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Category: Microbial Genetics and Molecular Biology
Gene Transfer in Mycobacterium tuberculosis: Shuttle Phasmids to Enlightenment, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555818845/9781555818838_Chap01-1.gif /docserver/preview/fulltext/10.1128/9781555818845/9781555818838_Chap01-2.gifAbstract:
Infectious diseases have plagued humankind throughout history and have posed serious public health problems. Yet vaccines have eradicated smallpox and antibiotics have drastically decreased the mortality rate of many infectious agents ( 1 ). Although the precise viral agents had not yet been characterized, the smallpox vaccine work of Edward Jenner was critical in demonstrating that inoculation with pus from cowpox lesions could protect from a subsequent challenge with smallpox. These pioneering transfer experiments laid the groundwork for the eventual eradication of smallpox, as announced by the World Health Organization in 1979. The discovery of DNA as genetic material and the understanding of how this information translates into specific phenotypes have changed the paradigm for developing new vaccines, drugs, and diagnostic tests. Knowledge of the mechanisms of immunity and mechanisms of action of drugs has led to new vaccines and new antimicrobial agents. For example, the discovery of the Australia antigen (HBsAg) led to the subsequent engineering of the first recombinant vaccine, whose remarkable efficacy offers hope that eradication of hepatitis B in humans is not an unreasonable expectation ( 1 , 2 ). Similarly, HIV infections, which not so long ago were uniformly fatal, are now controlled with drugs that were developed by understanding the HIV genome and gene products required for the HIV life cycle. The key to the acquisition of the knowledge of these mechanisms has been identifying the elemental causes (i.e., genes and their products) that mediate immunity and drug resistance. The identification of these genes is made possible by being able to transfer the genes or mutated forms of the genes into causative agents or surrogate hosts. Such an approach was limited in Mycobacterium tuberculosis by the difficulty of transferring genes or alleles into M. tuberculosis or a suitable surrogate mycobacterial host. The construction of shuttle phasmids, chimeric molecules that replicate in Escherichia coli as plasmids and in mycobacteria as mycobacteriophages, was instrumental in developing gene transfer systems for M. tuberculosis. This review will discuss M. tuberculosis genetic systems and their impact on tuberculosis (TB) research.
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Specialized transduction is outlined as follows: the center plasmid represents the shuttle phasmid phA159, which contains 90% TM4 phage DNA and 10% plasmid DNA. The stars mark the sites of the mutations in the TM4 genome. The nonessential genes that are deleted to create the shuttle phasmid are noted in the picture, flanked by PacI sites. This site can be replaced with one of three things: (i) a reporter gene such as green fluorescent protein (GFP), (ii) an allelic exchange substrate (AES) that contains an antibiotic resistance marker, or (iii) a transposase gene to facilitate transposon mutagenesis. Going counterclockwise in this schematic, the recombinant cosmid can be packaged into phage heads using an in vitro packaging mix, and the subsequent phages can be used to transduce E. coli to create E. coli transductant colonies. Going clockwise from the shuttle phasmid, one can transfect M. smegmatis mc2155 at 30°C to yield plaques on an M. smegmatis lawn, resulting from lysis of the cells. The plaques can then be purified and amplified to obtain a high-titer phage lysate that can subsequently be used to transduce any mycobacterial species.
Generation of mutants in the RD1 region of M. tuberculosis and M. bovis. (A) Schematic of M. tuberculosis H37Rv RD1 region showing predicted NcoI sites. Arrows at the top represent the genes in this region. Upstream flanking sequences (UFS) and downstream flanking sequences (DFS) used to generate the knockout are indicated as filled bars above the grid line. Each increment in the grid line represents 1 kbp. The RD1 sequence deleted from M. bovis BCG is represented by an open bar spanning from Rv3871 to Rv3879c. The site of the insertion of transposon Tn5370 is also indicated. (B) Southern analysis of the NcoI-digested genomic DNA isolated from the wild type and the ΔRD1 mutants generated by using specialized transduction in M. tuberculosis and M. bovis. Lane 1, M. tuberculosis H37Rv; lane 2, M. tuberculosis H37Rv ΔRD1; lane 3, M. tuberculosis Erdman; lane 4, M. tuberculosis Erdman ΔRD1; lane 5, M. tuberculosis CDC1551; lane 6, M. tuberculosis CDC1551 ΔRD1; lane 7, M. bovis Ravenel; lane 8, M. bovis Ravenel ΔRD1. The probe used in the Southern analysis was either DFS (left), demonstrating the deletion of RD1, or IS6110-specific (right). The IS6110 probe is used to characterize the four strains. Reprinted with permission.
(Top) Schematic representation of the specialized transducing phage. A replicating shuttle phasmid phAE2067 containing mabA, inhA carrying the S94A mutation, a hyg resistance cassette, and hemZ was used to transduce M. tuberculosis (M. tb). The two possible sites of recombination are marked 1 and 2. (Middle) The recombination can occur either before the point mutation (crossover type 1), resulting in an INH-resistant and ETH-resistant recombinant carrying the S94A mutation, or after the point mutation (crossover type 2; the strain contains a wild-type inhA gene). (Bottom) Individual M. tuberculosis H37Rv inhA(S94A) transductants (n = 150) were screened by picking and patching onto plates containing either hygromycin (50 µg/ml) or INH (0.2 µg/ml). Reprinted with permission.
Improvements for high-throughput specialized transduction