Isolation and Characterization of Acyl Homoserine Lactonase (AHL-lactonase) Producing Bacteria from Agricultural Field in Java

Abstract

Quorum sensing (QS) is a bacterial communication mechanism correlating with the number of bacterial population and accumulation of autoinducer (AI). Quorum sensing in Gram-negative bacteria involves a AI group of acyl homoserine lactone (AHL) to regulate the expression of several genes, including virulence genes. Anti-quorum sensing compounds can degrade AHL so that virulence genes can not be expressed. Acyl homoserine lactonase (AHL-lactonase) encoded by the aiiA gene is one enzyme that can act as an anti-quorum sensing. This study aimed to isolate and characterize the AHL-lactonase producing bacteria isolated from agricultural field in Java. Activity of AHL degradation bioassay using Chromobacterium violaceum as a bacterial indicator. Verification the presence of AHL-laktonase was performed by gene aiiA amplification. As many as 12 bacterial isolates showed AHL degradation activity and two isolates had the aiiA gene i.e. INT1c and SGT3g isolates. These isolates had bacil cells with chain arrangement and endospores. They were grouped in Gram-positive bacteria. AiiA gene sequence analysis showed that aiiA gene of INT1c had homology with aiiA gene of Bacillus cereus ATTCC 14579 while the SGT3g isolate had homology with aiiA gene of Bacillus thuringiensis BMB171. Molecular identification based on 16S rRNA gene showed that INT1c isolate had the closest similarity with the Bacillus cereus strain GM3 (98%) while SGT3g isolate had the closest similarity with the Bacillus thuringiensis strains A1-1 (99%).

Quorum sensing (QS) adalah mekanisme komunikasi bakteri yang bergantung pada jumlah populasi bakteri tersebut dan akumulasi senyawa autoinducer (AI). Quorum sensing pada kelompok bakteri Gram negatif melibatkan senyawa acyl homoserine lactone (AHL) untuk mengatur beberapa ekspresi gen, termasuk gen virulensi. Senyawa anti-QS merupakan senyawa yang dapat mendegradasi AHL sehingga gen virulensi tidak dapat diekspresikan. Acyl homoserine lactonase (AHL-laktonase) yang disandikan oleh gen aiiA merupakan salah satu enzim yang dapat berperan sebagai anti-QS. Penelitian ini bertujuan untuk mengisolasi dan mengkarakterisasi bakteri penghasil AHL-laktonase yang berasal dari lahan pertanian di Jawa. Bioesei aktivitas degradasi AHL menggunakan bakteri indikator Chromobacterium violaceum. Verifikasi keberadaan AHL-laktonase pada isolat-isolat yang menunjukkan hasil bioesei positif dilakukan dengan amplifikasi gen aiiA. Dari 12 isolat bakteri yang menunjukkan aktivitas degradasi AHL, terdapat dua isolat yang memiliki gen aiiA yaitu isolat INT1c dan SGT3g. Kedua isolat tersebut memiliki bentuk sel batang, penataan sel berantai, berendospora, dan termasuk dalam kelompok bakteri Gram positif. Analisis sekuen gen aiiA menunjukkan bahwa gen aiiA isolat INT1c memiliki homologi dengan gen aiiA bakteri Bacillus cereus ATTCC 14579 sedangkan isolat SGT3g memiliki homologi dengan gen aiiA bakteri Bacillus thuringiensis BMB171. Identifikasi molekuler berdasarkan gen 16S rRNA menunjukkan bahwa isolat INT1c memiliki kemiripan terdekat dengan bakteri Bacillus cereus strain GM3 (98%) sedangkan isolat SGT3g memiliki kemiripan terdekat dengan bakteri Bacillus thuringiensis strain A1-1 (99%).