Biosynthetic gene cluster profiling from North Java Sea Virgibacillus salarius reveals hidden potential metabolites

Abstract

Virgibacillus salarius 19.PP.SC1.6 is a coral symbiont isolated from Indonesia's North Java Sea; it has the ability to produce secondary metabolites that provide survival advantages and biological functions, such as ectoine, which is synthesized by an ectoine gene cluster. Apart from being an osmoprotectant for bacteria, ectoine is also known as a chemical chaperone with numerous biological activities such as maintaining protein stability, which makes ectoine in high demand in the market industry and makes it beneficial to investigate V. salarius ectoine. However, there has been no research on genome-based secondary metabolite and ectoine gene cluster characterization from Indonesian marine V. salarius. In this study, we performed a genomic analysis and ectoine identification of V. salarius. A high-quality draft genome with total size of 4.45 Mb and 4426 coding sequence (CDS) was characterized and then mapped into the Cluster of Orthologous Groups (COG) category. The genus Virgibacillus has an "open" pangenome type with total of 18 genomic islands inside the V. salarius 19.PP.SC1.6 genome. There were seven clusters of secondary metabolite-producing genes found, with a total of 80 genes classified as NRPS, PKS (type III), terpenes, and ectoine biosynthetic related genes. The ectoine gene cluster forms one operon consists of ectABC gene with 2190 bp gene cluster length, and is successfully characterized. The presence of ectoine in V. salarius was confirmed using UPLC-MS/MS operated in Multiple Reaction Monitoring (MRM) mode, which indicates that V. salarius has an intact ectoine gene clusters and is capable of producing ectoine as compatible solutes.

Figure 1

Ectoine and 5-hydroxyectoine biosynthesis pathway. This pathway consists of ectA, ectB, and ectC as main genes. Not every ectoine-producing bacteria has the ectD gene, whose main activity is to produce ectoine derivative 5-hydroxyectoine.

Figure 2

(a) Calculation results of ANI and dDDH of 14 Virgibacillus genomes for sample identification. (b). Phylogenomic analysis of the V. salarius 19.PP.SC1.6 genome compared to 43 other Virgibacillus genomes in total. Phylogenomic tree was constructed using OrthoFinder and Visualized with iTOL software.

Figure 3

Circular analysis and visualization of V. salarius 19.PP.SC1.6 genome. From the outside to the inside, the first and second circle represent genes with COG annotation. Circles 3 (green and purple) and 4 (black) show GC skew and GC content as the deviation from the average for the complete genome.

Figure 4

Pan- and core-genome estimation curves. (a) The size of the pan-genome increases for every included genome indicating an open pan-genome. (b) Core-genome size decreases with more genomes included in analysis.

Figure 5

Visualization of Genomic Island detected in the V. salarius 19.PP.SC1.6 Genome using IslandViewer 4.0. The genome of V. salarius 19.PP.SC1.6 contains a total of 18 genomic islands.

Figure 6

Ectoine gene cluster. (A) Comparative schematic structure of ectoine biosynthetic gene organization in Virgibacillus salarius and other ectoine producing bacteria. l-2,4-diaminobutyric acid N-γ-acetyltransferase genes (ectA) are red, diaminobutyric acid transaminase genes (ectB) are yellow, and ectoine synthase (ectC) are blue. (B) Ectoine gene cluster in Virgibacillus salarius from WGS result showed two operons comprised of ectABC genes with two promoter (red box) and one terminator (green box). (C) Putative structural features of V. bacillus ectoine biosynthetic gene cluster, consist of − 35 and − 10 boxes, with transcription start site (+ 1).

Figure 7

Phylogenetic analysis of Virgibacillus salarius' (A) ectA, (B) ectB, and (C) ectC and other ectoine-producing bacteria deposited in the NCBI Database. Sequences were aligned using MEGA 10 and phylogenetic trees were built using the neighbor-joining (NJ) method within the MEGA 10 software. V. salarius' ectABC amino acid sequences were closely related with V. salexigens.

Figure 8

Comparative schematic representation of the V. salarius ectoine gene cluster structure based on (A) WGS and (B) plasmid sequencing results. Plasmid sequencing revealed that ectoine gene cluster forms one operon consisting of ectABC gene with the promoter shown in the green box and terminator shown in the red triangle.

Figure 9

Ectoine biosynthesis analysis of Virgibacillus salarius. UPLC-MRM-MS/MS map (a) The UPLC map shows spectra of intracellular ectoine extracted from V. salarius. (b) The UPLC map of the authentic standard of ectoine. The number shown in the map indicates that intracellular ectoine from V. salarius has high similarity in structure as compared to pure ectoine used as control.