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 or spon taneous xylitol-resistant mutants, xylitol being a toxic fructose analogue. Five such mutants were obtained, but only one, xy13, was unable to use fru ctose and had no phosphoenolpuryvate :fructose phosphotransferase system (f ructose-PTS) activity. Amplification and sequencing of the fructose permeas e gene of mutant xy13 revealed the presence of an adenylic insertion leadin g to a truncated permease. The second method is based on inactivation of fr uA and/or fruK by homologous recombination involving one crossing-over betw een the chromosomal genes and inactivated genes carried by replicative plas mids. 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 E 53). In strain H31, Southern blot analysis and PCR showed that the result o f 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 trans cribed. This work describes for the first time the use of two methods to ob tain fructose-auxotrophic mutants of S. citri. The method involving homolog ous recombination is a general procedure for gene disruption in S. citri.