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Genome-wide internal tagging of bacterial exported proteins

Nature Biotechnology volume 20, pages 839–842 (2002)Cite this article

Abstract

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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Figure 1: Function of ISphoA/hah.
Figure 2: Western blot of tagged proteins.
Figure 3: Fractionation of internally tagged proteins.

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Acknowledgements

We thank B. Traxler, W.S. Reznikoff, V. deLorenzo, I. Behlau, and A. Wright for discussions and plasmids, A. Alwood, I. Thaipisutikul, E. Round, T. Nguyen, A. Gerum, and Cold Spring Harbor Advanced Bacterial Genetics course participants for experimental contributions, and the National Science Foundation (MCB-9905048) for support.

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  1. Department of Genome Sciences, 357730, University of Washington, Seattle, 98195, WA

    Jeannie Bailey & Colin Manoil

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  1. Jeannie Bailey
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  2. Colin Manoil
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Correspondence to Colin Manoil.

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The authors declare no competing financial interests.

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Bailey, J., Manoil, C. Genome-wide internal tagging of bacterial exported proteins. Nat Biotechnol 20, 839–842 (2002). https://doi.org/10.1038/nbt715

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