a family of highly related macrocyclic polyketides that exhibit immunosuppressive and cytotoxic properties. First discovered in 1999, they are the primary virulence factors produced by the environmental human pathogen Mycobacterium ulcerans, the causative agent of Buruli ulcer, and by some closely-related aquatic mycobacteria that cause disease in fish and frogs. Mycolactones are characterized by a common 12-membered lactone core to which is appended an unsaturated fatty acyl side-chain of variable length and oxidation state. This Highlight summarizes recent progress in understanding the structural diversity of the mycolactones, their biological activity and mode of action in mammalian cells, and the genetics, evolution, and enzymology of their biosynthesis.

Mycobacterium ulcerans is found in aquatic ecosystems and causes Buruli ulcer in humans, a neglected but devastating necrotic disease of subcutaneous tissue that is rampant throughout West and Central Africa. Here, we report the complete 5.8-Mb genome sequence of M. ulcerans and show that it comprises two circular replicons, a chromosome of 5632 kb and a virulence plasmid of 174 kb. The plasmid is required for production of the polyketide toxin mycolactone, which provokes necrosis. Comparisons with the recently completed 6.6-Mb genome of Mycobacterium marinum revealed >98% nucleotide sequence identity and genome-wide synteny. However, as well as the plasmid, M. ulcerans has accumulated 213 copies of the insertion sequence IS2404, 91 copies of IS2606, 771 pseudogenes, two bacteriophages, and multiple DNA deletions and rearrangements. These data indicate that M. ulcerans has recently evolved via lateral gene transfer and reductive evolution from the generalist, more rapid-growing environmental species M. marinum to become a niche-adapted specialist. Predictions based on genome inspection for the production of modified mycobacterial virulence factors, such as the highly abundant phthiodiolone lipids, were confirmed by structural analyses. Similarly, 11 protein-coding sequences identified as M. ulcerans-specific by comparative genomics were verified as such by PCR screening a diverse collection of 33 strains of M. ulcerans and M. marinum. This work offers significant insight into the biology and evolution of mycobacterial pathogens and is an important component of international efforts to counter Buruli ulcer.