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Buruli ulcer (BU) occurs in >30 countries. The causative organism, Mycobacterium ulcerans, is acquired from the environment, but the exact mode of transmission is unknown. We investigated an outbreak of [...]

It had been assumed that production of the cytotoxie polyketide mycolactone was strictly associated with Mycobacterium ulcerans, the causative agent of Buruli ulcer. However, a recent study has uncovered a broader distribution of mycolactone-producing mycobacteria (MPM) that includes mycobacteria cultured from diseased fish and frogs in the United States and from diseased fish in the Red and Mediterranean Seas. All of these mycobacteria contain versions of the M. ulcerans pMUM plasmid, produce mycolactones, and show a high degree of genetic relatedness to both M. ulcerans and Mycobacterium marinum. Here, we show by multiple genetic methods, including multilocus sequence analysis and DNA-DNA hybridization, that all MPM have evolved from a common M. marinum progenitor to form a genetically cohesive group among a more diverse assemblage of M. marinum strains. Like M. ulcerans, the fish and frog MPM show multiple copies of the insertion sequence IS2404. Comparisons of pMUM and chromosomal gene sequences demonstrate that plasmid acquisition and the subsequent ability to produce mycolactone were probably the key drivers of speciation. Ongoing evolution among MPM has since produced at least two genetically distinct ecotypes that can be broadly divided into those typically causing disease in ectotherms (but also having a high zoonotic potential) and those causing disease in endotherms, such as humans.

We analyzed a global collection of Mycobacterium tuberculosis strains using 212 single nucleotide polymorphism (SNP) markers. SNP nucleotide diversity was high (average across all SNPs, 0.19), and 96% of the SNP locus pairs were in complete linkage disequilibrium. Cluster analyses identified six deeply branching, phylogenetically distinct SNP cluster groups (SCGs) and five subgroups. The SCGs were strongly associated with the geographical origin of the M. tuberculosis samples and the birthplace of the human hosts. The most ancestral cluster (SCG-1) predominated in patients from the Indian subcontinent, while SCG-1 and another ancestral cluster (SCG-2) predominated in patients from East Asia, suggesting that M. tuberculosis first arose in the Indian subcontinent and spread worldwide through East Asia. Restricted SCG diversity and the prevalence of less ancestral SCGs in indigenous populations in Uganda and Mexico suggested a more recent introduction of M. tuberculosis into these regions. The East African Indian and Beijing spoligotypes were concordant with SCG-1 and SCG-2, respectively; X and Central Asian spoligotypes were also associated with one SCG or subgroup combination. Other clades had less consistent associations with SCGs. Mycobacterial interspersed repetitive unit (MIRU) analysis provided less robust phylogenetic information, and only 6 of the 12 MIRU microsatellite loci were highly differentiated between SCGs as measured by [G.sub.ST]. Finally, an algorithm was devised to identify two minimal sets of either 45 or 6 SNPs that could be used in future investigations to enable global collaborations for studies on evolution, strain differentiation, and biological differences of M. tuberculosis.

The general stress response in Neisseria gonorrhoeae was investigated. Transcriptional analyses of the genes encoding the molecular chaperones DnaK, DnaJ, and GrpE suggested that they are transcribed from [[sigma].sup.32] (RpoH)-dependent promoters upon exposure to stress. This was confirmed by mutational analysis of the [[sigma].sup.32] promoter of dnaK. The gene encoding the gonococcal RpoH sigma factor appears to be essential, as we could not isolate viable mutants. Deletion of an unusually long rpoH leader sequence resulted in elevated levels of transcription, suggesting that this region is involved in negative regulation of RpoH expression during normal growth. Transcriptional analyses and protein studies determined that regulation of the RpoH-mediated stress response is different from that observed in most other species, in which regulation occurs predominantly at the transcriptional and translational levels. We suggest that an increase in the activity of preformed RpoH is primarily responsible for induction of the stress response in N. gonorrhoeae.

The pili expression is under the control of the pilE gene in Neisseria gonorrhoeae. The regulation of this gene is analyzed in detail to determine which sequences function in the efficient genetic transcription from this gene. Results reveal the presence of an integration host factor binding domain located upstream from a segment rich in adenosine and thymidine. Both of these segments are essential to the activation of pilE.

The biochemical properties of the recA gene from Clostridium perfringens were analyzed to determine the genetic mechanisms involved in microbial homologous recombination and DNA repair. Analysis of Clostridium perfringens recA gene indicated the role of DNA damage in activating the RecA protein due to exposure of the cells to methyl methanesulphonate. Furthermore, induction of recA gene due to DNA damage in Clostridium perfringens was also mediated by the presence of an SOS-like Cheo box operator in the upstream region.

A second putative sigma70 promoter upstream of the pilE gene encoding the pilin subunit in Neisseria gonorrhoeae that has an activity with Escherichia coli has been identified. Its transcription start point (tsp) was identified at 37 bp downstream of the previously mapped tsp for pilE. Site-directed mutagenesis and fusion studies involving PpilE::cat constructs showed that promoter activity is regulated differently in the two species.

A study shows that there is a promoter associated with 26-bp neisserial repeat (NR) and this repetitive sequence, found in many copies in the genome of Neisseria gonorrhoeae, may be a transposable element. A functional promoter may be formed by insertion of this sequence into an upstream region of a gene. The last two bases are located outside of the NR sequences, in one of the potential -10 regions of mapped promoter, in gonococcal uvrB gene.

The development and application of two multiplex real-time PCR assays targeting three distinct repeated sequences in Mycobacterium ulcerans genome is presented. The assay improves the turnaround time for diagnosis and replaces conventional gel-based PCR as the routine method for laboratory confirmation of Mycobacterium ulcerans infection.