Physical mapping of 32 genetic markers on the Pseudomonas aeruginosa PAO1 chromosome.
The Pseudomonas aeruginosa chromosome was fractionated with the enzymes SpeI and DpnI, and genomic fragments were separated by PFGE and used for mapping a collection of 40 genes. This permitted the localization of 8 genes previously mapped and of 32 genes which had not been mapped. We showed that a careful search of databases and identification of sequences that were homologous to known genes could be used to design and synthesize DNA probes for the mapping of P. aeruginosa homologues by Southern hybridization with genomic fragments, resulting in definition of the locations of the aro-2, dapB, envA, mexA, groEL, oprH, oprM, oprP, ponA, rpoB and rpoH genetic markers. In addition, a combination of distinct DNA sources were utilized as radioactively labelled probes, including specific restriction fragments of the cloned genes (glpD, opdE, oprH, oprO, oprP, phoS), DNA fragments prepared by PCR, and single-stranded DNA prepared from phagemid libraries that had been randomly sequenced. We used a PCR approach to clone fragments of the putative yhhF, sucC, sucD, cypH, pbpB, murE, pbpC, soxR, ftsA, ftsZ and envA genes. Random sequencing of P. aeruginosa DNA from phagemid libraries and database searching permitted the cloning of sequences from the acoA, catR, hemD, pheS, proS, oprD, pyo and rpsB gene homologues. The described genomic methods permit the rapid mapping of the P. aeruginosa genome without linkage analysis.
Liao
Xiaowen
X
Department of Microbiology and Immunology, University of British Columbia, 300-6174 University Boulevard, Vancouver BC, Canada V6T 1Z3.
Charlebois
Isabelle
I
Microbiologie Moléculaire et Génie des Protéines, Département de Microbiologie, Faculté de Médecine, Pavillon Charles-Eugène-Marchand, Université Laval, Ste-Foy, Québec, Canada G1K 7P4.
Ouellet
Catherine
C
Microbiologie Moléculaire et Génie des Protéines, Département de Microbiologie, Faculté de Médecine, Pavillon Charles-Eugène-Marchand, Université Laval, Ste-Foy, Québec, Canada G1K 7P4.
Morency
Marie-Josée
MJ
Microbiologie Moléculaire et Génie des Protéines, Département de Microbiologie, Faculté de Médecine, Pavillon Charles-Eugène-Marchand, Université Laval, Ste-Foy, Québec, Canada G1K 7P4.
Dewar
Ken
K
Microbiologie Moléculaire et Génie des Protéines, Département de Microbiologie, Faculté de Médecine, Pavillon Charles-Eugène-Marchand, Université Laval, Ste-Foy, Québec, Canada G1K 7P4.
Lightfoot
Jeff
J
Microbiologie Moléculaire et Génie des Protéines, Département de Microbiologie, Faculté de Médecine, Pavillon Charles-Eugène-Marchand, Université Laval, Ste-Foy, Québec, Canada G1K 7P4.
Foster
Jennifer
J
Department of Microbiology, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
Siehnel
Richard
R
Department of Microbiology and Immunology, University of British Columbia, 300-6174 University Boulevard, Vancouver BC, Canada V6T 1Z3.
Schweizer
Herbert
H
Department of Medical Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada T2N 4N1.
Lam
Joseph S
JS
Department of Microbiology, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
Hancock
Robert E W
REW
Department of Microbiology and Immunology, University of British Columbia, 300-6174 University Boulevard, Vancouver BC, Canada V6T 1Z3.
Levesque
Roger C
RC
Microbiologie Moléculaire et Génie des Protéines, Département de Microbiologie, Faculté de Médecine, Pavillon Charles-Eugène-Marchand, Université Laval, Ste-Foy, Québec, Canada G1K 7P4.
D001483:Base Sequence; D002876:Chromosomes, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D016521:Electrophoresis, Gel, Pulsed-Field; D015870:Gene Expression; D005798:Genes, Bacterial; D005819:Genetic Markers; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D018076:DNA, Complementary; D005819:Genetic Markers; D015345:Oligonucleotide Probes; C020102:endodeoxyribonuclease DpnI; C056436:endodeoxyribonuclease SpeI; D015252:Deoxyribonucleases, Type II Site-Specific