Abstract
We report the construction of a novel Tn7 vector for the tagging and enumeration of target bacteria from complex microbial communities. The system utilises a cassette for inducible bioluminescence and tetracycline resistance that integrates at a defined neutral position present in most Gram-negative species. We used this approach to chromosomally tag Pectobacterium such that it could be enumerated in mixed consortia without placing a significant bioburden on the tagged strain. Two Pectobacterium strains, a carbapenem antibiotic producer and an isogenic knock-out strain were tagged using this system. The modified Pectobacterium strains were used to compare the extent to which potato tuber-associated and endophytic bacteria can gain advantage and multiply in planta, utilising the nutrients released by a Pectobacterium infection, when the infecting Pectobacterium is either an antibiotic producer (Car+) or a carbapenem knock-out (Car−) strain. We show that the ability to synthesise carbapenem has a significant effect upon Pectobacterium numbers throughout the course of the infection. Whilst limiting the number of other bacterial species, carbepenem production allows the Pectobacterium to replicate to higher titres in the rotting tuber. We anticipate that the Tn7 tagging vector will be of use to other researchers studying ecological interactions in complex environments.
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Acknowledgements
Financial support for Katalin Kovács from the European Commission (Marie Curie Fellowship QLK1-CT-2000-60022 “Training site in the microbiological safety of foods”) is gratefully acknowledged. We thank Gregory McKenzie for the donation of the pGRG25 vector.
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Kovács, K., Hill, P.J., Grierson, D. et al. Development of a novel inducible bioluminescent and antibiotic resistance tagging system and its use to investigate the role of antibiotic production by Pectobacterium carotovorum ssp. carotovorum during potato tuber infection. Eur J Plant Pathol 125, 655–664 (2009). https://doi.org/10.1007/s10658-009-9513-4
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DOI: https://doi.org/10.1007/s10658-009-9513-4