Genome Sequences of Comamonadaceae Bacteria OS-1 and OS-4, Two Highly H2O2-Sensitive Strains Isolated from Pond Water

ABSTRACT The Comamonadaceae bacterial strains OS-1 and OS-4 were isolated from pond water and were found to be highly sensitive to hydrogen peroxide in the agar plates. Here, we report the nearly complete and complete genome sequences, respectively, of these two strains.

T he Comamonadaceae bacterial strains OS-1 and OS-4 were isolated as microbes that show unprecedented sensitivity to low micromolar levels of H 2 O 2 in the agar plates (1). These strains were isolated on 12 July 2018 from the water of a pond belonging to Hokkaido University (Sapporo, Hokkaido, Japan) (43°04927.50N, 141°20929.20E). Their genome sequences are reported for further understanding of their high H 2 O 2 sensitivities.
From the original frozen glycerol stocks, both strains were cultured on peptoneyeast-glucose (PYG) agar medium (2), and then single colonies were picked and grown for 5 days at 20°C in PYG liquid medium. The genomic DNA was extracted using the phenol-chloroform method (3), and the same DNA was used to prepare short-and long-read sequencing libraries. Short-read sequencing was performed using an iSeq 100 system with paired-end 150-bp reads, and the Nextera DNA Flex library preparation kit (Illumina) was used for sequencing library preparation. The reads were quality trimmed with fastp v0.12.4 (-q 15, -n 10, -t 1, -T 1, -l 20, -w 16, -g, with other parameters at the defaults) (4), which resulted in 4,535,428 paired-end reads for OS-1 and 4,052,006 paired-end reads for OS-4. Long-read sequencing was performed using a MinION Mk1B system, and the library was prepared with the SQK-LSK109 kit (Oxford Nanopore Technologies). The reads were base called using the high-accuracy mode of MinKNOW v4.1.22, followed by quality trimming using GNU Parallel v20181022 (5) and Nanofilt v2.7.1 (-quality 10, -length 500, -headcrop 75, with other parameters at the defaults) (6), and the quality was assessed with NanoStat v1.5.0 (6). As a result, 610,015 reads for OS-1 (read N 50 value of 2,113 bases and total read length of 1,069,535,023 bases) and 393,661 reads for OS-4 (read N 50 value of 3,489 bases and total read length of 1,130,220,753 bases) were obtained.
The hybrid assembly of iSeq and MinION reads was performed using SPAdes v3.14.1 (-k 21,33,41,55,77, -nanopore, -careful, with other parameters at the defaults) (7,8). For OS-1, a single contig of .200 nucleotides (nt) was generated (length of 5,121,829 nt and coverage of 64Â). This contig is a single chromosome; however, due to the presence of long tandem repeats on the ends of the contig, the circularity was not confirmed. For OS-4, 2 contigs of .200 nt were generated, i.e., a single chromosome (length of 3,763,098 nt and coverage of 68Â) and a single plasmid (length of 318,875 nt and coverage of 109Â). The ends of the contigs overlapped, and thus the circularities were confirmed by PCR amplification of the overlapping regions. The assembled sequences of both strains were annotated using Prokka v1.14.6 (9) with default parameters, and the chromosomal sequence of OS-4 was rotated manually to the start of the dnaA gene. The tandem repeats on the ends of the OS-1 chromosome contig were annotated as an rRNA operon, and at least two copies are present. The characteristics and accession numbers of the assembled and annotated sequences are presented in Table 1.
Both OS-1 and OS-4 strains contain putative genes of catalases and alkyl hydroperoxide reductases, which degrade H 2 O 2 . The results again demonstrated that microbes could be sensitive to low micromolar levels of H 2 O 2 in the agar plate regardless of the possession of common H 2 O 2 -degrading genes, as we reported previously (10).
Data availability. The genome sequences have been deposited in DDBJ under the accession number AP026969 for OS-1 and the accession numbers AP026970 and AP026971 for OS-4. The raw sequence reads have been deposited in the DRA under the accession numbers DRX402456 (iSeq) and DRX402457 (MinION) for OS-1 and the accession numbers DRX402458 (iSeq) and DRX402459 (MinION) for OS-4.