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ISSN 1672-9145 Acta Biochim Biophys Sin 2005, 37(1): 19–24 CN 31-1940/Q

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Characterization of a Mutant Listeria monocytogenes Strain Expressing Green Fluorescent Protein

Ling-Li JIANG, Hou-Hui SONG¹, Xue-Yan CHEN, Chun-Lin KE, Jing-Jing XU, Ning CHEN, and Wei-Huan FANG*

Institute of Preventive Veterinary Medicine and Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310029, China;

¹Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China

Abstract To construct a recombinant strain of Listeria monocytogenes for the expression of heterologous genes, homologous recombination was utilized for insertional mutation, targeting its listeriolysin O gene (hly). The gene encoding green fluorescent protein (GFP) was used as the indicator of heterologous gene expression. The gene gfp was inserted into hly downstream from its promoter and signal sequence by an overlapping extension polymerase chain reaction, and was then cloned into the shuttle plasmid pKSV7 for allelic exchange with the L. monocytogenes chromosome. Homologous recombination was achieved by growing the electro-transformed L. monocytogenes cells on chloramphenicol plates at a non-permissive temperature. Sequencing analysis indicated correct insertion of the target gene in-frame with the signal sequence. The recombinant strain expressed GFP constitutively as revealed by fluorescence microscopy. The mutant strain L. monocytogenes hly-gfp lost its hemolytic activity as visualized on the blood agar or when analyzed with the culture supernatant samples. Such insertional mutation resulted in a reduced virulence of about 2 logs less than its parent strain L. monocytogenes 10403s as shown by the 50%-lethal-dose assays in the mouse and embryonated chicken egg models. These results thus demonstrate that mutated L. monocytogenes could be a potential carrier for the expression of heterologous passenger genes or could act as an indicator organism in the food industry.

Key words Listeria monocytogenes; homologous recombination; insertional mutation; heterologous gene expression; green fluorescent protein (GFP)

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Received: October 9, 2004 Accepted: November 23, 2004

This study is partly supported by the grant from the National Natural Science Foundation of China (No. 30270073)

*Corresponding author: Tel/Fax, 86-571-86971242; E-mail, [email protected]