https://www.abbs.info
E-mail: [email protected]
ISSN
1672-9145
Acta Biochim Biophys Sin
2005, 37(1): 19–24
CN 31-1940/Q
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)
—————–
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]