Complete genome sequences of nine isolates from microplastics in the Bow River, Calgary, Canada

TL;DR

This paper reports the full genomes of nine bacteria found on microplastics in a Canadian river, offering insights into their potential to break down plastic.

Contribution

The study provides complete genome sequences of nine bacterial isolates from microplastics in the Bow River, revealing their biodegradation potential.

Findings

Nine bacterial strains were isolated from microplastics in the Bow River.

The complete genome sequences of these isolates were determined.

The isolates may contribute to understanding plastic biodegradation in freshwater environments.

Abstract

We present the complete genome sequences of nine bacterial strains isolated from microplastics from water or sediments of the Bow River in Calgary, Alberta. These isolates provide insight into the freshwater microplastic microbiome and their plastic biodegradation potential.

Tables

TABLE 1. Summary of isolates and their genomes

Isolate ID	LS1	LW8	RS5	RS7	RS8	RS10	RS11	RW6	RW9
Culture media	LB	LB	R2A	R2A	R2A	R2A	R2A	R2A	R2A
Classification	Lysinibacillus fusiformis	Pseudomonas sp.	Lysinibacillus sp.	Peribacillus sp.	Paenibacillus sp.	Lysinibacillus sp.	Lysinibacillus sp.	Acinetobacter sp.	Stenotrophomonas lactitubi
Genome size (bp)	4,761,317	5,952,450	5,165,228	5,809,518	7,333,835	5,165,155	4,918,501	3,939,831	4,537,953
Status	Complete	Complete	Complete	Complete	Complete	Complete	Complete	Complete	Complete
Genome coverage	47×	21×	46×	22×	28×	52×	59×	182×	78×
Number of reads	37,364	15,924	23,584	75,412	115,168	27,450	63,631	81,173	117,616
N50 reads (bp)	18,682	20,361	18,511	10,091	4,146	18,060	14,863	19,989	8,298
GC%	37.6	60.0	37.1	39.8	43.6	37.1	37.0	38.8	66.1
No. of sequences	1	2	1	3	7	1	5	1	1
CDS	4,602	5,277	4,900	5,508	6,547	4,995	4,779	3,651	4,049
tRNA	107	70	109	83	88	109	119	73	72
CRISPR repeats	1	2	14	6	8	14	13	0	8
Plastic degradation genes		PHA depolymerase, lipase, esterase		Oxr1 Oxidoreductase				Phthalate dioxygenase reductase	PHA depolymerase, PHB depolymerase
Hydrocarbon degradation genes		LadA						AlkB (2), LadA, AlmA
GenBank accession	CP167119	CP168138 (chromosome), CP168137 (pLW8a)	CP169418	CP168135 (chromosome), CP168134 (pRS7b), CP168136 (pRS7a)	CP168131 (chromosome), CP168128 (pRS8c), CP168129 (pRS8d), CP168130 (pRS8a), CP168132 (pRS8b), CP168133 (pRS8e)	CP167122	CP168124 (chromosome), CP168123 (pRS11a), CP168125 (pRS11b), CP168126 (pRS11d), CP168127 (pRS11c)	CP167121	CP167120
SRA accession	SRR31131491	SRR31131486	SRR31131489	SRR31131487	SRR31131488	SRR31131490	SRR31131492	SRR31131493	SRR31131485

Full text

ANNOUNCEMENT

Microplastics are pervasive contaminants in environments throughout the globe, serving as hosts for microbial communities distinct from those in the surrounding environment (1). Here, we present genomes of bacteria isolated from microplastics in the Bow River, after the confluence with Nose Creek and Elbow River, within Calgary, Canada’s fourth largest city.

Microplastics were isolated from water (40 L) and sediment samples (0.5 Kg) (51.04344 °N, 114.01502 °W) through 20 µm filtration or CaCl_2_ density separation (1.4 Kg/L), respectively, followed by Nile red staining (2). Microplastics were rinsed twice with 1× PBS buffer and inoculated in either liquid LB (3) or R2A media (4) at room temperature (22°C). Upon visible growth (2–3 days), a 10^−4^ dilution of the liquid culture was spread-plated on the respective solid media (LB or R2A with 1.5% agar at room temperature), followed by 4–5 rounds of streaking to achieve isolation. One colony from each isolate was grown in respective liquid media for 48 hours and used for high-molecular-weight DNA extraction using the Lucigen MasterPure Complete DNA & RNA Purification Kit (Mandel Scientific) using the manufacturer-supplied protocol for Gram-positive bacteria. DNA (no shearing or size selection) was sequenced on an R10.4.1 Nanopore MinION flowcell using Native Barcoding Kit 24 (SQK-NBD114.24, Oxford Nanopore) and basecalled using Dorado (v7.3.11, e8.2_400bps_sup@v5.0.0 model).

Technical sequences, such as Nanopore adaptors and low-quality reads (≤1,000 bp, Q ≤10), were removed by Porechop ABI (v0.50, ABI mode) (5) and Chopper (v0.80) (6), respectively. Genomes were assembled with Flye (v2.9.3; nano-hq mode) (7) and polished with Medaka (v1.11.3). Genome completeness and contamination were calculated using CheckM2 (v1.0.1) (8). Genomes with ≥99.90% ANI were dereplicated using OrthoANIu (9) and annotated using NCBI Prokaryotic Genome Annotation Pipeline (PGAP, version 6.7) (10), and CRISPR regions were identified using CRISPR-CasFinder (subtype clustering model) (11). Taxonomy was assigned using GTDB-Tk (v2.4.0 with release 220 database) (12).

The nine isolate genomes with fully circularized chromosomes, determined by the Flye assembler, ranged in size from 3,939,831 to 7,333,835 base pairs, encoding 3,610 to 6,553 coding sequences (CDS) and 72 to 118 tRNAs (Table 1). Extrachromosomal elements (ECEs) were present in four genomes, with the RS8 genome containing six ECEs. Among the isolates, Lysinibacillus, Peribacillus, and Paenibacillus are gram-positive, while Pseudomonas, Stenotrophomonas, and Acinetobacter are gram-negative.

As these bacteria were isolated from microplastics, genomes were searched for plastic degradation genes using BLAST against PlasticDB and the Plastic Microbial Biodegradation Database (13, 14). Four genomes had hits for plastic-degrading genes (>80% identity). Isolate LW8 and RW9 have a gene for polyhydroxyalkanoate (PHA) depolymerase that can degrade poly(3-hydroxybutyrate-co-3-hydroxyvalerate), polycaprolactone, polyethersulfone, polyhydroxyalkanoate, and polylactic acid. RW9 also has a polyhydroxybutyrate (PHB) depolymerase gene involved in the biodegradation of polyhydroxybutyrate. RS7 and RW6 contained genes for polyurethane and phthalate biodegradation, respectively. Some plastics are hydrocarbon derivatives and can be degraded through hydrocarbon degradation pathways (15, 16). Hence, the genomes were searched for hydrocarbon metabolism markers using CANT-HYD (17). LW8 and RW6 contained long-chain alkane monooxygenase (LadA), with the latter containing two other alkane metabolism genes (AlkB and AlmA).