DNA transposition is an important biological phenomenon that mediates genome rearrangements, inheritance of antibiotic resistance determinants, and integration of retroviral DNA. Transposition has also become a powerful tool in genetic analysis, with applications in creating insertional knockout mutations, generating gene-operon fusions to reporter functions, providing physical or genetic landmarks for the cloning of adjacent DNAs, and locating primer binding sites for DNA sequence analysis. DNA transposition studies to date usually have involved strictly in vivo approaches, in which the transposon of choice and the gene encoding the transposase responsible for catalyzing the transposition have to be introduced into the cell to be studied (microbial systems and applications are reviewed in ref. 1). However, all in vivo systems have a number of technical limitations. For instance, the transposase must be expressed in the target host, the transposon must be introduced into the host on a suicide vector, and the transposase usually is expressed in subsequent generations, resulting in potential genetic instability. A number of in vitro transposition systems (for Tn5, Tn7, Mu, Himar1, and Ty1) have been described, which bypass many limitations of in vivo systems. For this purpose, we have developed a technique for transposition that involves the formation in vitro of released Tn5 transposition complexes (TransposomesTM) followed by introduction of the complexes into the target cell of choice by electroporation. In this report, we show that this simple, robust technology can generate high-efficiency transposition in all tested bacterial species (Escherichia coli, Salmonella typhimurium, and Proteus vulgaris) We also isolated transposition events in the yeast Saccharomyces cerevisiae.

The food-borne pathogen Listeria monocytogenes is notable for its ability to grow under osmotic stress and at low temperatures. It is known to accumulate the compatible solutes glycine betaine and carnitine from the medium in response to osmotic or chill stress, and this accumulation confers tolerance to these stresses. Two permeases that transport glycine betaine have been identified, both of which are activated by hyperosmotic stress and one of which is activated by low temperature. An osmotically activated transporter for carnitine, OpuC, has also been identified. We have isolated a Tn917-LTV3 insertional mutant that could not be rescued from hyperosmotic stress by exogenous carnitine. The mutant, LTS4a, grew indistinguishably from a control strain (DP-L1044) in the absence of stress or in the absence of carnitine, but DP-L1044 grew substantially faster under osmotic or chill stress in the presence of carnitine. LTS4a was found to be strongly impaired in KCl-activated as well as chill-activated carnitine transport. 13 C nuclear magnetic resonance spectroscopy of perchloric acid extracts showed that accumulation of carnitine by LTS4a was negligible under all conditions tested. Direct sequencing of LTS4a genomic DNA with a primer based on Tn917-LTV3 yielded a 487-bp sequence, which allowed us to determine that the opuC operon had been interrupted by the transposon. It can be concluded that opuC encodes a carnitine transporter that can be activated by either hyperosmotic stress or chill and that the transport system plays a significant role in the tolerance of L. monocytogenes to both forms of environmental stress. (28), and contamination of food is therefore common. Listeriosis occurs mainly in immunocompromised individuals, such as pregnant women, AIDS patients, and transplant recipients, and is approximately 25% fatal (19). L. monocytogenes has been shown to be tolerant to levels of osmotic stress as high as that afforded by 10% NaCl and grows at temperatures as low as Ϫ0.1°C (5). Thus, it is resistant to two major forms of food preservation. Listeria monocytogenes is a gram-positive, food-borne pathogen (15). It is widely distributed in the environmentA common response to hyperosmotic stress, after an initial loss of cellular water, is the intracellular accumulation of compatible solutes in order to restore osmotic pressure and turgor (29). The presence of the compatible solute glycine betaine or carnitine in the medium therefore confers a degree of osmotolerance (3,13,14,22). We have also found that L. monocytogenes accumulates glycine betaine and carnitine in response to chill stress and that the presence of these compatible solutes in the medium confers cryotolerance (11,13,22). These osmolytes are not synthesized by L. monocytogenes and must be transported from the environment. The rate and the extent of their transport can be controlled by regulation of the synthesis of the transport systems and by their biochemical activation by osmotic gradients, temperature, or other factors.In earlier work, we demon...

Pierce's disease, a lethal disease of grapevine, is caused by Xylella fastidiosa, a gram-negative, xylem-limited bacterium that is transmitted from plant to plant by xylem-feeding insects. Strains of X. fastidiosa also have been associated with diseases that cause tremendous losses in many other economically important plants, including citrus. Although the complete genome sequence of X. fastidiosa has recently been determined, the inability to transform or produce transposon mutants of X. fastidiosa has been a major impediment to understanding pathogen-, plant-, and insect-vector interactions. We evaluated the ability of four different suicide vectors carrying either Tn5 or Tn10 transposons as well as a preformed Tn5 transposase-transposon synaptic complex (transposome) to transpose X. fastidiosa. The four suicide vectors failed to produce any detectable transposition events. Electroporation of transposomes, however, yielded 6 x 10(3) and 4 x 10(3) Tn5 mutants per microg of DNA in two different grapevine strains of X. fastidiosa. Molecular analysis showed that the transposition insertions were single, independent, stable events. Sequence analysis of the Tn5 insertion sites indicated that the transpositions occur randomly in the X. fastidiosa genome. Transposome-mediated mutagenesis should facilitate the identification of X. fastidiosa genes that mediate plant pathogenicity and insect transmission.

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Base excision sequence scanning (BESS) is a new polymerase chain reaction (PCR)-based mutation scanning method that locates and identifies all DNA mutations. The BESS method consists of two procedures that generate "T" (BESS T-Scan) and "G" ladders (BESS G-Tracker) analogous to T and G ladders of dideoxy sequencing. The BESS procedures are simple to perform and require no special equipment or gels, no reaction optimization beyond PCR, and no heteroduplex formation. The samples are analyzed on standard sequencing gels or on automated DNA sequencers, and the data produced are easy to interpret, requiring a simple comparison of the sequence of normal and mutant DNA. The BESS method is versatile, having applications not only for mutation detection, but also single nucleotide polymorphism (SNP) discovery and analysis, DNA fingerprinting (including viral and bacterial typing), and clone identification. In this study, we utilize BESS in two of these applications: detection of a point mutation in BRCA1, and DNA typing of human papilloma virus (HPV).