Three different putative phosphate transport receptors are encoded by the Mycobacterium tuberculosis genome and are present at the surface of Mycobacterium bovis BCG

Abstract

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.

Porins Are Required for Uptake of Phosphates by Mycobacterium smegmatis

“…We show that the outer membrane represents a permeability barrier for phosphates in both assays, in contrast to previous claims that PhoA and PstS are cell surface proteins (18,19). Importantly, we demonstrate that Msp porins are required for the * Corresponding author.…”

Cross‐Reactive Immune Responses Against Mycobacterium bovis BCG in Mice Infected with Non‐Tuberculous Mycobacteria Belonging to the MAIS‐Group

“…After completion, proteins were electrophoretically transferred to nitrocellulose sheets. Strips were blocked with fetal calf serum (FCS) and incubated overnight in 1:50 dilution of the pooled serum samples collected at the successive time-points or in 1:10 dilution of monoclonal antibodies (MoAb) directed against the following antigens from M. bovis BCG: 30-32 kDa Ag85 (MoAb TD 32-15 [14,15]); 37-38 kDa PstS-2 (2A-1-2 [16]) and the 65 kDa hsp 65 (1A1 [17]). Antibodies were visualized by peroxidase-conjugated rabbit anti-mouse IgG antibody (Dakopatts, Copenhagen, Denmark) and a-chloronaphthol.…”

“…Antibodies in BALB/c sera were predominantly directed against the GroEL-related hsp 65 (and its degradation products) identified by the 1A1 MoAb [17]. Starting from about day 50, additional reactivities against the 37-38 kDa PstS-2 homologue [16], against a 35 kDa protein [13] and against the 30-32 kDa Ag85 complex [14,15] were observed in sera from M. intracellulare-infected BALB/c mice. Sera from M. aviumand M. scrofulaceum-infected BALB/c mice were essentially directed against the hsp 65 stress protein.…”

The Phosphate Starvation Stimulon ofCorynebacterium glutamicumDetermined by DNA Microarray Analyses

“…It was then realized that the genome of M. tuberculosis harbors three different phosphate-binding protein genes in three operons (pstBS1C1A2, pstS2, and pstS3C2A1) at one locus (12,27) and that the phosphate-binding proteins are surface attached (27). By using translational fusions, two P i starvation-responsive promoters were identified for the pstBS1C1A2 operon (43).…”