Epidemiologic evaluation of enterococci has been limited by the lack of a simple and effective method for comparing strains. In this study, we have compared chromosomal restriction endonuclease digestion patterns of 27 isolates of Enterococcus faecalis from three different locations by using pulsed-field electrophoresis of large chromosomal fragments (14 to 1,000 kilobases). AU but two isolates generated a clear, evaluable pattern with a single lysis and digestion, and the remaining two were visualized when a larger quantity of bacteria was used. All isolates from different locations generated different restriction patterns, as did most isolates within a single location; there was also evidence of spread of strains between individuals in each location. The ease with which this analysis can be performed, together with the clarity and polymorphism seen in the patterns, suggests that this technique will be very useful for epidemiological evaluations of nosocomial enterococcal infections.

A defined synthetic medium was used to determine the amino acid requirements of Enterococcus faecalis OG1 and to demonstrate the absence of a requirement for exogenous purines or pyrimidines. Genomic libraries prepared from strain OG1 were transduced into Escherichia coli auxotrophic mutants, and cloned DNAs which complemented pyrC, pyrD, purF, purL, and guaAB mutations were identified. These and other cloned DNAs with known functions were localized on a restriction map of OG1 which was generated with SfiI (5 fragments), AscI (9 fragments), and NotI (15 fragments); the size of the OG1 chromosome was revised from a previous estimate of approximately 2,750 kb to 2,825 kb. The synthetic medium and the restriction map should be useful for studying enterococcal metabolic functions and the relationships between chromosomally encoded genes.

Background: Global analysis of the genome, transcriptome, and proteome is facilitated by the recent development of tools for large-scale, highly parallel analysis. We describe a novel nucleic acid amplification system that generates products by several methods. 3-Ribo-SPIA TM primes cDNA synthesis at the 3 polyA tail, and whole transcript (WT)-Ribo-SPIA primes cDNA synthesis across the full length of the transcripts and thus provides whole-transcriptome amplification, independent of the 3 polyA tail. Methods: We developed isothermal linear nucleic acid amplification systems, which use a single chimeric primer, for amplification of DNA (SPIA) and RNA (Ribo-SPIA). The latter allows mRNA amplification from as little as 1 ng of total RNA. Amplification efficiency was calculated based on the delta threshold cycle between nonamplified cDNA targets and amplified cDNA. The amounts and quality of total RNA and amplification products were determined after purification of the amplification products. GeneChip ® array gene expression profiling and real-time PCR were used to test the accuracy and reproducibility of the method. Quantification of cDNA products (before and after amplification) at the 2 loci along the transcripts was used to assess product length (for evaluation of the 3-initiated Ribo-SPIA) and equal representation throughout the length of the transcript (for evaluation of the whole transcript amplification system, WT-Ribo-SPIA TM ). Results: Ribo-SPIA-based global RNA amplification exhibited linearity over 6 orders of magnitude of tran-

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...

Several approaches were used to construct a complete NotI restriction enzyme cleavage map of the genome of Escherichia coli MG1655. The approaches included use of transposable element insertions that created auxotrophic mutations and introduced a NotI site into the genome, hybridization of NotI fragments to the ordered lambda library constructed by Kohara et al. (BioTechniques 10:474-477, 1991), Southern blotting of NotI digests with cloned genes as probes, and analysis of the known E. coli DNA sequence for NotI sites. In all, 22 NotI cleavage sites were mapped along with 26 transposon insertions. These sites were localized to clones in the lambda library and, when possible, sequenced genes. The map was compared with that of strain EMG2, a wild-type E. coli K-12 strain, and several differences were found, including a region of about 600 kb with an altered restriction pattern and an additional fragment in MG1655. Comparison of MG1655 with other strains revealed minor differences but indicated that this map was representative of that for many commonly used E. coli K-12 strains.