Transformation of <i>Aggregatibacter actinomycetemcomitans</i> with Green Fluorescent Protein

• Hashizume-Takizawa Tomomi

  Department of Microbiology and Immunology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba, 271-8587, Japan

• Shinozaki-Kuwahara Noriko

  Department of Microbiology and Immunology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba, 271-8587, Japan

• Kurita-Ochiai Tomoko

  Department of Microbiology and Immunology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba, 271-8587, Japan

抄録

Green fluorescent protein(GFP)is one of the most useful biological markers for monitoring gene expression and the localization of particular cells. We constructed a GFP-expressing Aggregatibacter actinomycetemcomitans transformant by transposon insertion. This anaerobic, oral, gram-negative bacterium is thought to be the major causative agent in localized aggressive periodontitis and possesses the gene coding for leukotoxin(ltx). As this gene is stably expressed, the region extending from the ltx promoter to the initiation codon was amplified using PCR and temporarily cloned into a small Escherichia coli plasmid, pResKm. The DNA fragment containing the GFP gene(gfp)ORF was excised from the commercially available plasmid pAcGFP1-C1, purified by gel electrophoresis, and inserted downstream of the ltx initiation codon in the correct reading frame. This gene fusion was further cloned into the Not I site of the commercially available shuttle plasmid, pUTmini-Tn5, which contains the gene coding for the transposase, and then introduced into the chromosome of A. actinomycetemcomitans strain HK1651 by electroporation. Transformants were isolated by screening for the appropriate antibiotic resistance. FACS analysis revealed that the final A. actinomycetemcomitans-GFP integrant exhibited slightly more intense fluorescence than wild-type A. actinomycetemcomitans. These data demonstrate that transposon-mediated chromosomal integration is a useful approach for constructing mutant A. actinomycetemcomitans strains.

収録刊行物

• ＩＪＯＭＳ

  ＩＪＯＭＳ 14 (2-3), 41-47, 2016

  日本大学松戸歯学部 口腔科学研究所

キーワード

• green fluorescent protein
• <i>A. actinomycetemcomitans</i>
• leukotoxin

詳細情報

• CRID: 1390282680189517824
• NII論文ID: 130005126921
• DOI: 10.5466/ijoms.14.41
• ISSN: 21854254
• ISSN: 13479733
• Web Site: https://www.jstage.jst.go.jp/article/ijoms/14/2-3/14_41/_pdf
• Web Site: https://search.jamas.or.jp/link/ui/2016344052
• 本文言語コード: en
• データソース種別:
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