Bacterial genome sequences of uncharacterized Chitinophaga species isolated from the International Space Station

ABSTRACT

We report four Chitinophaga sp. strains isolated from wastewater collected onboard the International Space Station. Here, we present three finished and one draft genome. Taxonomic ranks established by genome-based analysis indicate that these Chitinophaga sp. strains represent candidates for a new species.

ANNOUNCEMENT

The genus Chitinophaga was described by Sangkhobol and Skerman when characterizing the type species Chitinophaga pinensis (1). Chitinophaga consists of Gram-negative, rod-shaped bacterium (2), and at the time of writing, there are 56 species listed on List of Prokaryotic Names with Standing in Nomenclature (www.bacterio.net) (3). There have been previous reports of Chitinophaga isolated from ground-based wastewater systems (4), and it has been identified by studies conducted onboard the International Space Station (ISS) (5).

Here, we present four whole-genome sequences of Chitinophaga recovered from ISS wastewater during expeditions 54 and 65. Samples 180180018-2 and 180180018-3 were collected on 30 October 2017, and 212800008-4 and 212800010-3 were collected on 6 September 2021. The ISS Water Processor Assembly manages water at various levels of cleanliness and handles water reclamation (6). Following microbiological sample collection, 250 mL was returned to Earth in a Teflon collection bag under ambient conditions. Upon arrival, samples were maintained under ambient conditions and transported to the Microbiology Laboratory at the Johnson Space Center in Houston.

The microbial composition of the wastewater samples was characterized as described previously through filtration, cultivation on Reasoner’s 2A agar, and isolate archival (7). Colonies were revived from frozen glycerol stocks and cultured in tryptic soy broth for 24 h at 35°C prior to isolation of genomic DNA, for both short- and long-read libraries, with the Circulomics Nanobind CBB Big DNA Kit [Pacific Biosciences (PacBio)] without shearing or size selection. DNA quality was assessed with a Qubit 4 fluorometer (Thermo Fisher Scientific) and TapeStation (Agilent). To obtain long reads, libraries were prepared with the Oxford Nanopore Technologies SQK-RAD004 kit for 180180018-2 and 180180018-3 and SQK-RBK004 kit for 212800008-4 and 212800010-3. Libraries were loaded into an R9.4.1 flow cell and ran on a MinION for 72 h. Raw reads were base called using Dorado v0.4.1, adapters were removed with Porechop (8) v0.2.4, and reads shorter than 500 bp were filtered using Filtlong (9) v0.2.1 with the following flags: --min_length 500 --target_bases 500,000,000. Corresponding paired-end 300 bp short reads were obtained from libraries prepared with the Illumina DNA Preparation Kit, with sequencing using v.3 reagents on an Illumina MiSeq. These reads were quality filtered using FastP (10) v0.23.4 with the following parameters: --detect_adapter_for_pe --trim_poly_g --cut_front --cut_tail --length_required 21 --low_complexity_filter --correction. Hybrid genome assembly was carried out using Unicycler (11) v0.5.0 with default parameters guided by a long-read-only assembly using Flye (12) v2.9.2 and the --nano-hq parameter. Resulting assemblies were short-read polished using Polypolish (13) v0.5.0 and PyPolca (14) v0.2.0. Annotations were performed using Bakta (15) v1.8.2 in compliant mode using its full database (5.0). Default parameters were used for all software unless otherwise specified.

For 180180018-2, 180180018-3, and 212800008–4, complete and circular genomes were assembled. For strain 212800010-3, a draft genome containing 12 contigs was assembled with an N₅₀ of 7,928,055 bp. All genomes have >99.7% completeness and <0.9% contamination, as assessed by CheckM2 (16) v1.0.1. Genome sequencing metrics are listed in Table 1. All genomes were classified to the genus of Chitinophaga using GTDBtk (17) v2.3.2.

TABLE 1.

Summary of genome sequencing, assembly, and annotation results of four bacteria isolated from ISS

NASA sample ID	No. of reads (Illumina/ Nanopore)	Read coverage (Illumina/ Nanopore)	Nanopore read N50 (bp)	Genome size (bp)	No. of contigs	Complete genome	G + C content (%)	CheckM2 completeness/ contamination	No. of coding sequences	GenBank accession	BioSample accession	SRA accession
180180018-2	12,970,158 57,193	419 36	9,033	8,131,931	1	Yes	46.3	99.83 0.86	6,484	CP140773	SAMN38502441	SRS19676642
180180018-3	13,868,428 167,836	406 175	17,666	8,131,986	1	Yes	46.3	99.85 0.86	6,484	CP140772	SAMN38502442	SRS19676643
212800008-4	4,251,176 41,424	148 53	20,039	8,110,254	1	Yes	46.3	99.8 0.86	6,467	CP140771	SAMN38502443	SRS19676644
212800010-3	3,179,880 60,934	111 22	5,508	7,933,844	12	No	46.3	99.77 0.77	6,353	JAXUID000000000	SAMN38502444	SRS19676645

DATA AVAILABILITY

This Whole Genome Shotgun project has been deposited in GenBank under the accession number PRJNA1046541. The version described in this paper is the first version.