ABSTRACT

ANNOUNCEMENT

The phylum Bacteroidetes is a proliferating but underexplored bioresource for natural product discovery (1, 2). The bacteria colonize diverse habitats and are among the most abundant groups of bacteria within marine ecosystems (3). In an effort to access new Bacteroidetes strains, strains PAP.12 and PAP.19 were isolated from Cenderawasih Bay National Park (Papua, Indonesia). The strains were deposited in the Fraunhofer strain collection (4) under the identifiers EXT111900 and EXT111901.

EXT111900 was retrieved from the upper layer (5 to 10cm) of marine sediment (1°47′45.6″S, 134°25′12.0″E). EXT111901 was isolated from a sponge (1°48′03.6″S, 134°06′03.6″E) by cutting using a diving knife while latex gloves were worn. The samples were put into sterile bags, brought to the surface to be kept at ambient seawater temperature, and processed within 2 hours after sampling, as follows. Sponge samples were cut into small pieces using a sterile scalpel, and all samples were rinsed with sterile seawater before being plated on artificial seawater (1.5% agar, 0.01% KBr, 2.3% NaCl, 1.1% MgCl₂·6H₂O, 0.1% CaCl₂·2H₂O, 0.1% KCl, 0.004% SrCl₂·6 H₂O, 0.4% Na₂SO₄, 0.02% NaHCO₃, and 0.003% H₃BO₃ in H₂O) with Escherichia coli prey and cycloheximide. Bacterial colonies were isolated, and sequencing samples were prepared by growing the strains aerobically for 24h at 30°C in marine broth (product number CP73.1; Carl Roth GmbH). Cell pellets were resuspended in buffer ATL (Qiagen) containing RNase A. ZR BashingBead lysis tubes (Zymo Research) were used for cell disruption. DNA was isolated using QIAmp 96 DNA QIAcube high-throughput (HT) kits with the addition of proteinase K (Qiagen). Libraries for short-read sequencing were prepared using the Illumina DNA preparation tagmentation kit with 500ng DNA as input and 5 cycles of indexing PCR. Library quality was evaluated (Agilent 2100 Bioanalyzer), and libraries were sequenced on an Illumina NovaSeq system using a NovaSeq 6000 SP v1 sequencing kit with 2 × 150 bp read length and a depth of 4.0 to 5.0 million reads per sample. For sequence processing and analysis, software tools were run with default settings unless otherwise stated. The sequencing was demultiplexed (Illumina bcl2fastq v2.19.0.316), quality checked (Fastp v0.20.1 [5], with additional parameters –detect_adapter_for_pe, –cut_by_quality5, –cut_by_quality3, –low_complexity_filter, –length_required 21, and –correction), and visualized (MultiQC v1.7 [6]). Paired-end reads were quality filtered (Fastp v0.20.1 [5]), assembled (Unicycler v0.4.8 [7]), and quality checked (CheckM v1.0.18 [8]), and genomes were annotated using Bakta v1.5.1 (9). The genome of strain EXT111900 consists of 5,032,044bp in 101 contigs (coverage, 405×; N₅₀, 235,524 bp), with a GC content of 39.4%, revealing completeness of 99.81% and contamination of 0.19%. The genome contains 4,286 protein-coding genes, 41 tRNAs, 1 transfer-messenger RNA (tmRNA), 3 rRNAs, and 3 noncoding RNAs (ncRNAs). The genome of strain EXT111901 consists of 4,582,827bp in 6 contigs (coverage, 362×; N₅₀, 3,058,286 bp), with a GC content of 42.1%, revealing completeness of 99.81% and no contamination. The genome contains 3,944 protein-coding genes, 47 tRNAs, 1 tmRNA, 6 rRNAs, and 3 ncRNAs.

Taxonomic ranks were established by using the Type Strain Genome Server (TYGS) (10) and autoMLST (11) and by determining the average nucleotide identity (ANI) (12). Roseivirga pacifica DY53 (13) represents the closest related type strain of EXT111901. Digital DNA-DNA hybridization (dDDH) values 86.6% (d0), 70.8% (d4) and 86.7% (d6) and an ANI of 96.65%, all exceeding the species delineation threshold, support affiliation of EXT111901 with the species R. pacifica. The closest related type strain for EXT111900 is Algoriphagus zhangzhouensis DSM 25035 (14). The dDDH values are 71.8% (d0), 35.8% (d4), and 62.9% (d6), and the ANI is 88.84%. Accordingly, EXT111900 possibly represents a candidate new Algoriphagus species.

Prediction of biosynthetic gene clusters (BGCs) was performed using antiSMASH v6.0 (15). Their grouping into gene cluster families (GCFs) together with the Minimum Information about a Biosynthetic Gene cluster (MIBiG) (16) reference clusters using BiG-SCAPE (17) with a similarity index cutoff value of 0.6 allowed BGC similarity determinations. EXT111900 carries one BGC clustering with BGC0000650, encoding the carotenoid flexixanthin (18). Although less similar, the same match was detected for EXT111901 (Fig. 1). A difference is the presence of a lycopene β-cyclase in EXT111900 and its absence in EXT111901.

Open in a separate window

FIG 1

BiG-SCAPE network (left) of strains EXT111901 (turquoise) and EXT111900 (dark blue). BGCs sharing similarity with the flexixanthin MIBiG cluster were aligned (right). EXT111901 carries two terpene BGCs, a ribosomally synthesized and posttranslationally modified peptide (RiPP) BGC, and a homoserine lactone (Other) BGC. Terpene BGCs of EXT111901 and EXT111900 each cluster with the flexixanthin BGC0000650 of Algoriphagus sp. strain KK10202C (similarity index values of 0.207 and 0.604, respectively). A difference is the fusion-type lycopene β-cyclase (pink open reading frame [ORF]) present in EXT111900 and absent in EXT111901. The structure of flexixanthin is depicted at the bottom.

ACKNOWLEDGMENTS

REFERENCES