Agrobacterium tumefaciens transfers a piece of its Ti plasmid DNA (transferred DNA or T-DNA) into plant cells during crown gall tumorigenesis. A. tumefaciens can transfer its T-DNA to a wide variety of hosts, including both dicotyledonous and monocotyledonous plants. We show that the host range of A. tumefaciens can be extended to include Saccharomyces cerevisiae. Additionally, we demonstrate that while T-DNA transfer into S. cerevisiae is very similar to T-DNA transfer into plants, the requirements are not entirely conserved. The Ti plasmid-encoded vir genes ofA. tumefaciens that are required for T-DNA transfer into plants are also required for T-DNA transfer into S. cerevisiae, as is vir gene induction. However, mutations in the chromosomal virulence genes of A. tumefaciens involved in attachment to plant cells have no effect on the efficiency of T-DNA transfer into S. cerevisiae. We also demonstrate that transformation efficiency is improved 500-fold by the addition of yeast telomeric sequences within the T-DNA sequence.Agrobacterium tumefaciens causes crown gall tumors in plants by transferring a segment of DNA (transferred DNA or T-DNA) from its tumor-inducing (Ti) plasmid to the nucleus of plant cells. The T-DNA becomes integrated into the plant nuclear genome where it functions to give rise to the characteristic tumor (reviewed in refs. 1 and 2). This process depends on the induction of a set of Ti plasmid-encoded virulence (vir) genes. vir genes are induced via the virA/virG two-component regulatory system which senses monosaccharides and phenolic compounds from wounded plants (reviewed in ref.3). The T-DNA is a single-stranded DNA molecule produced by a virDl/D2-encoded site-specific endonuclease that nicks within two 24-bp direct repeat sequences on the Ti plasmid (4). These repeats, termed border sequences, flank the T-DNA. Following cleavage and excision, the T-DNA is coated by the singlestranded DNA binding protein VirE2 (5), and the resulting T-DNA complex is transferred to the plant cell.The mechanism by which the T-DNA complex is transported through the inner and outer bacterial membranes and into the plant cell is not well understood. It is believed on the basis of several lines of evidence that the VirB proteins and VirD4 are involved in T-DNA transport (reviewed in ref. 6). Once the T-DNA complex enters the plant cell, it is targeted to the nucleus via nuclear localization sequences in the VirD2 and VirE2 proteins (7,8). Upon entering the nucleus, the T-DNA is integrated into the plant genome by illegitimate recombination, a process likely mediated by host factors (9).The study of host factors involved in T-DNA transfer has been difficult and would be greatly facilitated by the availability of a host model amenable to genetic manipulation. Given the similarities between T-DNA transfer and conjugative transfer of broad-host-range plasmids (reviewed in refs. 1, 6, and 10), we set out to determine if A. tumefaciens can transfer T-DNA to the yeast Saccharomyces cerevisiae. It has been demo...

Antimicrobial cationic peptides have been discovered in many different organisms and often possess a broad range of activity. In this study, we investigated the mechanisms of actions of melittin and two synthetic peptides, CEME (a cecropin-melittin hybrid) and CEMA, against gram-negative bacteria. CEMA was produced by recombinant DNA procedures and is an analog of CEME with a modified C terminus resulting in two additional positive charges. All three peptides showed good antimicrobial activity against four different gram-negative bacteria, but only CEMA was able to somewhat augment the activity of some conventional antibiotics in synergy studies. Studies using the bacteria Pseudomonas aeruginosa and Enterobacter cloacae showed that the peptides all possessed the ability to permeabilize bacterial outer membranes to the hydrophobic fluorophor 1-N-phenylnaphthylamine and the protein lysozyme, with CEMA being the most active. CEMA also had the strongest relative binding affinity for bacterial endotoxin (lipopolysaccharide).These data collectively indicated that these peptides all cross the outer membrane by the self-promoted uptake pathway and that CEMA is the peptide most effective at accessing this pathway.The past few decades have witnessed the emergence of many different peptide antibiotics, including defensins (19), insect cecropins (5), magainins (42), and melittin (12). Cecropins and melittin belong to the group of antimicrobial peptides that exist in a random-coil configuration in aqueous solutions but adopt a helix-turn-helix structure upon interaction with membranes (9, 34). Both of these peptides have one amphipathic a-helix and one hydrophobic a-helix, but the order of these helices in the two peptides is inverted. Many synthetic peptides have been created in attempts to improve antibacterial activity. Included in these are cecropin-melittin hybrid peptides which possess the amphipathic N-terminal a-helix of cecropin A followed by the hydrophobic N-terminal a-helix of melittin (40). These peptides have been shown to have a broad range of antibacterial activity against both gram-negative and grampositive bacteria (40). Cecropins (6), melittin (36), and the hybrid peptides (41) have all been shown to form ion-permeable channels in lipid membranes.Gram-negative bacteria pose an additional challenge to these peptides in the form of an outer membrane which constitutes a permeability barrier to antibiotics (14,24,37

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A cecropin/melittin hybrid peptide (CEME) produced by recombinant DNA procedures was tested for its ability to interact with the outer membrane of Pseudomonas aeruginosa and found to have identical biological properties to that of chemically synthesized CEME. CEME was shown to kill P. aeruginosa and permeabilize its outer membrane to lysozyme and 1-N-phenylnaphthlyamine, in some cases better than other antimicrobial agents and permeabilizers. CEME demonstrated a high-binding affinity to purified P. aeruginosa lipopolysaccharide (LPS) and LPS in whole-cell environments. These data provide information on the molecular mechanism of CEME antimicrobial activity and strongly suggest that it is taken up across the outer membrane by the self-promoted uptake pathway.