Genome sequence of Gaetbulibacter sp. PBL-D1 isolated from a Malaysian beach: insights into glycoside hydrolase repertoires

Abstract

Objectives

Marine bacteria are valuable reservoirs of novel carbohydrate-active enzymes (CAZymes) with industrial relevance. A halophilic marine bacterial strain PBL-D1, was isolated from Batu Laut beach in Selangor, Malaysia. The 16S rRNA gene sequence analysis identified strain PBL-D1 as a member of the genus Gaetbulibacter. To date, no enzymes from Gaetbulibacter spp. have been biochemically characterised. The present study reports on the annotated draft genome of Gaetbulibacter sp. PBL-D1 and comparative genome analyses of Gaetbulibacter glycoside hydrolase (GH) enzymes. These findings provide the first genomic insights into the GH repertoire of Gaetbulibacter spp., revealing unexplored enzymes with promising potential for industrial biocatalysis.

Data description

The draft genome of Gaetbulibacter sp. PBL-D1 is 3,433,971 bp in size, has a G+C content of 33.0%, and encodes 3,068 protein-coding genes. The CAZymes analysis revealed that Gaetbulibacter genomes harboured at least nine putative GH-encoding genes, grouped into starch-degrading and β-glucan-degrading enzymes. These enzymes are of industrial importance, particularly in food processing and biofuel production. Collectively, PBL-D1 and other available Gaetbulibacter genomes serve as attractive sources of GH candidates that may be useful in polysaccharide degradation.

Objective

Marine bacteria are promising sources of carbohydrase enzymes for various industrial applications [1]. Among them, Gaetbulibacter spp. remain understudied, with no reports available on their industrially relevant enzymes. These halophilic bacteria are predominantly found in seawater and tidal sediments [2]. As of October 2025, six species have been validly described and listed in the List of Prokaryotic Names with Standing in Nomenclature database (https://lpsn.dsmz.de): Gaetbulibacter aestuarii KYW382^T, Gaetbulibacter aquiaggeris KEM-8^T, Gaetbulibacter jejuensis CNURIC 14^T, Gaetbulibacter lutimaris D1-y4^T, Gaetbulibacter marinus IMCC1914^T, and Gaetbulibacter saemankumensis SMK-12^T [2–7]. Only nine draft genomes (including this work) are currently available in the NCBI Genome database (October 2025), highlighting the need for genomic studies to elucidate the biochemical pathways and biotechnological prospects of this genus.

Carbohydrate-Active enZYmes (CAZymes) catalyse the degradation, modification, or biosynthesis of polysaccharides (e.g., starch and lignocellulose) and are catalogued in the CAZy database [8]. They are grouped into glycoside hydrolases (GHs), glycosyltransferases (GTs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), and auxiliary activities (AAs) [8]. GHs such as α-amylase, type I pullulanase, oligo-1,6-glucosidase, and α-glucosidase are widely used in starch saccharification for sugar syrup production [9], whereas lignocellulose degradation involves a combination of GHs (i.e., β-glucosidase and endoglucanase), CEs, and AAs [10]. Although the ability of Gaetbulibacter spp. to hydrolyse starch or lignocellulose is unknown, preliminary biochemical tests indicated α-glucosidase and β-glucosidase activities [2–7].

The Batu Laut beach located in Selangor, Malaysia (2.6747°N, 101.5166°E) is an underexplored site for microbial and enzyme discovery. Previous isolates from this site (e.g., Nitratireductor sp. PBL-C9 and Vibrio sp. PBL-C16) revealed novel GHs associated with polysaccharide degradation [11, 12]. Here, we present an annotated draft genome of Gaetbulibacter sp. PBL-D1 isolated from Batu Laut beach and analyse GH repertoires in PBL-D1 and eight publicly available Gaetbulibacter genomes, providing the first insights into their potential for industrial applications.

Data description

An overview of the data analysis workflow is provided in Table 1 (Data File 1). Gaetbulibacter sp. PBL-D1 was isolated from wet sediment and mud (upper 15 cm layer) collected from Batu Laut beach in Selangor, Malaysia, using a previously described ex situ cultivation method [23]. Pure colonies of PBL-D1 were obtained by streaking the cells on marine agar (Condalab, Spain) at 30 °C (pH 7.5) for 48 h. Genomic DNA was extracted using the Monarch Genomic DNA Purification Kit (New England BioLabs, USA) following the manufacturer’s protocol. The 16S rRNA gene (Table 1, Data Set 1) was amplified using primers 27F and 1492R [24], sequenced, and taxonomically identified using NCBI BLASTn and the EzBioCloud 16S database [25]. The analyses showed that strain PBL-D1 was closely related (99.86%) to G. jejuensis CNURIC 14^T [5].

Table 1.

Overview of data files/data sets

Label	Name of data file/data set	File types (file extension)	Data repository and identifier (DOI or accession number)
Data file 1	Data analysis workflow for the isolation, identification, whole-genome sequencing, and carbohydrate-active enzymes mining of Gaetbulibacter sp. PBL-D1.	PDF file (.pdf)	Figshare: 10.6084/m9.figshare.30225265 [13]
Data file 2	Genome attributes of Gaetbulibacter spp. according to Minimum Information about a Genome Sequence (MIGS) specification.	Excel file (.xlsx)	Figshare: 10.6084/m9.figshare.30225280 [14]
Data file 3	Genomic comparison of strain PBL-D1 against all available genomes of Gaetbulibacter spp. using digital DNA-DNA hybridisation (dDDH) and average nucleotide identity (ANI).	Excel file (.xlsx)	Figshare: 10.6084/m9.figshare.30225295 [15]
Data file 4	Distribution of CAZymes in strain PBL-D1 and eight other known genomes of Gaetbulibacter spp.	PDF file (.pdf)	Figshare: 10.6084/m9.figshare.30225301 [16]
Data file 5	List of several glycoside hydrolases (GHs) identified in Gaetbulibacter genomes and their identity to the other genera.	Excel file (.xlsx)	Figshare: 10.6084/m9.figshare.30225313 [17]
Data set 1	16S rRNA gene sequence of Gaetbulibacter sp. PBL-D1	Fasta file (.fasta)	NCBI GenBank (http://identifiers.org/nucleotide:PV639090.1) [18]
Data set 2	NCBI BioProject entry for Gaetbulibacter sp. PBL-D1	NCBI BioProject (no file format)	NCBI BioProject (http://identifiers.org/bioproject:PRJNA1262674) [19]
Data set 3	NCBI BioSample entry for Gaetbulibacter sp. PBL-D1	NCBI BioSample (no file format)	NCBI BioSample (http://identifiers.org/biosample:SAMN48478941) [20]
Data set 4	NCBI whole-genome assembly and annotation record of Gaetbulibacter sp. PBL-D1	Fasta file (.fasta)	NCBI GenBank (http://identifiers.org/nucleotide:JBNWKB000000000.1) [21]
Data set 5	Illumina NovaSeq X raw sequence reads of Gaetbulibacter sp. PBL-D1	Fastq files (.fq.gz)	NCBI Sequence Read Archive (SRA) (http://identifiers.org/insdc.sra:SRR33593457) [22]

For whole-genome sequencing, PBL-D1 was grown on marine agar (pH 7.5) at 30 °C for 48 h. Genomic DNA was extracted from a single colony of the cells as described above. A paired-end library was prepared using the standard protocol of the NEBNext Ultra II DNA Library Preparation Kit for Illumina (New England BioLabs, USA). Sequencing was performed using the Illumina NovaSeq X system with 150 bp paired-end reads. Raw reads were trimmed using Fastp v0.24.1 [26], de novo assembled using SOAPdenovo v2.04 [27], and annotated using the NCBI Prokaryotic Genome Annotation Pipeline v6.10 [28]. Genome comparison between PBL-D1 and eight publicly available Gaetbulibacter genomes (NCBI, October 2025) was performed using digital DNA-DNA hybridisation (dDDH) via Genome-to-Genome Distance Calculator v3.0 [29] and average nucleotide identity (ANI) in EzBioCloud [30]. CAZymes were mined using dbCAN3 [31]. Default parameters were used for all software tools, unless otherwise specified.

Genomic features of PBL-D1 are summarised in Table 1 (Data File 2; Data Sets 2–5). Sequencing produced 1,218,647,100 bases from 4,062,157 paired-end reads. The assembled genome comprised 3,433,971 bp in 44 contigs, with 352× coverage, an N₅₀ of 183,456 bp, and a G + C content of 33.0%. Annotation identified 3,135 genes, including 3,068 protein-coding genes, 54 RNAs (4 5S rRNAs, 3 16S rRNAs, 3 23S rRNAs, 40 tRNAs, and 4 ncRNAs), and 13 pseudogenes. Genome comparative analyses showed that PBL-D1 is closely related to G. jejuensis CNURIC 14^T (dDDH, 76.10%; ANI, 97.45%) (Table 1, Data File 3).

Table 1 (Data File 4) presents the CAZymes distribution in Gaetbulibacter genomes. The Gaetbulibacter spp. encode 94–175 CAZymes, with GHs and GTs being the predominant groups (average 45.22% GH; ~40.11% GT). In PBL-D1, 108 CAZymes were identified, including 42 GHs, 50 GTs, 2 PLs, and 14 CEs. Industrially relevant GHs shared among Gaetbulibacter genomes are listed in Table 1 (Data File 5). Starch-degrading enzymes (i.e., α-amylase, type I pullulanase, oligo-1,6-glucosidase, and α-L-fucosidase) were detected in several Gaetbulibacter genomes, with PBL-D1 uniquely harbouring two α-amylase genes. These enzymes are widely applied in food processing [9]. Additionally, β-glucan-degrading GHs such as β-glucosidase, endo-1,4-β-mannosidase, glucan 1,3-β-glucosidase, and β-xylosidase were found in PBL-D1 and other Gaetbulibacter genomes; these enzymes are potentially used for biofuel production [10].

Limitations

The current genomic understanding of Gaetbulibacter spp. is limited, and their industrially relevant enzymes remain uncharacterised. This study presented the genomic features of Gaetbulibacter sp. PBL-D1 and provided the comparative analysis of the underexplored GHs within the genomes of Gaetbulibacter spp. The identified GHs suggest potential roles in starch and lignocellulose degradation; however, these insights are derived solely from genome-based predictions. In the future, studies involving gene cloning, expression, protein purification, and the functional biochemical characterisation of these GHs will be performed to validate their enzymatic activities and evaluate their potential biotechnological applications.

Abbreviations

AAs

Auxiliary activities

ANI

Average nucleotide identity

BLASTn

Nucleotide Basic Local Alignment Search Tool

CAZymes

Carbohydrate-Active enZYmes

CEs

Carbohydrate esterases

dDDH

Digital DNA-DNA hybridisation

DNA

Deoxyribonucleic acid

GHs

Glycoside hydrolases

GTs

Glycosyltransferases

NCBI

National Center for Biotechnology Information

PLs

Polysaccharide lyases

RNA

Ribonucleic acid

Author contributions

U.M.K., conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, software, supervision, validation, writing the original, review and editing the manuscript; N.A.M.N., data curation, formal analysis, investigation, methodology, validation, writing the original draft; W.N.I.W.I., data curation, formal analysis, investigation, methodology, validation, writing the original draft. All authors have read and agreed to the final version of the manuscript.

Funding

This research was supported in full or in part with the Kurita Asia Research Grant via the KWEF Overseas Research Grant Program 2024 (grant number 24Pmy033), provided by Kurita Water and Environment Foundation (KWEF), awarded to U.M.K. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Data availability

This whole-genome sequencing project has been deposited in NCBI GenBank under the BioProject accession number PRJNA1262674, BioSample accession number SAMN48478941, and GenBank accession number JBNWKB000000000.1. The raw sequencing reads have been deposited in the NCBI Sequence Read Archive (SRA) under the accession number SRR33593457. The 16S rRNA gene sequence of Gaetbulibacter sp. PBL-D1 has been deposited in NCBI GenBank under the accession number PV639090.1. Please see Table 1 and references [18–22] for details and links to the data.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.