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Chapter 1 : Gene Transfer in : Shuttle Phasmids to Enlightenment

Author: William R. Jacobs1
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Affiliations: 1: Howard Hughes Medical Institute, Albert Einstein College of Medicine, 1301 Morris Park Avenue, Bronx, NY 10461
Content Type: Monograph
Publication Year: 2014

Category: Microbial Genetics and Molecular Biology

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Abstract:

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 ( ). 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 ( ). 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 by the difficulty of transferring genes or alleles into or a suitable surrogate mycobacterial host. The construction of shuttle phasmids, chimeric molecules that replicate in as plasmids and in mycobacteria as mycobacteriophages, was instrumental in developing gene transfer systems for . This review will discuss genetic systems and their impact on tuberculosis (TB) research.

Citation: Jacobs W. 2014. Gene Transfer in : Shuttle Phasmids to Enlightenment, p 3-25. In Hatfull G, Jacobs W (ed), Molecular Genetics of Mycobacteria, Second Edition. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MGM2-0037-2013
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Gene Transfer in Mycobacterium tuberculosis: Shuttle Phasmids to Enlightenment
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Citation: Jacobs W. 2014. Gene Transfer in : Shuttle Phasmids to Enlightenment, p 3-25. In Hatfull G, Jacobs W (ed), Molecular Genetics of Mycobacteria, Second Edition. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MGM2-0037-2013
/content/10.1128/9781555818845.chap1.fig1-1
Figure 1
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 packaging mix, and the subsequent phages can be used to transduce to create transductant colonies. Going clockwise from the shuttle phasmid, one can transfect mc155 at 30°C to yield plaques on an 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.
10.1128/9781555818845/fig1-1_thmb.gif
10.1128/9781555818845/fig1-1.gif
Image of Figure 1
Figure 1

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 packaging mix, and the subsequent phages can be used to transduce to create transductant colonies. Going clockwise from the shuttle phasmid, one can transfect mc155 at 30°C to yield plaques on an 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.

Citation: Jacobs W. 2014. Gene Transfer in : Shuttle Phasmids to Enlightenment, p 3-25. In Hatfull G, Jacobs W (ed), Molecular Genetics of Mycobacteria, Second Edition. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MGM2-0037-2013
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Figure 2
Generation of mutants in the region of and Schematic of H37Rv 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 sequence deleted from BCG is represented by an open bar spanning from to . The site of the insertion of transposon Tn is also indicated. Southern analysis of the NcoI-digested genomic DNA isolated from the wild type and the Δ mutants generated by using specialized transduction in and . Lane 1, H37Rv; lane 2, H37Rv Δ; lane 3, Erdman; lane 4, Erdman Δ; lane 5, CDC1551; lane 6, CDC1551 Δ; lane 7, Ravenel; lane 8, Ravenel Δ. The probe used in the Southern analysis was either DFS (left), demonstrating the deletion of , or IS-specific (right). The IS probe is used to characterize the four strains. Reprinted with permission.
10.1128/9781555818845/fig1-2_thmb.gif
10.1128/9781555818845/fig1-2.gif
Image of Figure 2
Figure 2

Generation of mutants in the region of and Schematic of H37Rv 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 sequence deleted from BCG is represented by an open bar spanning from to . The site of the insertion of transposon Tn is also indicated. Southern analysis of the NcoI-digested genomic DNA isolated from the wild type and the Δ mutants generated by using specialized transduction in and . Lane 1, H37Rv; lane 2, H37Rv Δ; lane 3, Erdman; lane 4, Erdman Δ; lane 5, CDC1551; lane 6, CDC1551 Δ; lane 7, Ravenel; lane 8, Ravenel Δ. The probe used in the Southern analysis was either DFS (left), demonstrating the deletion of , or IS-specific (right). The IS probe is used to characterize the four strains. Reprinted with permission.

Citation: Jacobs W. 2014. Gene Transfer in : Shuttle Phasmids to Enlightenment, p 3-25. In Hatfull G, Jacobs W (ed), Molecular Genetics of Mycobacteria, Second Edition. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MGM2-0037-2013
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Figure 3
Schematic representation of the specialized transducing phage. A replicating shuttle phasmid phAE2067 containing , carrying the S94A mutation, a resistance cassette, and was used to transduce (). The two possible sites of recombination are marked 1 and 2. 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 gene). Individual H37Rv (S94A) transductants ( = 150) were screened by picking and patching onto plates containing either hygromycin (50 µg/ml) or INH (0.2 µg/ml). Reprinted with permission
10.1128/9781555818845/fig1-3_thmb.gif
10.1128/9781555818845/fig1-3.gif
Image of Figure 3
Figure 3

Schematic representation of the specialized transducing phage. A replicating shuttle phasmid phAE2067 containing , carrying the S94A mutation, a resistance cassette, and was used to transduce (). The two possible sites of recombination are marked 1 and 2. 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 gene). Individual H37Rv (S94A) transductants ( = 150) were screened by picking and patching onto plates containing either hygromycin (50 µg/ml) or INH (0.2 µg/ml). Reprinted with permission

Citation: Jacobs W. 2014. Gene Transfer in : Shuttle Phasmids to Enlightenment, p 3-25. In Hatfull G, Jacobs W (ed), Molecular Genetics of Mycobacteria, Second Edition. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MGM2-0037-2013
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Tables

Gene Transfer in Mycobacterium tuberculosis: Shuttle Phasmids to Enlightenment
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Citation: Jacobs W. 2014. Gene Transfer in : Shuttle Phasmids to Enlightenment, p 3-25. In Hatfull G, Jacobs W (ed), Molecular Genetics of Mycobacteria, Second Edition. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MGM2-0037-2013
/content/10.1128/9781555818845.chap1.tab1-1
Table 1
Improvements for high-throughput specialized transduction
Generic image for table
Table 1

Improvements for high-throughput specialized transduction

Citation: Jacobs W. 2014. Gene Transfer in : Shuttle Phasmids to Enlightenment, p 3-25. In Hatfull G, Jacobs W (ed), Molecular Genetics of Mycobacteria, Second Edition. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MGM2-0037-2013

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