Improved Electroporation andCloning Vector Systemfor Gram-Positive Bacteria

cells grownininhibitory concentrations ofglycine andtheuseofanacidic sucrose electroporation solution. Frequencies of>2 x 105 transformants per,ugofplasmid DNA wereobtained forE.faecalis cells, whereas various strains of streptococci andBacillus anthracis weretransformed atfrequencies of103to104transformants perpgof plasmid DNA withthesameprotocol. A novel Escherichia coli-Streptococcus andEnterococcus shuttle cloning vector, pDL276, wasconstructed foruseinconjunction withtheelectroporation system. Thisvector features amultiple cloning site region flanked byE.coli transcription termination sequences, arelatively small size (<7 kb),anda kanamycin resistance determinant expressed inbothgram-positive andgram-negative hosts. Various enterococcal andstreptococcal DNA sequences werecloned inE.coli (including sequences thatcould notbecloned onother vectors) andwerereturned totheoriginal hostbyelectroporation. Thevector and electroporation system wasalso usedtoclone directly into E.faecalis. Because oftheir importance aspathogens andtheir widespread useinvarious industrial processes, considerable effort hasbeendirected toward thedevelopment ofmodern genetic andmolecular techniques toanalyze andmanipulate gram-positive bacteria. Ourlaboratories areinvolved ina variety ofgenetic studies ofstreptococci (for thepurposes of this discussion, weinclude closely related organisms suchas enterococci andlactococci inthis group). Oneimportant aspect ofthisworkinvolves theconstruction ofcloning vectors andtheimprovement ofmethodstointroduce recombinant DNA molecules, constructed invitro, into host strains that arenotnaturally competent. Recombinant DNA manipulations intheseorganisms typically haveinvolved initial cloning andmolecular analyses inEscherichia coli, followed byreintroduction ofthecloned DNA (ormutated derivatives thereof) into theoriginal streptococcal hostfor studies ofexpression, complementation, genereplacement, etc.A numberofshuttle vectors havebeendeveloped for these purposes (10, 20,32). Although host-vector systems suchasthese havegreatly facilitated recombinant DNA studies instreptococci, there arestill significant limitations tothepotential application of this technology. Twoproblems associated withthestreptococcal shuttle vectors described thusfararetheir rather large size andtheir limited numberofuseful cloning sites. Another significant problem relates todifficulty ingenerating random, representative genebanksofstreptococcal DNA in