Published online 28 October 2004
Nucleic Acids Research, Vol. 32 No. 19 © Oxford University Press 2004; all rights reserved
Acinetobacter sp. ADP1: an ideal model organism for genetic analysis and genome engineering
1 The Scripps Research Institute, BCC-379, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA and 2 Evologic SA, 2 rue Gaston Crémieux, 91000 Evry, France
* To whom correspondence should be addressed at Department of Microbiology and Cell Science, University of Florida, P.O. Box 110700, Gainesville, FL 32611-0700, USA. Tel: +1 352 392 9416; Fax: +1 352 392 5922; Email: vcrecy{at}ufl.edu
Received July 1, 2004; Revised August 30, 2004; Accepted September 21, 2004
Acinetobacter sp. strain ADP1 is a naturally transformable gram-negative bacterium with simple culture requirements, a prototrophic metabolism and a compact genome of 3.7 Mb which has recently been sequenced. Wild-type ADP1 can be genetically manipulated by the direct addition of linear DNA constructs to log-phase cultures. This makes it an ideal organism for the automation of complex strain construction. Here, we demonstrate the flexibility and versatility of ADP1 as a genetic model through the construction of a broad variety of mutants. These include marked and unmarked insertions and deletions, complementary replacements, chromosomal expression tags and complex combinations thereof. In the process of these constructions, we demonstrate that ADP1 can effectively express a wide variety of foreign genes including antibiotic resistance cassettes, essential metabolic genes, negatively selectable catabolic genes and even intact operons from highly divergent bacteria. All of the described mutations were achieved by the same process of splicing PCR, direct transformation of growing cultures and plating on selective media. The simplicity of these tools make genetic analysis and engineering with Acinetobacter ADP1 accessible to laboratories with minimal microbial genetics expertise and very little equipment. They are also compatible with complete automation of genetic analysis and engineering protocols.
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