Cloning and expression of quorum sensing N-acyl-homoserine synthase (LuxI) gene detected in Acinetobacter baumannii

Abstract

Objectives: In present study we aimed to clone the luxI gene encoding N-acyl-homoserine synthase detected in biofilm forming clinical isolates of Acinetobacter baumannii and study its expression in Escherichia coli transformants. Materials and Methods: Four A. baumannii hospital strains which demonstrated strong biofilm activity were selected in this investigation. The presence of luxI gene was detected using PCR technique. Purified PCR product DNA was initially cloned to pTG19 plasmid embedded with overhang 3'dT residue and transformed to Escherichia coli K12 DH5α (luxI- mutant). The gene was then recovered from agarose gel after digestion after digestion with DraI restriction enzyme and ligated by T4 DNA ligase into pET28a expression vector using NdeI and XhoI enzymes. Recombinant (pET28a + luxI) was transformed into E. coli BL21 (DE3) containing knockout luxI- gene. The luxI putative gene was further detected in transformants by colony PCR. Expression of the luxI gene in the recombinant E. coli BL21 cells was studied by quantitative real time PCR (qRT-PCR) and the presence of N-acyl-homoserine lactone (AHL) in wild types and the transformants were checked by colorimetric assay and Fourier Transform Infra- Red (FT-IR). Results: In our study, we found successful cloning of AHL from A. baumannii strain 23 which showed high biofilm. The presence of luxI gene in recombinant E. coli BL21 was confirmed by PCR. There was four fold increases in expression of luxI in the transformants (P ≤ 0.05). To verify the AHL synthesis, it was found that, strain 23 and the transformants showed highest amount of AHL activity (OD = 1.524). The FT-IR analysis indicated stretching C=O bond of the lactone ring and primary amides (N=H) at 1764.69 cm-1 and 1659.23 cm-1 respectively. Conclusion: From above results we concluded that, luxI and AHL are the only quorum sensing elements existed in A. baumannii and pET28a vector allows efficient AHL expression in E. coli BL21 transformants.

Keywords

• Acinetobacter baumannii
• luxI gene
• quantitative real time PCR
• cloning