Combined Physical and Genetic Map of the Pseudomonas putida KT2440 Chromosome
AUTOR(ES)
Ramos-Díaz, M. Angeles
FONTE
American Society for Microbiology
RESUMO
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 SwaI, 9 PmeI, 6 PacI, and 6 I-CeuI 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 PmeI, SwaI, I-CeuI, and PacI 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-Tn5-Km mutants of P. putida KT2440 were used as a source of DNA, and the band carrying the mini-Tn5 in each mutant was identified after PFGE of a series of complete chromosomal digestions and hybridization with the kanamycin resistance gene of the mini-Tn5 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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=107723Documentos Relacionados
- Cloning, Sequencing, and Phenotypic Characterization of the rpoS Gene from Pseudomonas putida KT2440
- Hydroxyectoine Is Superior to Trehalose for Anhydrobiotic Engineering of Pseudomonas putida KT2440
- Cell Density-Dependent Gene Contributes to Efficient Seed Colonization by Pseudomonas putida KT2440
- General method of rapid Smith/Birnstiel mapping adds for gap closure in shotgun microbial genome sequencing projects: application to Pseudomonas putida KT2440
- A combined genetic and physical map of the Streptomyces coelicolor A3(2) chromosome.