Global spread and genetic monomorphism are hallmarks of Mycobacterium tuberculosis, the agent of human tuberculosis. In contrast, Mycobacterium canettii, and related tubercle bacilli that also cause human tuberculosis and exhibit unusual smooth colony morphology, are restricted to East-Africa. Here, we sequenced and analyzed the genomes of five representative strains of smooth tubercle bacilli (STB) using Sanger (4-5x coverage), 454/Roche (13-18x coverage) and/or Illumina DNA sequencing (45-105x coverage). We show that STB are highly recombinogenic and evolutionary early-branching, with larger genome sizes, 25-fold more SNPs, fewer molecular scars and distinct CRISPR-Cas systems relative to M. tuberculosis. Despite the differences, all tuberculosis-causing mycobacteria share a highly conserved core genome. Mouse-infection experiments revealed that STB are less persistent and virulent than M. tuberculosis. We conclude that M. tuberculosis emerged from an ancestral, STB-like pool of mycobacteria by gain of persistence and virulence mechanisms and we provide genome-wide insights into the molecular events involved.

Levels of three major dehydrins of 65, 60, and 14 kDa have been observed to increase in blueberry (Vaccinium smpp.) floral buds during chill unit accumulation and cold acclimation and decrease during deacclimation and resumption of growth. Indeed, levels of the 65‐, 60‐, and 14‐kDa dehydrins increase such that they become the most predominant proteins visible on sodium dodecyl sulfate (SDS)‐polyacrylamide gels. The peptide sequence information from the 65‐ and 60‐kDa dehydrins was used to synthesize degenerate DNA primers for amplification of a part of the gene(s) encoding the dehydrins. One pair of primers amplified a 174‐bp fragment. The 174‐bp fragment was used to screen a cDNA library (prepared from RNA from cold‐acclimated blueberry floral buds) and resulted in the isolation of a clone with a 2.0‐kb insert. The cDNA was sequenced and found to be a full‐length clone encoding a K5‐type dehydrin (5 K boxes). Five high‐confidence peptide sequences, ranging from 9 to 25 amino acids long, obtained from the 60‐kDa dehydrin exactly matched sequences encoded within the cDNA clone. Furthermore, amino acid composition of the 60‐kDa dehydrin agreed well with the expected amino acid composition based on the cDNA sequence. However, the DNA sequence and coupled in vitro transcription/translation reactions of the cDNA clone indicated that it encodes a dehydrin with a native molecular mass of ∼40 kDa instead of 60 kDa. Experiments to determine if the dehydrins undergo post‐translational modifications revealed that the 65‐ and 60‐kDa dehydrins are glycosylated. Thus, our results indicate that the 2.0‐kb dehydrin cDNA encodes the native version of the 60‐kDa dehydrin. The dehydrin cDNA hybridized on RNA blots to two chilling/cold‐responsive messages of 2.0 and 0.5 kb. Both the 2.0‐ and 0.5‐kb messages increased to higher levels more quickly in the cold‐hardy cultivar Bluecrop than in the less hardy cultivar Tifblue. In addition, the 0.5‐kb message remained at a higher level longer in Bluecrop than in Tifblue.

Three blueberry (Vaccinium section Cyanococcus) genotypes, that have different chilling requirements and levels of cold hardiness, were studied. Depth of dormancy was evaluated and water status was determined, using nuclear magnetic resonance (NMR), throughout the accumulation of chilling that leads to release from dormancy. Among the two highbush cultivars studied, `Bluecrop' (Vaccinium corymbosum L.) was the most dormant and `Gulfcoast' (Vaccinium corymbosum L. x Vaccinium darrowi Camp) was the least dormant. The rabbiteye cultivar `Tifblue' (Vaccinium ashei Reade) had an intermediate dormancy. It appeared that the cultivar with the deepest dormancy had also the highest chilling requirement (CR). The NMR results showed that `Bluecrop' buds had the lowest relaxation times (T2), indicating that water was relatively more bound in `Bluecrop' buds than in the buds of the two other cultivars. Whatever the cultivar, no significant variation of T2s and water content of the buds was noted throughout the accumulation of chilling, even after CRs were satisfied. Within 1 day of forcing (24 °C, long day), there was a shift towards freer water but no change in the water content. Forcing was ineffective in freeing water until the plants received enough chilling to satisfy their CRs.

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