Restriction enzymes PacI and PmeI cut in regions rich in adenine and thymine regions that are not very common for Pseudomonas genus, as their percentage of guanine and cytosine is more than 55 % (1), as a cut-off frequency lower than predicted on the basis of this decreased mean percentage in AT, and a higher GC content, as might be the case for Psm ICMP921 and Psm GSPB2146.
PFGE for 8 Psm strains with PacI (A) and PmeI (B), electrophoresis conditions are described in materials and methods section.
Restriction frequency for PacI y PmeI enzymes in the eight genomes of Psm strains Enzyme ICMP921 GSPB2146 438 M2 307 308 CFBP1637 438* PacI 4 11 9 12 11 12 12 9 PmeI 11 8 14 13 13 14 14 14 Table 3.
To construct the physical map of the chromosome of Psm, three rare-cutting restriction endonucleases were chosen based on G+C content of P syringae strains, several enzymes with A+T rich recognition sequences were tested, among these enzymes PacI (TTAATTAA), PmeI (GTTTAAAC) and SwaI (ATTTAAAT) were selected which originated 14, 15 and 16 fragments, respectively.
The size of the chromosome of Psm was the result of the summation of the estimated size of individual DNA restriction fragments generated with PacI, PmeI and Swal and separated by PFGE.
Mutants M2, M7 and 9 were digested with PacI and PmeI (Fig.
The rare cutting restriction endonucleases PacI, PmeI, and SwaI were previously reported for construction physical maps in bacteria (21,22,25,26).
Artificial transposable elements pTn5cat1 and pTn5Spcat endowed with restriction sites for PacI, PmeI and SwaI (unpublished) were used to obtain a collection of insertional mutants, each with an extra restriction site for these endonucleases.
maculicola M2 chromosome digested with PacI, PmeI and SwaI, with 14, 15 and 16 fragments, respectively.
PFGE analysis of Psm insertional mutants digested with PacI and PmeI. (M2, M7) Psm pTn5cat1 mutants, (9) Psm pTn5cat mutant and (WT) Psm wild type strain.