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ISSN 1672-9145 Acta Biochim Biophys Sin 2004, 36(12): 832–839 CN 31-1940/Q

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Identification and Analysis of Genes Present in Leptospira interrogans serovar lai but Absent in L. biflexa serovar monvalerio

Ping HE, Xiang-Yan ZHANG, Xiao-Kui GUO*, Bao-Yu HU, Xiao-Tian HUANG, Yang YANG, and Guo-Ping ZHAO^1,2*

Department of Microbiology and Parasitology, Shanghai Second Medical University, Shanghai 200025, China;

¹Research Center of Biotechnology, Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China;

²Chinese National Human Genome Center at Shanghai, Zhangjiang High Tech Park, Shanghai 201203, China

Abstract Genes present in virulent bacterial strains but absent in avirulent close relatives can be of great biologic and clinical interest. This project aimed to identify strain specific DNA sequences of Leptospira interrogans serovar lai, which is absent in the saprophytic L. biflexa serovar monvalerio, via suppression subtractive hybridization with the former as the tester while the latter as the driver. The mixture of PCR amplified DNA fragments from two subtractive hybridization experiments were cloned into pMD18-T vector and the positive clones were identified by dot blotting against the chromosome DNA of the two strains individually. After DNA sequencing and analysis, the distribution of these genomic fragment sequences in a panel of pathogenic and nonpathogenic leptospires was investigated employing dot blot analysis. Among the 188 positive clones randomly chosen, 24 contained the tester strain specific genomic regions, of which, 5 were non-coding fragments while the others contained 23 distinct protein coding sequences. Besides 9 genes encoding functional proteins, 12 genes encode unknown proteins and the rest two genes encode proteins with recognizable domain structures, one for a putative leucine-rich repeats (LRR) family protein while the other as an outer-membrane protein. Our experiment results indicated that suppression subtractive hybridization is effective for screening specific DNA sequences between two leptospiral strains, and some of these sequences might be responsible for virulence determination. Further analysis of these DNA sequences will provide important information on the pathogenesis of Leptospira.

Key words Leptospira interrogans serovar lai; suppression subtractive hybridization (SSH); specific genes

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Received: October 9, 2004 Accepted: November 16, 2004

This work was supported in part by the grants from the National Natural Science Foundation of China (No. 30370071) and the National High Technology Research and Development Program of China (No. 2003AA223031)

*Corresponding authors:

Xiao-Kui GUO: Tel, +86-21-64453285; Fax, +86-21-64453285; E-mail, [email protected]

Guo-Ping ZHAO: Tel, +86-21-50801919; Fax, +86-21-50801922; E-mail, [email protected]