Genetic Transformation in Pseudomonas

Abstract

S U h l M A R Ylntraspecific and interspecific transformation in Pseudomonas has been investigated. Two Isoleucine + valiiie requiring mutants of Pseidotnonus neruginosa have been transformed t o prototrophy. By using gelatin liquifaction as a marker character it was observcd that two strains of P.J/iiorescens. a phytopathogen P. niangjferaeirzdic,ric. ii nd ii strain of Pseudon?onas originally isolated from fish were transformable. When P. solanacearum w a s used as a donor, the transformation frequency of P. niangijeraeindicae was vcry high. DNA from fish Pseudomonas 1 z+ax capable of transforming two strains of P. J-luorescens and P. iiiaizSiJerireiiiLli("(iC in addition to fish Pseudoinonas 2 I . When P. jluorescens was treated with the D N A from P. aei.zigiuzo.w transformation frequency was lower ; the cii p c i ty of pigment production, however, could not be transferred to any of tlw strains. The highest transformation frequency was obtained towards the end of the log phase of growth of the receptor bacteria, with a DNA concentration of about 10 ,ug./nil. Optimum temperature for transformation \+a\ 25-30 ' and agitation and star\ ;I tion in diluted media increased the number ol' transformants. The significmce of the observations has been discussed from the point of view of relationships among the strains and species studied.

Further Observations on Genetic Transformation in Pseudomonas

“…In the absence of actinomycin D, 4.6 % of recipient cells were transformed to the property of gelatin liquefaction ; after pretreatment with actinomycin D, only 0.46 were transformed. are in good agreement with the results of interspecific experiments involving I hese two strains reported previously (Khan & Sen, 1967); however, neither of these pathogens could transform or be transformed by the DNA of any other pseudomonad. The marker phenotype chosen is an important determinant of success in transformation experiments.…”

“…The transformation ;procedure, including the control experiments, was as described by Khan & Sen (1967). The sterility of the DNA preparation was checked before each experiment.…”

“…The method of scoring gelatin-liquefying transformants has been described by Khan & Sen (1967). To isolate antibiotic resistant transformants, dilution plates of the transformation mixture were incubated at 30 "C for I h and then slowly overlayered with 5 ml of nutrient agar containing the antibiotic, followed by incubation at the same temperature.…”

Genetic transformation of Pseudomonas aeruginosa with extracellular DNA.

“…However, no transformant could be obtained from a lysine-requiring mutant using intracellular DNA of the strain KYU-l, even applying the procedure of CHAKRABARTY et al (13). No transformant of chromosomal markers could be detected using the procedure of KHAN and SEN (8,9) with the whole bacterial DNA. This was also reported by SANO and KAGE-YAMA (10).…”

The order of replication of chromosomal markers inPseudomonas aeruginosastrain 1: I. Marker frequency analysis by transduction

“…Of the various methods devised (Yoshikawa & Sueoka, 1963;Erickson & Braun, 1968;Cerda-Olmedo, Hanawalt & Guerola, 1968;Masters, 1970;Masters & Broda, 1971;Caro & Berg, 1968;Wolfe, Pato, Ward & Glaser, 1968;Vielmetter, Messer & Schutte, 1968;Bird, Louarn, Martuscelli & Caro, 1972), that of Yoshikawa and Sueoka was chosen first since genetic transformation had been reported in P. aeruginosa strain 78 obtained from this laboratory (Khan & Sen, 1967). Since we could not repeat this work, however, the method of Masters (1970), which relied on transduction to measure gene frequencies, was selected.…”