Affiliation:
1. Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidı́n, Consejo Superior de Investigaciones Cientı́ficas, 18008 Granada, Spain
Abstract
ABSTRACT
A combined physical and genetic map of the
Pseudomonas putida
KT2440 genome was constructed from data obtained by pulsed-field gel electrophoresis techniques (PFGE) and Southern hybridization. Circular genome size was estimated at 6.0 Mb by adding the sizes of 19
Swa
I, 9
Pme
I, 6
Pac
I, and 6 I-
Ceu
I fragments. A complete physical map was achieved by combining the results of (i) analysis of PFGE of the DNA fragments resulting from digestion of the whole genome with
Pme
I,
Swa
I, I-
Ceu
I, and
Pac
I as well as double digestion with combinations of these enzymes and (ii) Southern hybridization analysis of the whole wild-type genome digested with different enzymes and hybridized against a series of probes obtained as cloned genes from different pseudomonads of rRNA group I and
Escherichia coli
, as
P. putida
DNA obtained by PCR amplification based on sequences deposited at the GenBank database, and by labeling of macrorestriction fragments of the
P. putida
genome eluted from agarose gels. As an alternative, 10 random mini-Tn
5
-Km mutants of
P. putida
KT2440 were used as a source of DNA, and the band carrying the mini-Tn
5
in each mutant was identified after PFGE of a series of complete chromosomal digestions and hybridization with the kanamycin resistance gene of the mini-Tn
5
as a probe. We established a circular genome map with an average resolution of 160 kb. Among the 63 genes located on the genetic map were key markers such as
oriC
, 6
rrn
loci (
rnnA
to -
F
),
recA
,
ftsZ
,
rpoS
,
rpoD
,
rpoN
, and
gyrB
; auxotrophic markers; and catabolic genes for the metabolism of aromatic compounds. The genetic map of
P. putida
KT2440 was compared to those of
Pseudomonas aeruginosa
PAO1 and
Pseudomonas fluorescens
SBW25. The chromosomal backbone revealed some similarity in gene clustering among the three pseudomonads but differences in physical organization, probably as a result of intraspecific rearrangements.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
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