VIRGIBACILLUS SALARIUS’S ECTOINE BIOSYNTHETIC GENE CLUSTER (ECTA, ECTB, ECTC) CHARACTERIZATION AND ISOLATION

• Main Author: Humaira, Zalfa
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/67839
• Institution: Institut Teknologi Bandung

Bacteria can synthesize a variety of compatible solutions that function as osmoprotectants, one of which is ectoine. Ectoine is an aspartate-derived amino acid with chemical characteristics that have high affinity for water, biomolecular protection, and UV light protection. Ectoine is also useful in industrial fields such as cosmetic ingredients to hydrate the skin, and in the medical field to protect the lung epithelium from inflammation. Ectoine is synthesized by a gene cluster that transcribed diaminobutyric acid acetyltransferase (EctA), diaminobutyrate-2-oxoglutarate transaminase (EctB), and ectoine synthase (EctC). Because of its value, ectoine is used in bioindustry and recombinant plasmids can be used in the production of ectoine. The moderate halophilic bacterium Virgibacillus salarius isolated from marine coral has an ectoine biosynthetic gene cluster in its genome, and it has the potential to produce ectoine by isolating the V. salarius ectoine gene cluster. However, there has been no research on the characterization of ectoine gene clusters in V. salarius, as well as gene cluster comparisons to see whether the ectoine biosynthetic gene cluster is sustainable in the Bacillaceae family. Therefore, this study aimed to characterize and isolate the ectoine biosynthetic gene cluster of V. salarius, as well as to compare the ectoine gene cluster in the Bacillaceae family. Genome sequences from Whole Genome Sequencing (WGS) of V. salarius were annotated using KEGG Onthology, and ectoine gene cluster prediction was performed using AntiSMASH. Characterization and visualization of the ectABC gene cluster structure in V. salarius included sequence length, promoter location, terminator, and transcription factors using FGENESB, FindTerm, and BPROM. Comparative analysis was carried out by looking for the percentage similarity of EctA, EctB, and EctC V. salarius protein sequences with 6 ectoine-producing bacteria from the Bacillaceae family and Halomonas elongata as outgroups based on sequences obtained from NCBI using ClustalX, identification of motifs and domains on each putative protein by MEME-Suites and CD-Search, as well as phylogenetic tree construction of putative protein sequences EctA, EctB, and EctC with MEGA-X. Isolation of the ectABC V. salarius gene was carried out by ligating the gene into the pGEMT cloning plasmid which was transformed into E. coli DH? bacteria. The isolated ectABC gene was then sequenced, and the sequence was confirmed through Pairwise Sequence Alignment with the sequence predicted by WGS. The results of the analysis include ectoine gene clusters consisting of ectA, ectB, and ectC genes which in all bacteria have a length of ± 2200 base pair (bp), predicted domain NAT_SF superfamily on EctA protein, diaminobutyrate-2-oxoglutarate transaminase on protein EctB, and ectoine synthase on the protein EctC. The phylogenetic tree showed that the EctABC protein from V. salarius was closely related to the EctABC protein from V. salexigens, and Multiple Sequence Alignment showed that the EctABC protein was highly conserved. The ectA, ectB, and ectC genes isolated from V. salarius were 480 bp, 1245 bp, and 387 bp, respectively.