Expanding the set of rhodococcal Baeyer–Villiger monooxygenases by high-throughput cloning, expression and substrate screening

To expand the available set of Baeyer–Villiger monooxygenases (BVMOs), we have created expression constructs for producing 22 Type I BVMOs that are present in the genome of Rhodococcus jostii RHA1. Each BVMO has been probed with a large panel of potential substrates. Except for testing their substrate acceptance, also the enantioselectivity of some selected BVMOs was studied. The results provide insight into the biocatalytic potential of this collection of BVMOs and expand the biocatalytic repertoire known for BVMOs. This study also sheds light on the catalytic capacity of this large set of BVMOs that is present in this specific actinomycete. Furthermore, a comparative sequence analysis revealed a new BVMO-typifying sequence motif. This motif represents a useful tool for effective future genome mining efforts.

embCAB Sequence Variation Among Ethambutol-Resistant Mycobacterium Tuberculosis Isolates Without embB306 Mutation

Mechanisms of resistance to ethambutol in Mycobacterium tuberculosis remain inadequately described. Although there is mounting evidence that mutations of codon 306 in embB play a key role, a significant number of phenotypically ethambutol-resistant strains do not carry mutations in this codon. Here, other mutations in the embCAB operon are suggested to be involved in resistance development

Genotyping Analyses of Tuberculosis Cases in U.S.- and Foreign-Born Massachusetts Residents

We used molecular genotyping to further understand the epidemiology and transmission patterns of tuberculosis (TB) in Massachusetts. The study population included 983 TB patients whose cases were verified by the Massachusetts Department of Public Health between July 1, 1996, and December 31, 2000, and for whom genotyping results and information on country of origin were available. Two hundred seventy-two (28%) of TB patients were in genetic clusters, and isolates from U.S-born were twice as likely to cluster as those of foreign-born (odds ratio [OR] 2.29, 95% confidence interval [CI] 1.69, 3.12). Our results suggest that restriction fragment length polymorphism analysis has limited capacity to differentiate TB strains when the isolate contains six or fewer copies of IS6110, even with spoligotyping. Clusters of TB patients with more than six copies of IS6110 were more likely to have epidemiologic connections than were clusters of TB patients with isolates with few copies of IS6110 (OR 8.01, 95%; CI 3.45,18.93)

Novel Mycobacterium tuberculosis Complex Pathogen, M. mungi

Seven outbreaks involving increasing numbers of banded mongoose troops and high death rates have been documented. We identified a Mycobacterium tuberculosis complex pathogen, M. mungi sp. nov., as the causative agent among banded mongooses that live near humans in Chobe District, Botswana. Host spectrum and transmission dynamics remain unknown

Mapping of Mycobacterium tuberculosis Complex Genetic Diversity Profiles in Tanzania and Other African Countries

The aim of this study was to assess and characterize Mycobacterium tuberculosis complex (MTBC) genotypic diversity in Tanzania, as well as in neighbouring East and other several African countries. We used spoligotyping to identify a total of 293 M. tuberculosis clinical isolates (one isolate per patient) collected in the Bunda, Dar es Salaam, Ngorongoro and Serengeti areas in Tanzania. The results were compared with results in the SITVIT2 international database of the Pasteur Institute of Guadeloupe. Genotyping and phylogeographical analyses highlighted the predominance of the CAS, T, EAI, and LAM MTBC lineages in Tanzania. The three most frequent Spoligotype International Types (SITs) were: SIT21/CAS1-Kili (n = 76; 25.94%), SIT59/LAM11-ZWE (n = 22; 7.51%), and SIT126/EAI5 tentatively reclassified as EAI3-TZA (n = 18; 6.14%). Furthermore, three SITs were newly created in this study (SIT4056/EAI5 n = 2, SIT4057/T1 n = 1, and SIT4058/EAI5 n = 1). We noted that the East-African-Indian (EAI) lineage was more predominant in Bunda, the Manu lineage was more common among strains isolated in Ngorongoro, and the Central-Asian (CAS) lineage was more predominant in Dar es Salaam (p-value<0.0001). No statistically significant differences were noted when comparing HIV status of patients vs. major lineages (p-value = 0.103). However, when grouping lineages as Principal Genetic Groups (PGG), we noticed that PGG2/3 group (Haarlem, LAM, S, T, and X) was more associated with HIV-positive patients as compared to PGG1 group (Beijing, CAS, EAI, and Manu) (p-value = 0.03). This study provided mapping of MTBC genetic diversity in Tanzania (containing information on isolates from different cities) and neighbouring East African and other several African countries highlighting differences as regards to MTBC genotypic distribution between Tanzania and other African countries. This work also allowed underlining of spoligotyping patterns tentatively grouped within the newly designated EAI3-TZA lineage (remarkable by absence of spacers 2 and 3, and represented by SIT126) which seems to be specific to Tanzania. However, further genotyping information would be needed to confirm this specificity

Supervised learning for the automated transcription of spacer classification from spoligotype films

<p>Abstract</p> <p>Background</p> <p>Molecular genotyping of bacteria has revolutionized the study of tuberculosis epidemiology, yet these established laboratory techniques typically require subjective and laborious interpretation by trained professionals. In the context of a Tuberculosis Case Contact study in The Gambia we used a reverse hybridization laboratory assay called spoligotype analysis. To facilitate processing of spoligotype images we have developed tools and algorithms to automate the classification and transcription of these data directly to a database while allowing for manual editing.</p> <p>Results</p> <p>Features extracted from each of the 1849 spots on a spoligo film were classified using two supervised learning algorithms. A graphical user interface allows manual editing of the classification, before export to a database. The application was tested on ten films of differing quality and the results of the best classifier were compared to expert manual classification, giving a median correct classification rate of 98.1% (inter quartile range: 97.1% to 99.2%), with an automated processing time of less than 1 minute per film.</p> <p>Conclusion</p> <p>The software implementation offers considerable time savings over manual processing whilst allowing expert editing of the automated classification. The automatic upload of the classification to a database reduces the chances of transcription errors.</p

Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages

There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC) might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP) are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the 'Beijing' sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae. Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster for confirmation. Furthermore, both methods are rapid, flexible and comparably inexpensive

Investigating the coenzyme specificity of phenylacetone monooxygenase from Thermobifida fusca

Type I Baeyer–Villiger monooxygenases (BVMOs) strongly prefer NADPH over NADH as an electron donor. In order to elucidate the molecular basis for this coenzyme specificity, we have performed a site-directed mutagenesis study on phenylacetone monooxygenase (PAMO) from Thermobifida fusca. Using sequence alignments of type I BVMOs and crystal structures of PAMO and cyclohexanone monooxygenase in complex with NADP+, we identified four residues that could interact with the 2′-phosphate moiety of NADPH in PAMO. The mutagenesis study revealed that the conserved R217 is essential for binding the adenine moiety of the nicotinamide coenzyme while it also contributes to the recognition of the 2′-phosphate moiety of NADPH. The substitution of T218 did not have a strong effect on the coenzyme specificity. The H220N and H220Q mutants exhibited a ~3-fold improvement in the catalytic efficiency with NADH while the catalytic efficiency with NADPH was hardly affected. Mutating K336 did not increase the activity of PAMO with NADH, but it had a significant and beneficial effect on the enantioselectivity of Baeyer–Villiger oxidations and sulfoxidations. In conclusion, our results indicate that the function of NADPH in catalysis cannot be easily replaced by NADH. This finding is in line with the complex catalytic mechanism and the vital role of the coenzyme in BVMOs

Immunogenicity of antigens from the TbD1 region present in M. africanum and missing from "modern" M. tuberculosis: a cross- sectional study

<p>Abstract</p> <p>Background</p> <p>Currently available tools cannot be used to distinguish between sub-species of the <it>M. tuberculosis </it>complex causing latent tuberculosis (TB) infection. <it>M. africanum </it>causes up to half of TB in West- Africa and its relatively lower progression to disease suggests the presence of a large reservoir of latent infection relative to <it>M. tuberculosis</it>.</p> <p>Methods</p> <p>We assessed the immunogenicity of the TbD1 region, present in <it>M. africanum </it>and absent from "modern" <it>M. tuberculosis</it>, in an ELISPOT assay using cells from confirmed <it>M. africanum </it>or <it>M. tuberculosis </it>infected TB patients without HIV infection in the Gambia.</p> <p>Results</p> <p>Antigens from the TbD1 region induced IFNγ responses in only 35% patients and did not discriminate between patients infected with <it>M. africanum </it>vs. <it>M. tuberculosis</it>, while PPD induced universally high responses.</p> <p>Conclusions</p> <p>Further studies will need to assess other antigens unique to <it>M. africanum </it>that may induce discriminatory immune responses.</p