A genuine mycobacterial thermophile: Mycobacterium hassiacum growth, survival and GpgS stability at near-pasteurization temperatures.

Mycobacterium hassiacum is so far the most thermophilic among mycobacteria as it grows optimally at 50 °C and up to 65 °C in a glycerol-based medium, as verified in this study. Since this and other nontuberculous mycobacteria (NTM) thrive in diverse natural and artificial environments, from where they may access and infect humans, we deemed essential to probe M. hassiacum resistance to heat, a strategy routinely used to control microbial growth in water-supply systems, as well as in the food and drink industries. In addition to possibly being a threat in its own right in rare occasions, M. hassiacum is also a good surrogate for studying other NTM species more often associated with opportunistic infection, namely Mycobacterium avium and Mycobacterium abscessus as well as their strictly pathogenic counterparts Mycobacterium tuberculosis and Mycobacterium leprae. In this regard, this thermophilic species is likely to be useful as a source of stable proteins that may provide more detailed structures of potential drug targets. Here, we investigate M. hassiacum growth at near-pasteurization temperatures and at different pHs and also characterize its thermostable glucosyl-3-phosphoglycerate synthase (GpgS), an enzyme considered essential for M. tuberculosis growth and associated with both nitrogen starvation and thermal stress in different NTM species.

Decoding the similarities and differences among mycobacterial species.

Mycobacteriaceae comprises pathogenic species such as Mycobacterium tuberculosis, M. leprae and M. abscessus, as well as non-pathogenic species, for example, M. smegmatis and M. thermoresistibile. Genome comparison and annotation studies provide insights into genome evolutionary relatedness, identify unique and pathogenicity-related genes in each species, and explore new targets that could be used for developing new diagnostics and therapeutics. Here, we present a comparative analysis of ten-mycobacterial genomes with the objective of identifying similarities and differences between pathogenic and non-pathogenic species. We identified 1080 core orthologous clusters that were enriched in proteins involved in amino acid and purine/pyrimidine biosynthetic pathways, DNA-related processes (replication, transcription, recombination and repair), RNA-methylation and modification, and cell-wall polysaccharide biosynthetic pathways. For their pathogenicity and survival in the host cell, pathogenic species have gained specific sets of genes involved in repair and protection of their genomic DNA. M. leprae is of special interest owing to its smallest genome (1600 genes and ~1300 psuedogenes), yet poor genome annotation. More than 75% of the pseudogenes were found to have a functional ortholog in the other mycobacterial genomes and belong to protein families such as transferases, oxidoreductases and hydrolases.

The crystal structure of M. leprae ML2640c defines a large family of putative S-adenosylmethionine-dependent methyltransferases in mycobacteria.

Mycobacterium leprae protein ML2640c belongs to a large family of conserved hypothetical proteins predominantly found in mycobacteria, some of them predicted as putative S-adenosylmethionine (AdoMet)-dependent methyltransferases (MTase). As part of a Structural Genomics initiative on conserved hypothetical proteins in pathogenic mycobacteria, we have determined the structure of ML2640c in two distinct crystal forms. As expected, ML2640c has a typical MTase core domain and binds the methyl donor substrate AdoMet in a manner consistent with other known members of this structural family. The putative acceptor substrate-binding site of ML2640c is a large internal cavity, mostly lined by aromatic and aliphatic side-chain residues, suggesting that a lipid-like molecule might be targeted for catalysis. A flap segment (residues 222-256), which isolates the binding site from the bulk solvent and is highly mobile in the crystal structures, could serve as a gateway to allow substrate entry and product release. The multiple sequence alignment of ML2640c-like proteins revealed that the central alpha/beta core and the AdoMet-binding site are very well conserved within the family. However, the amino acid positions defining the binding site for the acceptor substrate display a higher variability, suggestive of distinct acceptor substrate specificities. The ML2640c crystal structures offer the first structural glimpses at this important family of mycobacterial proteins and lend strong support to their functional assignment as AdoMet-dependent methyltransferases.

Towards the integration of foreign DNA into the chromosome of Mycobacterium leprae.

Integrative plasmid vectors based on the pSAM2 system of Streptomyces ambofaciens offer great potential for the genetic analysis of Mycobacterium leprae. To assess this, the chromosomal attachment site of M. leprae, att-pSAM2, has been cloned, mapped and characterized. Nucleotide sequence analysis shows att-pSAM2 to correspond to a putative tRNA(pro) gene identical in sequence to those of S. ambofaciens and M. tuberculosis. In addition, it is shown that the genes encoding aspartate semialdehyde dehydrogenase, asd, and an anonymous protein antigen recognized by sera from leprosy patients, are linked to the M. leprae att-pSAM2 locus.