Fructose operon mutants of Spiroplasma citri.
Fructose-negative mutants of Spiroplasma citri wild-type strain GII-3 were selected by two methods. The first method is based on the selection of spontaneous xylitol-resistant mutants, xylitol being a toxic fructose analogue. Five such mutants were obtained, but only one, xyl3, was unable to use fructose and had no phosphoenolpuryvate:fructose phosphotransferase system (fructose-PTS) activity. Amplification and sequencing of the fructose permease gene of mutant xyl3 revealed the presence of an adenylic insertion leading to a truncated permease. The second method is based on inactivation of fruA and/or fruK by homologous recombination involving one crossing-over between the chromosomal genes and inactivated genes carried by replicative plasmids. Fructose-negative mutants were obtained at a frequency of about 10%. Fructose-PTS activity and 1-phosphofructokinase activity were not detected in four representative mutants that were characterized (H31, H45, E38 and E53). In strain H31, Southern blot analysis and PCR showed that the result of homologous recombination was, as expected, the presence in the chromosome of two mutated fruA-fruK copies with the plasmid sequence in between. Only the mutated copy, under control of the fructose operon promoter, was transcribed. This work describes for the first time the use of two methods to obtain fructose-auxotrophic mutants of S. citri. The method involving homologous recombination is a general procedure for gene disruption in S. citri.
Gaurivaud
Patrice
P
Laboratoire de Biologie Cellulaire et Moléculaire, Institut de Biologie Végétale Moléculaire, Institut National de la Recherche Agronomique and Université Victor Segalen Bordeaux 2, Domaine de la Grande Ferrade, BP 81, 33883 Villenave d'Ornon cedex, France1.
Laigret
Frédéric
F
Laboratoire de Biologie Cellulaire et Moléculaire, Institut de Biologie Végétale Moléculaire, Institut National de la Recherche Agronomique and Université Victor Segalen Bordeaux 2, Domaine de la Grande Ferrade, BP 81, 33883 Villenave d'Ornon cedex, France1.
Verdin
Eric
E
Laboratoire de Biologie Cellulaire et Moléculaire, Institut de Biologie Végétale Moléculaire, Institut National de la Recherche Agronomique and Université Victor Segalen Bordeaux 2, Domaine de la Grande Ferrade, BP 81, 33883 Villenave d'Ornon cedex, France1.
Garnier
Monique
M
Laboratoire de Biologie Cellulaire et Moléculaire, Institut de Biologie Végétale Moléculaire, Institut National de la Recherche Agronomique and Université Victor Segalen Bordeaux 2, Domaine de la Grande Ferrade, BP 81, 33883 Villenave d'Ornon cedex, France1.
Bové
Joseph M
JM
Laboratoire de Biologie Cellulaire et Moléculaire, Institut de Biologie Végétale Moléculaire, Institut National de la Recherche Agronomique and Université Victor Segalen Bordeaux 2, Domaine de la Grande Ferrade, BP 81, 33883 Villenave d'Ornon cedex, France1.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004352:Drug Resistance, Microbial; D005632:Fructose / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D008969:Molecular Sequence Data; D009154:Mutation; D009876:Operon; D011995:Recombination, Genetic; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D013149:Spiroplasma / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014993:Xylitol / Q000494:pharmacology; D056968:Lac Repressors
D001426:Bacterial Proteins; D005632:Fructose; D012097:Repressor Proteins; D014157:Transcription Factors; D014993:Xylitol; C104848:FruA protein, Myxococcus xanthus; C068837:FruR protein, Bacteria; D056968:Lac Repressors