Heterologous Phi X174 gene E-expression in Ralstonia eutropha: E-mediated lysis is not restricted to gamma-subclass of proteobacteria

E-lysis of Ralstonia eutropha H16, which belongs to the beta-subclass, was undertaken to verify whether transmembrane tunnel formation is possible in bacteria which do not belong to the enterobacteriaceae. For this purpose, a new gene E expression plasmid, pKG12, with two origins of replication, ori V and oriT, from plasmid pRP4, chloramphenicol and kanamycin resistance gen es and a casette composed of lambda c1857 and lambda pR gene E was construc ted. Temperature upshift of R. eutropha H16 (pKG12) from 28 to 45 degrees C during exponential growth resulted in lysis of the strain with features ch aracteristic of E-mediated lysis of Escherichia coli. The cytoplasmic conte nts released can easily be separated from the still intact envelope fractio n by centrifugation or filtration. As R. eutropha H16 represents an importa nt industrial organism, E-mediated lysis could facilitate procedures for th e recovery of intracellular mediators or products like polyhydroxyalkanoate s. (C) 1998 Elsevier Science B.V. All rights reserved.