As a result of the explosive growth of bacterial genomic and
postgenomic information, there is a pressing need for efficient, inexpensive
strategies for characterizing the in vivo behavior and function of newly
identified gene products. We describe here an internal tagging procedure, based
on transposon technology1,
2, to facilitate the analysis of
membrane-bound and secreted proteins in Gram-negative bacteria. The technique
is based on a broad−host range transposon (ISphoA/hah), which may
be used to generate both alkaline phosphatase (AP) gene fusions and 63-codon
in-frame insertions in the genome. The 63-codon insertion encodes an influenza
hemagglutinin epitope and a hexahistidine sequence, permitting sensitive
detection and metal affinity purification of tagged proteins. For each gene
targeted, it is thus possible to monitor the disruption of phenotype (using the
transposon insertion), the gene's transcription and translation (using the AP
reporter activity), and the behavior of the unfused protein (using the internal
tag). Studies on a sequence-defined collection of Escherichia coli
strains generated using the transposon showed that the synthesis and
subcellular localization of tagged proteins could be readily monitored. The use
of ISphoA/hah should provide a cost-effective approach for genome-wide
in vivo studies of the behavior of exported proteins in a number of
bacterial species.
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