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 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 M13 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 M13 phage; segregants lacking M13 sequences were then selected as deoxycholate-resistant (DocR) ones. An improved method for recombination of mutations in pir-dependent, oriR6K vectors involved the use of plasmids containing genes for tetracycline resistance (TcR). Merodiploid recombinants were selected by conjugative transfer of such plasmids into a recipient lacking pir (encoding the pi 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Metcalf
W W
WW
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907.
Wanner
B L
BL
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005966:Glucuronidase / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D010957:Plasmids; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics
D001616:beta-Galactosidase; D005966:Glucuronidase