Directed introduction of DNA cleavage sites to produce a high-resolution genetic and physical map of the Acinetobacter sp. strain ADP1 (BD413UE) chromosome

Abstract

The natural transformability of the soil bacterium Acinetobacter sp. ADPl (BD413UE), formerly classified as A. calcoaceticus, has facilitated previous physiological and biochemical investigations. In the present studies, the natural transformation system was exploited to generate a physical and genetic map of this strain's 378021 91 kbp circular chromosome. Previously isolated Acinetobacter genes were modified in vitro to incorporate a recognition sequence for the restriction endonuclease Notl. Following transformation of the wild-type strain by the modified DNA, homologous recombination placed each engineered Not1 cleavage site at the chromosomal location of the corresponding gene. This allowed precise gene localization and orientation of more than 40 genes relative to a physical map which was constructed with transverse alternating field electrophoresis (TAFE) and Southern hybridization methods. The positions of Notl, Ascl and I-Ceul recognition sites were determined, and the latter enzyme identified the presence of seven ribosomal RNA operons. Multiple chromosomal copies of insertion sequence IS1236 were indicated by hybridization. Several of these copies were concentrated in one region of the chromosome in which a spontaneous deletion of approximately 100 kbp occurred. Moreover, contrary to previous reports, ColEl-based plasmids appeared to replicate autonomously in Acinetobacter sp. ADPI.

mucK, a gene in Acinetobacter calcoaceticus ADP1 (BD413), encodes the ability to grow on exogenous cis,cis-muconate as the sole carbon source

“…An additional result which adds some extra weight to a physiological role for mucK involving some substrate other than muconate is its chromosomal location. Whereas the ben-cat genes involved in benzoate, catechol, and muconate utilization are clustered on the ADP1 chromosome (29,32), mucK has been shown to map within 10 kbp of the pca-qui-pob cluster (2,8,9), encoding the quinate and p-hydroxybenzoate branch of the ␤-ketoadipate pathway, but about 300 kbp distant from the ben-cat cluster (13).…”

“…The genes for the two convergent, biochemically and genetically homologous branches of the pathway are found in two supraoperonic clusters, the ben-cat cluster (29,32), comprising the genes for the conversion of benzoate to tricarboxylic acid cycle intermediates, and the pca-qui-pob cluster (2,8,9), comprising the genes for the catabolism of p-hydroxybenzoate and quinate. Both clusters are Ͼ20 kbp and yet are separated on the chromosome by about 290 kbp (13).…”

“…Investigation of the genetics of the pathway in strain ADP1 has been greatly advanced by the ease with which it undergoes natural transformation and recombination; this facilitates the cloning of DNA which complements mutations (2), the accurate mapping of mutations within a gene (11), the recovery of DNA from the chromosome by gap repair (14), and the physical mapping of genes on the chromosome (13).…”

benK encodes a hydrophobic permease-like protein involved in benzoate degradation by Acinetobacter sp. strain ADP1

“…1). Methods for its construction have been described previously (10,31). ␤-Galactosidase (LacZ) activity (31) with or without the addition of possible inducers was measured.…”

“…strain ADP1, formerly Acinetobacter calcoaceticus (10,13), utilizes benzoate as a carbon source. The conversion of benzoate to tricarboxylic acid cycle intermediates has been studied (27), but the mechanism of benzoate transport has not been investigated.…”