Genome sequences of a Paenibacillus sp. and two Sporosarcina spp. isolated from New Hampshire soil

ABSTRACT

We present the genomes of a Paenibacillus sp. and two Sporosarcina spp. isolated from soil samples collected in New Hampshire, USA. Selective culturing targeted bacteria that could perform microbial-induced carbonate precipitation via the urease pathway at 4°C and below.

ANNOUNCEMENT

Microbial induced carbonate precipitation (MICP) is a biogeochemical process where microbial metabolism facilitates calcium carbonate formation. One mechanism is ureolysis, whereby urease hydrolyzes urea, increasing pH and inducing carbonate precipitation (1). Selective culturing targeted bacteria with MICP potential (2) at cold temperatures (≤4°C). Organisms were isolated from soil collected at the US Army Cold Regions Research and Engineering Laboratory (CRREL) field site in Hanover, NH, USA. A sterilized trowel removed 4 cm of topsoil from a 20×16 cm area; soil was collected in a Whirl-Pak bag and stored at 4°C. A soil sample of 4.533 g was inoculated into 100 mL of filter-sterilized Christensen’s urea broth (CUB) (3, 4), pre-chilled at 4°C. The enrichment was incubated at 4°C on a shaker (100 rpm). During the soil enrichment, the medium turned yellow to pink, indicating increased pH and potential urease activity. After 13 days, 20 mL was transferred into 80 mL of fresh CUB, and incubation continued. After 40 days, 100 µL of a 1:10 dilution was spread onto 2× Reasoner’s 2 agar (R2A) (Difco, 218263) plates and incubated at 4°C for 53 days. Individual colonies were streaked for isolation on 2× R2A at 25°C and Christensen’s urea agar to confirm pH change.

To prepare cell pellets, isolates were revived from glycerol stocks on Luria Broth (LB) (Difco, 244620) agar plates. 25 mL LB cultures were grown at room temperature for 2–3 days, shaking at 175 rpm (early log phase). The equivalent of 8–12 OD₆₀₀ units was collected, cells were washed with sterile phosphate buffered saline, resuspended in 500 µL DNA/RNA Shield (Zymo Research, R1100-50), and stored at 4°C prior to shipping to Plasmidsaurus (Eugene, OR, USA) for Oxford Nanopore Technology (ONT) and Illumina library preparation, sequencing, and hybrid assembly.

Plasmidsaurus extracted DNA using the Quick-DNA miniprep plus kit (Zymo, D4068) with a lysozyme pre-treatment. Long-read sequencing libraries were constructed using v14 chemistry (ONT rapid sequencing DNA kit, SQK-RBK114.96) with proprietary modifications to the manufacturer’s protocol. Libraries were sequenced on an ONT PromethION P24 using a primer-free protocol on R10.4.1 flow cells. Base calling was conducted using ONT’s Dorado basecaller in super-accurate mode. Short-read sequencing libraries were constructed using the ExpressPlex 96 library prep kit (SeqWell, 301098) and sequenced on an Illumina NextSeq2000 (2×159 bp).

Hybrid assembly was performed by Plasmidsaurus following their standard pipeline. ONT long reads were processed by removing the 5% worst reads using Filtlong v0.2.1 (5), then downsampled to 100× coverage based on a de novo assembly sketch from Miniasm v0.3 (6). Downsampled reads were assembled and overlapping ends trimmed using Flye v2.9.1 (7), followed by polishing using medaka v1.8.0 (8). Circularization was visualized using Bandage v0.8.1 (9). Raw Illumina reads were aligned to the ONT assembly using BWA-MEM (10), and the resulting SAM files were polished with polypolish v0.6.0 (11) to generate the hybrid assembly. Genome completion and contamination were assessed using CheckM2 v1.0.2 (12), and annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (Table 1; 13–15). Genome annotation identified the urease operon in all three isolates. Default parameters were used except where otherwise noted.

TABLE 1.

Summary of assembled genomes

Characteristic	FA6	FA9	FA15
Source	Soil	Soil	Soil
Location	Hanover, NH, USA	Hanover, NH, USA	Hanover, NH, USA
ONT total reads	121,062	172,378	177,674
ONT total bases	608,186,242	737,406,775	722,240,319
ONT N50 (bp)	5,978,692	3,707,078	4,622,437
Assembled coverage (%)	95	100	97
Circular Genomes	Yes	Yes	Yes
Illumina total reads	716,056	991,365	1,532,240
Illumina read length (bp)	159	159	159
Illumina total bases	113,852,904	157,627,035	243,626,160
Assembly level	Complete genome	Complete genome	Complete genome
GC content (%)	42	39	41
No. of contigs	1	1	1
Genome size (bp)	5,978,707	3,707,084	4,622,452
Assembled N50 (bp)	5,978,707	3,707,084	4,622,452
Check M2 completeness (%)	99.94	99.99	99.99
Check M2 contamination (%)	0.81	1.66	0.39
Organism	Paenibacillus sp.	Sporosarcina sp.	Sporosarcina sp.
Nearest Match using Extended 16S Analysis from Type (Strain) Genome Server (16, 17)	Paenibacillus crassostreae (KU057345)	Sporosarcina siberiensis (KF258678)	Sporosarcina globispora X68415, Sporosarcina psychrophila X54968
BioSample	SAMN47621321	SAMN47621322	SAMN47621323
SRA accession no. (ONT)	SRR32921792	SRR32921790	SRR32921788
SRA accession no. (Illumina)	SRR32921791	SRR32921789	SRR32921787
GenBank assembly no.	GCA_051550225.1	GCA_051550205.1	GCA_051550195.1
CDSs (total)	5,271	3,705	4,320
Genes (coding)	5,177	3,659	4,259
5S rRNA	10	8	11
16S rRNA	10	8	10
23S rRNA	10	8	10
tRNA	86	69	69
CRISPR arrays	3	0	0

DATA AVAILABILITY

All sequence data are available under NCBI BioProject PRJNA1243441 and via the accession numbers listed in Table 1.