A gene encoding a protein homologous to the periplasmic ABC phosphate binding receptor PstS from Escherichia coli was cloned and sequenced from a gt11 library of Mycobacterium tuberculosis by screening with monoclonal antibody 2A1-2. Its degree of similarity to the E. coli PstS is comparable to those of the previously described M. tuberculosis phosphate binding protein pab (Ag78, Ag5, or 38-kDa protein) and another M. tuberculosis protein which we identified recently. We suggest that the three M. tuberculosis proteins share a similar function and could be named PstS-1, PstS-2, and PstS-3, respectively. Molecular modeling of their three-dimensional structures using the structure of the E. coli PstS as a template and their inducibility by phosphate starvation support this view. Recombinant PstS-2 and PstS-3 were produced and purified by affinity chromatography. With PstS-1, these proteins were used to demonstrate the specificity of three groups of monoclonal antibodies. Using these antibodies in flow cytometry and immunoblotting analyses, we demonstrate that the three genes are expressed and their protein products are present and accessible at the mycobacterial surface as well as in its culture filtrate. Together with the M. tuberculosis genes encoding homologs of the PstA, PstB, and PstC components we cloned before, the present data suggest that at least one, and possibly several, related and functional ABC phosphate transporters exist in mycobacteria. It is hypothesized that the mycobacterial gene duplications presented here may be a subtle adaptation of intracellular pathogens to phosphate starvation in their alternating growth environments.

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.

We previously identified a 70-kDa serine/threonine protein kinase (MbK or PknD) from Mycobacterium tuberculosis Erdman containing a transmembrane domain and bearing a 270-amino acid N-terminal kinase domain. With the use of a polyclonal serum, Mbk has now been identified by Western blotting in protein extracts from M. tuberculosis and confirmed to be localised in the envelope. An identical mbk gene has been found by sequencing different M. tuberculosis and M. africanum strains. Surprisingly, in two virulent M. bovis strains and four different strains of M. bovis BCG, an additional adenine after position 829 of the open reading frame was found that produces a frame shift resulting in a predicted truncated, presumably free cytoplasmic protein, encoding only the N-terminal 30-kDa Mbk kinase domain. This sequence polymorphism has been confirmed by Western blot analysis of M. bovis BCG protein extracts.

We have cloned and sequenced the genes coding for the 856 antigen from M. bovis BCC and M. tuberculosis. Within this gene, the only difference in sequence between M. bovis BCC and M. tuberculosis corresponds, respectively, to a C+T yielding a Leu-tPhe replacement at position 100 of the mature 856 protein. Therefore as we described previously for the 85A gene, there is also very little variation between these two species within the 856 gene. KEY WORDS: antigen 85, Mycobacterium bovis BCC, Mycobacterium tuberculosis, secreted protein

Proteins of the antigen 85 complex are abundantly secreted into the culture supematant of a variety of mycobacteria (1, 2). These proteins are known to be responsible for the high affinity of mycobacteria to fibronectin (3). The 32 kDa protein (antigen 85A) appears to be a major stimulant of cellular and humoral immunity towards mycobacteria both in mice and man (4-6). The gene encoding the 32 kDa protein of Mycobacterium tuberculosis has recently been identified by us (7).Comparison of this gene with the a-antigen from M. bovis BCG (Tokyo) (also referred to as antigen 85B or as MPB 59 protein (8)) revealed 73.8% homology between coding regions of the DNA sequences (7, 9). This figure should be corrected to 77.5% considering that both sequences have been revised since their publication (10, and this report). It is of interest that the homology is limited to the region coding for the mature protein and the carboxy-terminal part of the signal sequences.After screening a Xgtl 1 BCG library (prepared from M. bovis BCG strain 1173P2) with a 5'-230 bp PstI fragment from our M. tuberculosis 32 kDa protein gene (7) (see Figure 2)