CONSTRUCTION OF NEW BETA-GLUCURONIDASE CASSETTES FOR MAKING TRANSCRIPTIONAL FUSIONS AND THEIR USE WITH NEW METHODS FOR ALLELE REPLACEMENT

Five cassettes carrying uidA, encoding beta-glucuronidase, were made f or the construction of insertion mutants with transcriptional fusions to uidA. Three uidA cassettes contain antibiotic-resistance genes, for chloramphenicol (Cm), for kanamycin (Km) and neomycin (Nm), or for st reptomycin (Sm) and spectinomycin (Sp). Some cause polar insertions wh ile others provide a promoter for downstream gene expression. The expr ession of these uidA cassettes was compared to the expression of lacZ at the same site in phnD, a phosphate-regulated gene for phosphonate u se. Several phn::uidA or phn::lacZ insertions were recombined onto the chromosome to test mutational effects and to measure gene expression in single copy. This was done using one of three methods for allele re placement. A new method involved recombination of mutations in M13 ont o the chromosome by infection of an Escherichia coli rep mutant that f ails to propagate single-stranded DNA phages. Merodiploid recombinants were selected using a resistance marker carried by the M13 phage; seg regants lacking M13 sequences were then selected as deoxycholate-resis tant (Doc(R)) ones. An improved method for recombination of mutations in pir-dependent, ori(R6K) vectors involved the use of plasmids contai ning genes for tetracycline resistance (Tc(R)) . Merodiploid recombina nts were selected by conjugative transfer of such plasmids into a reci pient lacking pir (encoding the PI protein of the R6K plasmid); segreg ants lacking vector sequences were subsequently selected as Tc-sensiti ve ones. Both procedures are efficient and allow for recombining marke d as well as unmarked mutations onto the chromosome. In addition, some insertions with an antibiotic-resistance marker were directly recombi ned onto the chromosome by transformation of a recD mutant with linear DNA. New E. coli mutants deleted for uidA, lacZ and phoA are also des cribed, including ones that allow for the simultaneous use of multiple reporter genes.