Cold Spring Harbor Laboratory Courses in Phage and Bacterial Genetics: A Catalyst for Innovation in Molecular Biology, Genetics, and Microbiology.

The ability to answer complex scientific questions depends on the experimental methods available. New methods often allow scientists to answer questions that were previously intractable, leading to discoveries that often dramatically change a field. Beginning with Max Delbrück's famous summer phage course at Cold Spring Harbor Laboratory in 1945, the Phage, Bacterial Genetics, and Advanced Bacterial Genetics courses have provided hands-on experiences to generations of scientists that facilitated the broad adoption of new experimental methods into laboratories around the world. These methods have led to discoveries that changed the way we think about genetics, bacteria, and viruses, transforming our understanding of biology. The impact of these courses has been further amplified by published laboratory manuals that provide detailed protocols for the evolving experimental toolkit. These courses catalyzed intensive and critical discourse about ideas that were previously intractable and provided novel experimental approaches to answer new questions-a process that epitomizes Thomas Kuhn's concepts of Scientific Revolution, spinning off the new field of Molecular Biology and dramatically changing the field of microbiology.

Fitness effects of replichore imbalance in Salmonella enterica.

A fitness cost due to imbalanced replichores has been proposed to provoke chromosome rearrangements in Salmonella enterica serovars. To determine the impact of replichore imbalance on fitness, the relative fitness of isogenic Salmonella strains containing transposon-held duplications of various sizes and at various chromosomal locations was determined. Although duplication of certain genes influenced fitness, a replichore imbalance of up to 16° did not affect fitness.

Transport of phage P22 DNA across the cytoplasmic membrane.

Although a great deal is known about the life cycle of bacteriophage P22, the mechanism of phage DNA transport into Salmonella is poorly understood. P22 DNA is initially ejected into the periplasmic space and subsequently transported into the host cytoplasm. Three phage-encoded proteins (gp16, gp20, and gp7) are coejected with the DNA. To test the hypothesis that one or more of these proteins mediate transport of the DNA across the cytoplasmic membrane, we purified gp16, gp20, and gp7 and analyzed their ability to associate with membranes and to facilitate DNA uptake into membrane vesicles in vitro. Membrane association experiments revealed that gp16 partitioned into the membrane fraction, while gp20 and gp7 remained in the soluble fraction. Moreover, the addition of gp16, but not gp7 or gp20, to liposomes preloaded with a fluorescent dye promoted release of the dye. Transport of (32)P-labeled DNA into liposomes occurred only in the presence of gp16 and an artificially created membrane potential. Taken together, these results suggest that gp16 partitions into the cytoplasmic membrane and mediates the active transport of P22 DNA across the cytoplasmic membrane of Salmonella.

Macrophages influence Salmonella host-specificity in vivo.

The generalist Salmonella enterica serovar Typhimurium causes disease in many animal species, but the closely related host-specific serovar Typhi only causes disease in humans. Typhi and Typhimurium share major virulence loci; hence it is not known exactly why Typhi does not cause disease in mice. We tested the hypothesis that macrophages contribute to Salmonella host-specificity in mice. No significant difference in survival of the two serovars was observed in vitro in mouse macrophage cell lines and primary murine peritoneal and bone marrow-derived macrophages after 24h. In contrast, differential survival was observed following infection in vivo. When BALB/c mice were infected intraperitoneally (i.p.), both Typhi and Typhimurium induced neutrophil influx into the peritoneum and macrophages were the major cell type containing internalized bacteria at 0.5 and 4h post-infection for both serovars. The number of Typhimurium in macrophages remained high at 4h post-infection, but the number of Typhi in macrophages decreased substantially within 4h after i.p. infection. These results indicate that macrophages are able to distinguish Typhi from Typhimurium when infected in vivo but no significant differences were observed after 24h in vitro, suggesting that the differential killing of the two serovars by macrophages requires additional factors within the host.

Use of antibiotic-resistant transposons for mutagenesis.

One of the greatest advances in molecular genetics has been the application of selectable transposons in molecular biology. After 30 years of use in microbial genetics studies, transposons remain indispensable tools for the generation of null alleles tagged with selectable markers, genetic mapping, manipulation of chromosomes, and generation of various fusion derivatives. The number and uses of transposons as molecular tools continues to expand into new fields such as genome sciences and molecular pathogenesis. This chapter outlines some of the many uses of transposons for molecular genetic analysis and strategies for their use.

Isolation of transposon insertions.

Transposon insertions in and near a gene of interest facilitate the genetic characterization of a gene in vivo. This chapter is dedicated to describing the isolation of mini-Tn10 insertions in any desired nonessential gene in Salmonella enterica, as well as the isolation of mini-Tn10 insertions near particular genes. The protocols describe use of a tetracycline-resistant Tn10 derivative, but similar approaches can be used for derivatives resistant to other antibiotics. In addition, these approaches are directly applicable to other bacteria that have generalized transducing phages.

Localized mutagenesis.

Localized mutagenesis can be used to obtain mutants in genes of interest based on linkage to selectable markers. Mutagens diethylsulfate and hydroxylamine are used to obtain predominantly transition mutations in the DNA either by whole chromosomal mutagenesis or mutagenesis of DNA isolated as purified plasmid or packaged in transducing phage particles. Selectable markers can include those based on auxotrophic requirements, carbon or nitrogen source utilization, or antibiotic resistance markers, such as those encoded in transposons.

Strain collections and genetic nomenclature.

The ease of rapidly accumulating a large number of mutants requires careful bookkeeping to avoid confusing one mutant with another. Each mutant constructed should be assigned a strain number. Strain numbers usually consist of two to three capital letters designating the lab where they were constructed and a serial numbering of the strains in a central laboratory collection. Every mutation should be assigned a name that corresponds to a particular gene or phenotype, and an allele number that identifies each specific isolate. When available for a particular group of bacteria, genetic stock centers are the ultimate resources for gene names and allele numbers. Examples include the Salmonella Genetic Stock Centre ( http://www.ucalgary.ca/~kesander/), and the E. coli Genetic Stock Center (http://cgsc.biology.yale.edu/). It is also important to indicate how the strain was constructed, the parental (recipient) strain, and the source of any donor DNA transferred into the recipient strain (Maloy et al., 1996).

Use of operon and gene fusions to study gene regulation in Salmonella.

Coupling the expression of a gene with an easily assayable reporter gene provides a simple genetic trick for studying the regulation of gene expression. Two types of fusions between a gene and a reporter gene are possible. Operon fusions place the transcription of a reporter gene under the control of the promoter of a target gene, but the translation of the reporter gene and target gene are independent; gene fusions place the transcription and translation of a reporter gene under the control of a target gene, and result in a hybrid protein. Such fusions can be constructed in vitro using recombinant DNA techniques or in vivo using transposon derivatives. Many different transposon derivatives are available for constructing operon and gene fusions, but two extremely useful fusion vectors are (1) Mu derivatives that form operon and gene fusions to the lacZ gene, and (2) Tn5 derivative that forms gene fusions to the phoA gene.

Dissecting nucleic acid-protein interactions using challenge phage.

The bacteriophage P22-based challenge system is a sophisticated genetic tool for the characterization of sequence-specific recognition of DNA and RNA in vivo. The construction of challenge phage follows simple phage lysate preparations and detection of constructs by positive selection methods for plaques on selective strains. The challenge phage system is a powerful tool for the characterization of protein-DNA and protein-RNA interactions in vivo. The challenge phage has been further developed to characterize the interactions of multiple proteins in heteromultimeric complexes that are required for DNA binding. Under appropriate conditions, expression of the ant gene determines the lysis-lysogeny decision of P22. This provides a positive selection for and against DNA binding: repression of ant can be selected by requiring growth of lysogens, and mutants that cannot repress ant can be selected by requiring lytic growth of the phage. Thus, placing ant gene expression under the control of a specific DNA-protein interaction provides very strong genetic selections for regulatory mutations in the DNA-binding protein and DNA-binding site that either increase or decrease the apparent strength of a DNA-protein interaction in vivo. Furthermore, the challenge phage contains a kanamycin-resistance element that can be used to either directly select for lysogeny or to determine the frequency of lysogeny for a given protein-DNA interaction to measure the efficiency of DNA binding in vivo. Selection for lysogeny can be used to isolate DNA-binding proteins with altered or enhanced DNA-binding specificities. The challenge phage selection provides a general method for identifying critical residues involved in DNA-protein interactions. Challenge phage selections have been used to genetically dissect many different prokaryotic and eukaryotic DNA-binding interactions.

Mud-P22.

Mud-P22 derivatives are hybrids between phage Mu and P22 that can be inserted at essentially any desired site on the Salmonella chromosome (Benson and Goldman, 1992; Youderian et al., 1988). Induction of Mud-P22 insertions yields phage particles that, as a population, carry chromosomal DNA from the region between 150 and 250Kb on one side of the insertion. Thus, phage lysates from a representative set of Mud-P22 insertions into the S. typhimurium chromosome yield an ordered library of DNA that provides powerful tools for the genetic and physical analysis of the Salmonella genome. Although Mud-P22 has not yet been used in other species, this approach should be applicable in a variety of other bacteria as well.

Facile approach for constructing TEV insertions to probe protein structure in vivo.

The tobacco etch virus (TEV) protease has been used as a tool to examine protein structure in vivo. TEV cleavage sites (TEVcs) have been introduced via cloning into unique restriction sites or random transposon mutagenesis. We describe a facile, efficient method for introducing TEVcs at precise locations in a gene to test specific predictions about protein structure. The method uses the lamda Red recombination system to construct seamless, in-frame insertions of the TEVcs at any desired location within an open reading frame (ORF). The system was tested using the multifunctional PutA protein Salmonella enterica sv. Typhimurium. The first step involved insertion of a chloramphenicol resistance (Cam(R)) cassette with a transcriptional terminator at the desired location. A second swap then replaces the Cam(R) insertion with the TEVcs. Placing a copy of the lac operon downstream of the putA gene provides a simple counterselection for replacement of the Cam(R) insertion and also provides a reporter gene for monitoring transcription of the mutated gene.