Construction of new β-glucuronidase cassettes for making transcriptional fusions and their use with new methods for allele replacement

Five cassettes carrying uidA, encoding β-glucuronidase, were made for 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 streptomycin (Sm) and spectinomycin (Sp). Some cause polar insertions while others provide a promoter for downstream gene expression. The expression of these uidA cassettes was compared to the expression of lacZ at the same site in phnD, a phosphate-regulated gene for phosphonate use. 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 replacement. A new method involved recombination of mutations in M 13 onto the chromosome by infection of an Escherichia coli rep mutant that fails to propagate single-stranded DNA phages. Merodiploid recombinants were selected using a resistance marker carried by the M 13 phage; segregants lacking M 13 sequences were then selected as deoxycholate-resistant (Doc^R) ones. An improved method for recombination of mutations in pir-dependent, ori_R6K vectors involved the use of plasmids containing genes for tetracycline resistance (Tc^R). Merodiploid recombinants were selected by conjugative transfer of such plasmids into a recipient lacking pir (encoding the n protein of the R6K plasmid); segregants lacking vector sequences were subsequently selected as Tc-sensitive ones. Both procedures are efficient and allow for recombining marked as well as unmarked mutations onto the chromosome. In addition, some insertions with an antibiotic-resistance marker were directly recombined onto the chromosome by transformation of a recD mutant with linear DNA. New E. coli mutants deleted for uidA, lacZ and phoA are also described, including ones that allow for the simultaneous use of multiple reporter genes.