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Applied and Environmental Microbiology, August 2008, p. 4727-4736, Vol. 74, No. 15
0099-2240/08/$08.00+0     doi:10.1128/AEM.00297-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

High-Throughput Identification and Validation of In Situ-Expressed Genes of Lactococcus lactis{triangledown}

Herwig Bachmann,1,2 Michiel Kleerebezem,1,2,3,4 and Johan E. T. van Hylckama Vlieg1,2,3*

NIZO food research, P.O. Box 20, 6710 BA Ede, The Netherlands,1 Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands,2 TI Food & Nutrition, P.O. Box 557, 6700 AN Wageningen, The Netherlands,3 Wageningen University, Microbiology Department, Dreijenplein 10, 6703 HB Wageningen, The Netherlands4

Received 4 February 2008/ Accepted 24 April 2008

Understanding the functional response of bacteria to their natural environment is one of the current challenges in microbiology. Over the past decades several techniques have been developed to study gene expression in complex natural habitats. Most of these methods, however, are laborious, and validation of results under in situ conditions is cumbersome. Here we report the improvement of the recombinase-based in vivo expression technology (R-IVET) by the implementation of two additional reporter genes. The first one is an {alpha}-galactosidase gene (melA), which facilitates the rapid identification of in vivo-induced genes. Second, the bacterial luciferase genes (luxAB) are transcriptionally coupled to the resolvase gene, which allows rapid validation and characterization of in vivo-induced genes. The system is implemented and validated in the industrially important lactic acid bacterium Lactococcus lactis. We demonstrate the applicability of the advanced R-IVET system by the identification and validation of lactococcal promoter elements that are induced in minimal medium compared to the commonly used rich laboratory medium M17. R-IVET screening led to the identification of 19 promoters that predominantly control expression of genes involved in amino acid and nucleotide metabolism and in transport functions. Furthermore, the luciferase allows high-resolution transcription analysis and enabled the identification of complex medium constituents and specific molecules involved in promoter control. Rapid target validation exemplifies the high-throughput potential of the extended R-IVET system. The system can be applied to other bacterial species, provided that the reporter genes used are functional in the organism of interest.

* Corresponding author. Mailing address: NIZO food research, P.O. Box 20, 6710 BA Ede, The Netherlands. Phone: 31-318659511. Fax: 31-318650400. E-mail: Johan.van.hylckama.vlieg{at}nizo.nl

{triangledown} Published ahead of print on 6 June 2008.

Applied and Environmental Microbiology, August 2008, p. 4727-4736, Vol. 74, No. 15
0099-2240/08/$08.00+0     doi:10.1128/AEM.00297-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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