Identification of genes encoding exported Mycobacterium tuberculosis proteins using a Tn552'phoA in vitro transposition system

Abstract

Secreted and cell envelope-associated proteins are important to both Mycobacterium tuberculosis pathogenesis and the generation of protective immunity to M. tuberculosis. We used an in vitro Tn552'phoA transposition system to identify exported proteins of M. tuberculosis. The system is simple and efficient, and the transposon inserts randomly into target DNA. M. tuberculosis genomic libraries were targeted with Tn552'phoA transposons, and these libraries were screened in M. smegmatis for active PhoA translational fusions. Thirty-two different M. tuberculosis open reading frames were identified; eight contain standard signal peptides, six contain lipoprotein signal peptides, and seventeen contain one or more transmembrane domains. Four of these proteins had not yet been assigned as exported proteins in the M. tuberculosis databases. This collection of exported proteins includes factors that are known to participate in the immune response of M. tuberculosis and proteins with homologies, suggesting a role in pathogenesis. Nine of the proteins appear to be unique to mycobacteria and represent promising candidates for factors that participate in protective immunity and virulence. This technology of creating comprehensive fusion libraries should be applicable to other organisms.

FIG. 1

Tn552′phoA transposons. (A) A representative Tn552′phoA element is shown. The 48-bp Tn552 ends are represented by arrowheads. The nucleotide sequence of the modified end with the mutation to create the ORF is shown with its amino acid sequence below it. The mutation of A to T at position 41 from the outside end is shown by the underlined nucleotide. The original sequence of the stop codon is shown below it, with an arrow indicating the changed nucleotide. The first two nucleotides are in lowercase letters, indicating that they require an additional nucleotide from the target sequence to form a codon. (B) Schematic representations of the three elements used in this study. The asterisk indicates the presence of the mutation in the Tn552 sequences (arrowheads). (C) Insertion into the target DNA is accomplished by transposase (TnpA), transposon, and target DNA under the appropriate reaction conditions. The ′phoA gene will be in frame when it inserts after the first nucleotide of a codon in the proper orientation. The target site is shown by an uppercase “N,” and it is duplicated upon insertion. The asterisk indicates the presence of the mutation in the Tn552 sequences (arrowheads).

FIG. 2

Quantitative alkaline phosphatase activity of M. tuberculosis DNA-phoA fusions. Alkaline phosphatase activity was measured as described in Materials and Methods. The values presented are the average of at least three independent experiments, with error bars indicating standard deviation. The ORF fused to PhoA is indicated for each strain, ′PhoA represents the negative control strain, mc²2757. (A) Representative “early” blue strains from left to right: mc²2716, mc²2720, mc²2721, mc²2722, mc²2725, mc²2727, mc²2733, mc²2734, mc²2745, mc²2749, and mc²2750. (B) Representative “late” blue strains from left to right: mc²2719, mc²2730, mc²2731, mc²2732, mc²2735, mc²2736, mc²2737, mc²2738, mc²2739, mc²2740, mc²2741, mc²2742, mc²2743, mc²2744, mc²2712, mc²2748, mc²2752, mc²2753, mc²2754, mc²2755, and mc²2756. The asterisk indicates the level of background alkaline phosphatase activity.