We describe the identification of the gene encoding an immunodominant 32-kilodalton (kDa) protein of Mycobacterium tuberculosis. The 32-kDa antigen is abundantly secreted into the culture supernatant of a variety of mycobacteria and appears to be a major stimulant of cellular and humoral immunity against mycobacteria. Recombinant clones expressing a 140or 125-kDa j8-galactosidase fusion protein reactive with rabbit polyclonal anti-32 kDa protein serum were detected. The corresponding DNA sequence contains a 1,008-base-pair coding region. The deduced amino acid sequence corresponds to a 336-residue protein including the previously determined NH2-terminal sequence of the 32-kDa protein (Microb. Pathog. 2:351-366, 1987). Upstream of this NH2-terminal region, the gene codes for a signal peptide required for the secretion of a 294-amino-acid-long mature protein. A putative promotor sequence could be located upstream of the open reading frame. Comparison of the M. tuberculosis 32-kDa antigen with the Mycobacterium bovis BCG a-antigen (K. 170:3847-3854, 1988) revealed 73.8% homology between DNA sequences and 72.8% homology between amino acid sequences (signal and mature protein). Finally, the 140-kDa fusion protein could selectively be recognized by human tuberculous sera. This result confirms our previous finding that the 32-kDa antigen could be a valuable tool for the serological diagnosis of tuberculosis. Moreover, the availability of recombinant proteins opens perspectives for the localization of relevant B-and T-cell epitope regions on the 32-kDa antigen.

Nine monoclonal antibodies (MoAbs) were produced against Mycobacterium bovis BCG antigen 85 complex. Using isoelectric focusing combined with Western (immunoblot) blot analysis, antigenically related proteins could be identified in culture filtrates from M. tuberculosis, M. bovis, M. Kansasii, M. avium, M. xenopi, M. gordonae, M. fortuitum, M. phlei and M. smegmatis. Most of the MoAbs were found to be broadly cross-reactive between the various mycobacterial species, albeit some minor differences were observed. These MoAbs reacted generally, in each species, with different components. One MoAb (VID1-14) was found to be specifically directed only against antigen 85B from M. bovis, M. tuberculosis and M. kansasii.

A previous study of the effect of zinc deprivation on Mycobacterium bovis BCG pointed out the potential importance of an alcohol dehydrogenase for maintaining the hydrophobic character of the cell envelope. In this report, the effect of the overexpression of the M. bovis BCG alcohol dehydrogenase (ADH) in Mycobacterium smegmatis and M. bovis BCG is described. The purification of the enzyme was performed to apparent homogeneity from overexpressing M. bovis BCG cells and its kinetic parameters were determined. The enzyme showed a strong preference for both aromatic and aliphatic aldehydes while the corresponding alcohols were processed 100±1000-fold less efficiently. The best k cat /K m values were found with benzaldehyde . 3-methoxybenzaldehyde . octanal . coniferaldehyde. A phylogenetic analysis clearly revealed that the M. bovis BCG ADH together with the ADHs from Bacillus subtilis and Helicobacter pylori formed a sister group of the class C medium-chain alcohol dehydrogenases, the plant cinnamyl alcohol dehydrogenases (CADs). Comparison of the kinetic properties of our ADH with some related class C enzymes indicated that the mycobacterial enzyme substrate profile resembled that of the CADs involved in plant defence rather than those implicated in lignification. A possible role for the M. bovis BCG ADH in the biosynthesis of the lipids composing the mycobacterial cell envelope is proposed.Keywords: Mycobacterium bovis BCG; alcohol dehydrogenase; cinnamyl alcohol dehydrogenase.Alcohol dehydrogenases (ADHs) display a wide range of substrate specificities and fulfil several key physiological functions. They can be divided into three major categories. The first category is composed of the NAD(P)-dependent ADHs subdivided into three subgroups according to their metal dependence: (a) the medium-chain zinc-dependent enzymes; (b) the short-chain zinc-independent enzymes; and (c) the iron-activated enzymes. The NAD(P)-independent enzymes form the second category and the third category contains the oxidases which catalyse an essentially irreversible oxidation of alcohols [1].The vaccine strain Mycobacterium bovis BCG grows poorly on zinc-deprived Sauton medium, and forms a thin, pale, unfolded pellicle that gets wet and often sinks. This phenotype suggested a cell envelope with a reduced hydrophobic content. The cell composition and the products excreted into the medium, including aldehydes, can, to some extent, be correlated with a decrease of the specific activity of a soluble zinc-dependent alcohol dehydrogenase. This enzyme was partially purified from BCG cells and characterized as a dimeric NADP-dependent protein of broad specificity with higher affinities towards aldehydes than towards alcohols [2,3].The corresponding gene has been cloned and sequenced [4]. Comparison with other ADHs allowed classification of this enzyme into the zinc-containing, medium-chain alcohol/polyol dehydrogenase family which had been primarily described in eukaryotes [5]. Currently, this family is composed of the tetrameric and the dimeric...

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