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Chapter 51 : Genetic Tools for Use with Listeria monocytogenes

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Abstract:

The number and sophistication of genetic tools that have become available in recent years for the molecular characterization of have continued to increase. Plasmid vectors, reporter genes, systems designed for transposon mutagenesis, heterologous expression systems, integration vectors, and transducing phage have all greatly advanced the experimental capacity to generate, characterize, and complement mutations within and to define functional roles of gene products. This chapter provides a brief description of the genetic tools currently available for use with . Key references are given throughout this description to provide sources for expanded details on plasmid constructions, assay conditions, and other technical aspects. The variety of genetic tools described is meant to be representative of the resources available to those interested in genetics. More widely used tools for studying expression profiles of bacteria are whole-genome DNA macro- and microarrays, which provide a comprehensive transcriptional analysis enabling researchers to view the organism as a system. Several reporter genes developed for use in other systems have proven useful for monitoring transcriptional gene regulation. Transcriptional fusions to reporter genes such as , , , , and have all been constructed in and have been used successfully to monitor patterns of bacterial gene expression in culture and within infected cells and animals. The advantages and/or disadvantages of some of these reporter systems are discussed briefly.

Citation: Higgins D, Buchrieser C, Freitag N. 2006. Genetic Tools for Use with Listeria monocytogenes, p 620-633. In Fischetti V, Novick R, Ferretti J, Portnoy D, Rood J (ed), Gram-Positive Pathogens, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816513.ch51

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Image of FIGURE 1
FIGURE 1

Plasmid vectors for gene expression-complementation in . (A) pAM401 (88) contains the p15A origin of replication, chloramphenicol (), and tetracycline () resistance genes for selection in . A chloramphenicol resistance gene () and origin of replication ( ori) gene allow selection in . Unique cloning sites present within the gram-negative replicon portion are indicated. (B) pMK4 (79) contains a ColE1 origin of replication, β-lactamase gene (), and /− resistance gene for selection in . The and /− resistance genes provide selection in . pMK4 harbors a fragment of the gene () that is capable of α-complementation and contains five unique restriction sites for cloning.

Citation: Higgins D, Buchrieser C, Freitag N. 2006. Genetic Tools for Use with Listeria monocytogenes, p 620-633. In Fischetti V, Novick R, Ferretti J, Portnoy D, Rood J (ed), Gram-Positive Pathogens, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816513.ch51
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Image of FIGURE 2
FIGURE 2

Inducible expression-integration vectors for . (A) pLIV1 (20) contains the following sequences: a temperature-sensitive origin of replication (ts ori) and a chloramphenicol resistance gene () for plasmid selection in , the ColE1 origin of replication and β-lactamase gene () for cloning and selection in , an origin of transfer (oriT) to allow conjugal mating of plasmid derivatives from to , a unique XbaI restriction site for cloning genes under the transcriptional control of the SPAC/Oid IPTG-inducible promoter (20), tandem copies of the T1 transcription terminator (T1 terminators) upstream of the SPAC/Oid region to ensure that transcription of cloned genes initiates only from the SPAC promoter, the p60 gene () promoter to allow constitutive expression of the repressor gene (), and an erythromycin resistance determinant within the expression cassette ( ) for selection of inducible constructs on the chromosome. The inducible expression cassette can be integrated into the chromosome within the gene (Z′) via allelic exchange. (B) pLIV2 ( Table 1 ) is a new inducible expression vector derived from pLIV1 and the site-specific phage integration vector pPL2 ( Fig. 4 ) (52). pLIV2 contains the inducible expression cassette region from pLIV1 with eight unique restriction sites available for cloning. pLIV2 contains the p15A origin of replication and a chloramphenicol resistance gene ( ) for selection in . The PSA bacteriophage integrase gene () and attachment site () allow site-specific integration of the vector within an tRNAArg gene, following conjugal transfer from facilitated by the region. The resistance gene allows selection of integrated plasmids in .

Citation: Higgins D, Buchrieser C, Freitag N. 2006. Genetic Tools for Use with Listeria monocytogenes, p 620-633. In Fischetti V, Novick R, Ferretti J, Portnoy D, Rood J (ed), Gram-Positive Pathogens, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816513.ch51
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Image of FIGURE 3
FIGURE 3

Schematic diagram for construction of chromosomal mutations in . The method depicted and described in the text is suitable for the introduction of insertions, deletions, or single- or multiple-nucleotide substitutions within the chromosome. (A) Chromosomal integration of the temperature sensitive plasmid vector by homologous recombination between plasmid encoded genes A or B and the chromosomal alleles. The designated crossover points are arbitrary and can occur on either side of gene X. (B) Growth of bacterial cultures in the presence of drug selection at temperatures nonpermissive for plasmid replication selects for the merodiploid intermediates that result from plasmid integration into the chromosome. (C) Merodiploid intermediate strains are then passed for several generations without selective pressure at temperatures permissive for plasmid replication. Spontaneous excision of the integrated plasmid from the chromosome occurs. (D) Excised plasmids are cured at temperatures nonpermissive for plasmid replication in the absence of drug selection.

Citation: Higgins D, Buchrieser C, Freitag N. 2006. Genetic Tools for Use with Listeria monocytogenes, p 620-633. In Fischetti V, Novick R, Ferretti J, Portnoy D, Rood J (ed), Gram-Positive Pathogens, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816513.ch51
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Image of FIGURE 4
FIGURE 4

Site-specific phage integration vectors for . (A) pPL1 (52) contains the following features: a p15A origin of replication and chloramphenicol resistance gene () for selection in , an origin of transfer () for conjugal mating from to , the U153 bacteriophage integrase gene ( ) and attachment site () to allow site-specific integration of the vector within the gene, and a chloramphenicol resistance gene ( ) for selection of plasmid maintenance in . A multiple cloning site region (MCS) contains 12 unique restriction sites for cloning. (B) Features of pPL2 (52) are as described for pPL1 with the exception that pPL2 contains the PSA bacteriophage integrase gene () and attachment site () for site-specific integration of pPL2 within an tRNA gene. pPL2 contains 13 unique restriction sites within the MCS region.

Citation: Higgins D, Buchrieser C, Freitag N. 2006. Genetic Tools for Use with Listeria monocytogenes, p 620-633. In Fischetti V, Novick R, Ferretti J, Portnoy D, Rood J (ed), Gram-Positive Pathogens, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816513.ch51
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Image of FIGURE 5
FIGURE 5

Tn delivery plasmids pLTV1 and pLTV3. ColE1, replication functions derived from pBR322 (10); pE194Ts, temperature-sensitive gram-positive origin of replication (83); , neomycinphosphotransferase II; , bleomycin resistance gene; , Tn ribosomal methyltransferase gene; , tetracycline resistance gene from pAMα1Δ1 (65); , pC194-derived chloramphenicol acetyltransferase gene (45); , pBR322 β-lactamase gene; , promoterless gene from with translation initiation signals derived from gene .

Citation: Higgins D, Buchrieser C, Freitag N. 2006. Genetic Tools for Use with Listeria monocytogenes, p 620-633. In Fischetti V, Novick R, Ferretti J, Portnoy D, Rood J (ed), Gram-Positive Pathogens, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816513.ch51
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Image of FIGURE 6
FIGURE 6

A method for isolating a transposon insertion linked to a particular mutant locus in (transposon tagging, as described by D. Kaiser) (49). As an example, a mutant strain is depicted with a single base pair change within the bacterial chromosome that confers a mutant phenotype (mutation indicated by X). A library of random transposon insertions (Tn) is generated within the mutant strain, and a U153 bacteriophage lysate is prepared from the mutant strain transposon insertion library. Wild-type is incubated with the phage lysate, and transductants are selected based on the presence of the antibiotic resistance marker provided by Tn and the mutant phenoytpe conferred by mutation X. The Tn transposon is now genetically linked to mutation X, and the distance between the two can be determined based on the cotransduction frequency of Tn and X (see reference 59 for an experimental example and additional details). This approach can theoretically be used to map any unmarked mutation that confers a phenotype within the chromosome.

Citation: Higgins D, Buchrieser C, Freitag N. 2006. Genetic Tools for Use with Listeria monocytogenes, p 620-633. In Fischetti V, Novick R, Ferretti J, Portnoy D, Rood J (ed), Gram-Positive Pathogens, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816513.ch51
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Tables

Generic image for table
TABLE 1

Representative plasmid vectors

Presence of plasmid origin for replication function in gram-negative (gram−) or gram-positive (gram+) bacteria.

Presence or absence of origin of transfer, allowing plasmid conjugation.

Antibiotic resistance markers for drug selection in : Cm, chloramphenicol; Kn, kanamycin; Em, erythromycin; Sp, spectinomycin; Ble, bleomycin; Tet, tetracycline.

ts, temperature-sensitive plasmid origin of replication.

Citation: Higgins D, Buchrieser C, Freitag N. 2006. Genetic Tools for Use with Listeria monocytogenes, p 620-633. In Fischetti V, Novick R, Ferretti J, Portnoy D, Rood J (ed), Gram-Positive Pathogens, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816513.ch51

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