Biotechnologically potential genes in a polysaccharide-degrading epibiont of the Indonesian brown algae Hydroclathrus sp

Abstract

Background: Marine bacteria have recently attracted increasing attention to be harnessed for the production of valuable enzymes, vitamins, and bioactive compounds. Bacteria associated with the surfaces of marine macroalgae, called epibionts, are particularly interesting from ecological and biotechnological points of view, as they often exhibit antimicrobial activities to compete with pathogenic bacteria for nutrients and spaces. In search for biotechnologically potential genes from marine bacteria, we sequenced and analysed the genome of the epibiont HI03-3b, a polysaccharide-degrading bacterium associated with the surface of the Indonesian brown algae Hydroclathrus sp.

Results: The algal epibiont HI03-3b has a genome of approximately 4,860,704 bp in size with 42.02 mol% G + C content, consisting of 5655 open reading frames (ORFs), 4409 genes coding for proteins (CDSs), 94 genes for tRNAs, and 32 genes for rRNAs. The genome sequence of HI03-3b was most closely related to that of Cytobacillus firmus NCTC10335 with the average amino acid identity (AAI) of 95.0 %, average nucleotide identity (ANI) of 94.1 %, and a recommended DNA-DNA hybridization (DDH) of 57.60 %. These scores are lower than the most frequently used standard for species demarcation (95% ANI cutoff) and the new species threshold (DDH > 70.0% for the same bacterial species). Some differences in genome features and gene composition were observed between HI03-3b and NCTC10335, such as genes encoding carbohydrate active enzymes. These suggest that HI03-3b is unique and likely a novel species within Cytobacillus genus, and we therefore proposed its name as Cytobacillus wakatobiense HI03-3b. Genome sequence analyses indicated the presence of genes involved not only in polysaccharide and protein degradation but also in vitamin and secondary metabolite biosynthesis. Some of them encode enzymes and compounds with biotechnological interest, such as protease, chitinase, subtilisin, pullulanase, and bacillolysin, which are often associated with antimicrobial or antibiofilm activities. This antimicrobial potential is supported by our finding that the extracellular protein fraction of this epibiont inhibited the growth of the bacterial pathogen Staphylococcus aureus.

Conclusion: The epibiont Cytobacillus HI03-3b harbours genes for polysaccharide and protein degradation as well as for natural product biosynthesis, suggesting its potential ecological roles in outcompeting other bacteria during biofilm formation as well as in protecting its algal host from predation. Due to the presence of genes for vitamin biosynthesis, it might also provide the algal host with vitamins for growth and development. Some of these metabolic genes are biotechnologically important, as they could become a platform for bioengineering to generate various seaweed-derived substances sustainably, such as antibiofilm agents and vitamins, which are beneficial for human health.

Keywords: Antimicrobial activity; Bacterial epibiont; Biotechnologically potential genes; Ecological role; Genome sequencing; Marine macroalgae.

Fig. 1

Isolation of the potential algae-associated epibiont HI03-3b. The Indonesian brown algae Hydroclathrus sp. (photo taken by S. N. Ethica) where HI03-3b was isolated (A) and colony morphology of HI03-3b (B). Bioassays of HI03-3b showing a clearing zone surrounding colonies (duplo) on an alginate-containing MS plate after Lugol staining (C) and inhibition zone by the extracellular culture against the lawn bacterial pathogen S. aureus (D)

Fig. 2

The phylogenetic analysis of HI03-3b 16S-rRNA gene sequence using the UPGMA method [43]. The bootstrap consensus tree inferred from 1000 replicates represents the evolutionary history of the taxa analyzed [19]. The evolutionary distances were calculated according to the maximum composite likelihood method [62]. Evolutionary analyses were performed in MEGA X [32], which involved 45 16S-rRNA gene sequences from representative members of some genera within Bacillaceae family. There were a total of 1593 positions in the final dataset. The phylogenetic tree covers representative members of some genera within Bacillaceae family (Bacillus, Cytobacillus, Mesobacillus, Neobacillus, and Peribacillus). Neobacillus clade is as follows: 1, N. bataviensis NBRC 102449; 2, N. drentensis IDA1967; 3, N. novalis NBRC 102450; 4, N. niacini NBRC 15566; and 5, N. pocheonensis Gsoil 420. Mesobacillus clade is as follows: 1, M. foraminis CV53; 2, M. zeae JJ-247; 3, M. campisalis SA2-6; 4, M. stamsii BoGlc83; 5, M. thioparans BMP-1; 6, M. boroniphilus T-15Z; 7, M. subterraneus COOI3B; and 8, M. jeotgali YKJ-10. Peribacillus clade is as follows: 1, P. kribbensis BT080; 2, P. cavernae L5; 3, P. asahii A001; 4, P. psychrosaccharolyticus 23296; 5, P. muralis LMG 20238; 6, P. frigoritolerans DSM 8801; 7, P. implex NBRC 15720; and P. simplex LMG 11160. Bacillus clade is as follows: 1, B. aerophilus 28K; 2, B. stratosphericus 1KF2a; 3, B. licheniformis DSM 13; 4, B. licheniformis ATCC 14580; 5, B. velezensis FZB42; 6, B. vallismortis DSM 11031; 7, B. subtilis IAM 12118; 8, B. inaquosorum BGSC 3A28; 9, B. halotolerans DSM 8802; and 10, B. niacini IFO15566

Fig. 3

Pairwise genome sequence comparison to determine the order and direction of HI03-3b contigs. A HI03-3b contigs were mapped onto with the genome sequence of C. firmus NCTC10335 (Acc. Nu. NZ_UFTC01000001.1) with the ANI score of 94.15 %. B HI03-3b contigs were compared with the complete genome sequence of C. oceanisediminis 2691 (NCBI Acc. Nu. GCA_000294775.2) with the ANI score of 88.6 %

Fig. 4

Visualization of HI03-3b genome features. A Identification of CDSs, rRNA genes, and tRNA genes (indicated by blue, purple, and green colours) using PROKSEE on the CGView Server [58, 59] with Prokka annotation [54]. The order and direction of contigs generated after genome sequence assembly were determined based on blastn pairwise alignment [2] using the genome sequences of C. oceanisediminis 2691 and C. firmus NCTC10335 as the references. B Prediction of genomic islands using IslandViewer 4 based on three different methods (indicated by red, orange, and blue colours) [5] with the genome sequence of C. oceanisediminis YPW-V2 as the reference. Note: CDS, coding sequence; GI, genomic island

Fig. 5

The position of biotechnologically potential genes on the HI03-3b genome map. A Certain loci encoding biotechnologically potential enzymes and secondary metabolite biosynthesis were determined based on PROKSEE analysis on the CGView Server [58, 59]. B Natural product BGCs were identified in the HI03-3b genome sequence based on antiSMASH analysis [7] supported by BLASTx [2], showing the presence of core and additional biosynthetic genes, transport-related genes, regulatory genes, and other genes. The core biosynthetic genes of RiPP-like terpene BGC (from left to right) are predicted to code for squalene-hopene cyclase, leader peptide (SagB-type dehydrogenase domain), YcaO cyclodehydratase, and thiazole-containing bacteriocin maturation protein. PKS type III BGC contains a chalcone synthase gene (indicated by red colour) as the core biosynthetic gene. The siderophore BGC harbours the core biosynthetic genes encoding lucA/lucC family proteins. Based on RiPPMiner prediction [1] the core lassopeptide BGC encodes a precursor peptide that consists of a leader (VKAPGSTGEGHWKLGNLSAEEKSGIPRVAVKCVEMWRNTSGE) and core peptide sequence (GDSLVCN). A crosslink may occur between serine (S) and cysteine (C) residues in the core peptide [1]. Note: PKS, polyketide synthase; BGC, biosynthetic gene cluster; RiPP, ribosomally synthesized and post-translationally modified peptides