Genome Sequences of Bacteria Isolated from the International Space Station Water Systems

ABSTRACT

We report draft genomes of five bacteria recovered from the U.S. and Russian water systems onboard the International Space Station. The five genera include Ralstonia, Burkholderia, Cupriavidus, Methylobacterium, and Pseudomonas. These sequences will help further the understanding of water reclamation and environmental control and life support systems in space.

ANNOUNCEMENT

Water-associated bacteria capable of biofilm formation onboard the International Space Station (ISS) have represented a challenge for the operation of the environmental control and life support system (ECLSS) (1). Bacteria isolated from the water recovery system, which recycles urine, humidity condensate, and other nonpotable water to water for crew consumption and hygiene, have been used in multiple ground studies that pertain to ECLSS biofouling (2–5). Similarly, isolates from the Russian SRV-K, which recovers water from humidity condensate, and the SVO-ZV, which supplies ground water delivered to the ISS, have been and continue to be investigated (6). Several NASA-affiliated studies conceived of the idea of using a defined consortium of bacteria that are most frequently found in the ISS water to evaluate different means of biofouling control (7, 8). A subsequent microbial control report suggested using five specific spaceflight isolates of Ralstonia insidiosa, Burkholderia multivorans, Cupriavidus metallidurans, Methylobacterium fujisawaense, and Pseudomonas aeruginosa, based on available 16S rRNA identifications (3). All except P. aeruginosa have been recovered on multiple occasions from ISS water systems since 2002. These five specific organisms have been provided to other investigators assessing methods of microbial control within spaceflight water systems. Therefore, in this study, we established their complete genomes to facilitate further investigation.

Following sample collection onboard the ISS, water was returned to Earth in a Teflon collection bag and processed in the Microbiology Laboratory at the Johnson Space Center (JSC). For all isolates, bacteria were cultured on Reasoner’s 2A agar plates for 48 h at 35°C. The colonies obtained were subcultured, and glycerol stocks were archived at −80°C. Initial identifications were completed through 16S rRNA sequencing following the JSC Sanger sequencing standard procedures with primers 5F and 531R (9, 10). To obtain the full genome sequences, samples were cultured in tryptic soy broth for 24 h at 35°C. High-molecular-weight genomic DNA (gDNA) was obtained with the Circulomics Nanobind CCB Big DNA kit (Pacific Biosciences [PacBio]) without shearing or size selection and was assessed with a Qubit fluorometer (Thermo Fisher Scientific) and TapeStation (Agilent). To obtain long reads, libraries were prepared with the Oxford Nanopore Technologies (ONT) SQK-RBK004 kit. Libraries were loaded into an R9.4.1 flow cell and run on a MinION Mk1C system for 72 h. Raw reads were base called using the high-accuracy configuration of Guppy v.4.3.4, adapters were removed with Porechop v.0.2.4, and reads shorter than 2 kb were filtered using Filtlong v.0.2.0 with the following flags: –min_length 2000 –keep_percent 80 –trim –split 750. Corresponding paired-end 300-bp short reads were obtained from libraries prepared with the Illumina DNA preparation kit, with consecutive sequencing with v.3 reagents on an Illumina MiSeq system. These reads were filtered using BBduk (BBTools v.38.54) and quality filtered with the following parameters: minlen=50 qtrim=rl trimq=25 hdist=1 tpe tbo. Hybrid de novo genome assembly was carried out using Unicycler v.0.5.0 (11). Default parameters were used for all software unless otherwise specified. The genome assembly metrics for each isolate are listed in Table 1. Annotations were performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v.6.4 (12). The genomes obtained have >99.6% completeness, as assessed using CheckM v.1.0.18 (13).

TABLE 1.

Summary of draft genome sequences belonging to the five bacteria isolated from ISS water

Bacterial identification	NASA sample name	Sourcea	GenBank accession no.	Genome size (bp)	No. of Nanopore reads	Nanopore read N50 (bp)	No. of Illumina reads	No. of contigs	G+C content (%)	Genome N50 (bp)	No. of coding sequences
Ralstonia insidiosa	171870003-1	ISS WPA wastewater	JAQPZM000000000	6,271,672	166,983	28,078	1,374,153	5	63.33	3,668,357	5,878
Burkholderia contaminans	172630038-1	ISS WPA wastewater	JAQPZL000000000	8,711,463	42,546	23,803	1,902,557	6	66.23	3,246,788	7,851
Cupriavidus metallidurans	162430002-4	ISS WPA wastewater	JAQPZK000000000	7,278,803	79,657	21,481	926,800	5	63.47	3,954,298	6,809
Methylobacterium sp.	092160098−2	ISS SVO-ZV	JAQPZJ000000000	7,859,068	13,825	21,791	778,677	10	69.23	5,338,800	7,450
Pseudomonas aeruginosa	0201761-1	ISS SRV-K	CP117300	6,901,248	80,489	29,628	695,299	1	65.94	6,901,248	6,443

^aWPA, water processor assembly.

Data availability.

The complete genome sequences were deposited in GenBank under BioProject accession number PRJNA929559. The raw reads were deposited in the SRA database under BioProject accession number PRJNA929559. The versions described in this paper are the first versions.