TnblaM: a transposon for directly tagging bacterial genes encoding cell envelope and secreted proteins.
A transposon, TnblaM, designed for the direct selection of bacterial mutants with insertions in genes encoding cell envelope and secreted proteins, was constructed and subcloned into plasmid and bacteriophage lambda delivery vectors. TnblaM is a spectinomycin-resistant derivative of Tn5 with an unexpressed open reading frame encoding mature beta-lactamase (BlaM) at its left end. Therefore, when it inserts into genes in the correct orientation and reading frame, gene fusions encoding hybrid proteins are generated. By introducing TnblaM into bacterial cells and selecting ampicillin-resistant (ApR) colonies, the subset of isolates producing extracytoplasmic BlaM, and hence containing TnblaM inserted in genes encoding secreted proteins and cell envelope proteins, can be directly selected. TnblaM, like TnphoA, can therefore be used to preferentially mutagenise genes encoding extracytoplasmic proteins, but it has the advantage over TnphoA that the desired mutants can be isolated by direct selection (as ApR colonies) rather than by phenotypic screening. Isolates in which TnblaM occupies sites in the chromosome from which it can transpose at high frequency are readily identifiable, and constitute TnblaM donors, with which to simply and efficiently generate rare types of insertion mutants. Moreover, the ApR selection that is used with TnblaM can be fine-tuned to obtain blaM fusions to poorly or well-expressed genes.
Tadayyon
M
M
Microbial Genetics Group, School of Biological Sciences, University of Sussex, Falmer, Brighton, U.K.
Broome-Smith
J K
JK
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002876:Chromosomes, Bacterial / Q000378:metabolism; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016973:Pectobacterium carotovorum / Q000235:genetics; D010957:Plasmids; D015183:Restriction Mapping
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial