Draft genome assemblies from seven Bacillaceae isolates from woodland soil

Abstract

We isolated 7 endospore forming bacteria from campus woodland and sequenced their genomes using Illumina NextSeq. We share the draft genome assemblies for strains Bacillus wiedmanii_SC129, Bacillus pseudomycoides_SC131, Bacillus pumilis_SC133, Peribacillus butanolivorans_SC135, Bacillus thuringiensis_SC136, Priestia megaterium_SC138, and Bacillus wiedmanii_SC141. Draft genomes are between 3645032-5969865 bp and 34.8-41.2 % GC.

Introduction

Endospore forming bacteria are frequently found in a variety of environments including terrestrial soils and many are considered to be plant growth promoting bacteria (1). The publication by Anna L. McLoon, Julia Barker, Gillian Churan, Anthony Cucca, Lillian Gardner, Justin Gejo, Francesca Gerbasi, Michaela Higgins, Lillian Kronau, Jalin Le, Alyssa Lunman, Kelly Maune, Veezen Denise Mondelo, Madeline Naef, Caitlin Rigby, Caitlin Spiliotis can be found here: [URL]

Table of Contents

1. Background and Experimental Methods
2. Import and annotation
3. QC, Assembly, and Annotation
4. Taxonomic Classification
5. References

Narratives and data were generated by: Anna L. McLoon, Julia Barker, Gillian Churan, Anthony Cucca, Lillian Gardner, Justin Gejo, Francesca Gerbasi, Michaela Higgins, Lillian Kronau, Jalin Le, Alyssa Lunman, Kelly Maune, Veezen Denise Mondelo, Madeline Naef, Caitlin Rigby, Caitlin Spiliotis This umbrella narrative was created by Anna L. McLoon

Note to Authors

The publication may not be available at the time of the static Narrative creation. This can be added after the fact; please contact [email protected] to update the DOI landing page when this is done.

Background and Experimental Methods

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Sample Collection

We collected two soil cores from wooded areas of a suburban college campus, one a sloped, wooded area between buildings (42.7205, -73.7534), and another in a wooded area near a small wetland (42.7199, -73.7488) on January 23, 2024. Strain Bacillus wiedmannii SC129 was isolated from the organic horizon and strains Priestia megaterium SC138 and Bacillus wiedmannii SC141 from the mineral horizon from location 42.7205, -73.7534 (the wooded slope between campus buildings). Strains Bacillus pumilus SC133 and Peribacillus butanolivorans SC135 were isolated from the organic soil horizon and strains Bacillus pseudomycoides SC131 and Bacillus thuringiensis SC136 were isolated from the mineral horizon from location 42.7199, -73.7488 (the wooded area near a small wetland). We separated mineral and organic horizons, and to isolate individual endospores, we mixed approximately 100 μl of soil with 1 ml of sterile water, vortexed, and heated at 95°C for 10 minutes.

We characterized the sites and soil samples by measuring temperature, leaf litter depth, soil moisture, and soil pH using both water and calcium chloride solution (2-4):

 

Table 1: Characterization of soil samples

Site 1	Coordinates	Temperature	leaf litter depth	% moisture	pH (water)	pH (calcium chloride)
organic	42.7204552, -73.7533970	3C/37.4F	2 cm	38.9	7.45	6.48
mineral	42.7204552, -73.7533970	3C/37.4F	2 cm	18.72	6.3	5.19
Site 2	Coordinates	Temperature	leaf litter depth	% moisture	pH (water)	pH (calcium chloride)
organic	42.7198559, -73.7488097	2.0C/37.4F	4 cm	33.4	7.04	6.22
mineral	42.7198559, -73.7488097	2.0C/37.4F	4 cm	20.9	6.04	5.01

 

Isolation

Suspended cells were spread onto Tryptic soy agar + 5% sheep blood using a sterile swab and incubated overnight at 37° C as described previously (5,6). Isolates were colony purified on Tryptic Soy Agar plates. Tryptic soy broth cultures were grown for approximately 4 hours, then DNA was isolated using a Promega DNA wizard kit, following peptidoglycan digestion for an hour (1.15 μg/μl) in 50 mM EDTA.

Genome Sequencing

Genomic DNA libraries were prepared using the tagmentation based Illumina DNA prep kit and IDT 10 bp unique dual indices and sequenced as paired end 151 bp reads by SeqCenter (Pittsburgh, PA) using an Illumina NovaSeq X Plus sequencer. Demultiplexing, quality control, and adapter trimming were performed by the SeqCenter with bcl-convert v4.2.4. Accession numbers and individual KBase narratives are in table 2.

Table 2: Access to Narrative workflows and sequence data

Strain	Kbase narrative used for analyses	Bioproject accession number	SRA Biosample	GenBank accession
Bacillus wiedmannii strain SC129	https://kbase.us/n/172257/32/	PRJNA862062	SAMN43273684	JBLOJC000000000
Bacillus pseudomycoides strain SC131	https://kbase.us/n/172258/23/	PRJNA862062	SAMN43273685	JBLOJB000000000
Bacillus pumilis strain SC133	https://kbase.us/n/172259/45/	PRJNA862062	SAMN43273686	JBLOJA000000000
Peribacillus butanolivorans strain SC135	https://kbase.us/n/172260/62/	PRJNA862062	SAMN43273687	JBLOIZ000000000
Bacillus thuringiensis strain SC136	https://kbase.us/n/172261/23/	PRJNA862062	SAMN43273688	JBLOIY000000000
Priestia megaterium strain SC138	https://kbase.us/n/172262/51/	PRJNA862062	SAMN43273689	JBLOIX000000000
Bacillus wiedmanii strain SC141	https://kbase.us/n/172263/26/	PRJNA862062	SAMN43273690	JBLOIW000000000

QC, Assembly, and Annotation, and taxonomic clasification.

Sequence reads from each isolate were imported into separate narratives in the KBase environment for analysis (7,8). We checked read quality with FastQC v0.12.1, trimmed reads with Trimmomatic v0.36, assembled genomes de novo using SPAdes v3.15.3, and annotated the assemblies using RASTtk v1.073, Prokka v1.14.5, and DRAM v0.1.2 (9-18). We determined probable species identities using TYGS and GTDB-Tk v1.7.0, and results were concordant between programs (19-23). All programs were run using default parameters. The draft genomes range in completeness consisting of between 27 and 100 contigs, and range in size from 3,645,032 to 5,969,865 base pairs of sequence (table 2). For relative placement of species into a phylogenetic tree, see tree built below in this umbrella narrative.We also ran the draft genome assemblies through the antiSMASH secondary metabolite prediction program (24, 25).

Draft genomes range in size from 3,645,032 bp to 5,969,865 bp and from 34.8 - 41.2 % GC. Each strain is predicted to make 8 to 16 unique secondary metabolites including terpenes, RiPPs, non-ribosomally synthesized peptides, and NI-siderophores among others. While many strains are predicted to make at least one well characterized natural product including paeninodin and petrobactin, many operons identified have low or no similarity to known secondary metabolites (24–28).Therefore, these strains and genomes represent useful additions to our knowledge of soil microbes and potential sources of beneficial natural products.

 

Table 2: Data summary

strain	SC129	SC131	SC133	SC135	SC136	SC138	SC141
Site	42.7204552, -73.7533970	42.7198559, -73.7488097	42.7198559, -73.7488097	42.7198559, -73.7488097	42.7198559, -73.7488097	42.7204552, -73.7533970	42.7204552, -73.7533970
soil horizon	organic	mineral	organic	organic	mineral	mineral	mineral
species	Bacillus wiedmannii	Bacillus pseudomycoides	Bacillus pumilis	Peribacillus butanolivorans	Bacillus thuringiensis	Priestia megaterium	Bacillus wiedmannii
number of reads	5,545,784	6,877,022	6,439,688	6,974,014	6,648,606	5,923,568	5,481,716
number of contigs	31	100	40	46	31	41	27
Total length (bp)	5,485,886	5,216,294	3,645,032	5,859,099	5,969,865	5,853,104	5,671,044
N50	781,638	195,779	218,892	299,378	634,513	903,089	927,123
% GC	35.2	35.5	41.2	37.9	34.8	37.5	35.1
Predicted genes (Prokka via KBase)	5,742	5,258	3,695	5,635	5,953	6,063	5,762
Number of predicted secondary operons (antiSMASH)	12	12	10	8	16	8	10
predicted natural product types (specific known cluster name and similarity)	terpene (molybdenum cofactor 17%), 4 NRPS (anachelin 10%, bacillibactin, 85%), 3 RiPP-like, betalactone (fengycin 40%), LAP, NI-siderophore (petrobactin 100%), cyclic lactone autoinducer/ thiopeptide	lassopeptide (paeninodin 100%), LAP, 5 NRPS (bacillibactin 85%, desmamide A/B/C 18%), lanthipeptide class II (plantaricin W 66%), terpene, betalactone (fengycin 40%), 2 RiPP-like	2 betalactone (fengycin 53%, other (bacilysin 85%), T1PKS (zwittermicin A 18%), NRPS (lichenysin 92%), NI-siderophore (schizokinen 60%), terpene, T3PKS, RRE containing, RiPP-like	2 terpenes, 2 lassopeptide (paeninodin 100%), betalactone (fengycin 46%), NI-siderophore (schizokinen 60%), LAP, T3PKS	LAP, 6 NRPS (bacillibactin 85%), NI-siderophore (petrobactin 100%), betalactone (fengycin 40%), 2 RiPP-like, NRPS-like, terpene (molybdenum cofactor 17%), ranthipeptide, 2 RRE containing (thuricin CD 100%)	3 terpene (50% carotenoid, 13% surfactin), phosphonate, RRE-containing, T3PKS, NI-siderophore (62% schizokinen), lanthipeptide class i	LAP, 3 NRPS (bacillibactin 85% ), NI-siderophore (petrobactin, 100%), betalactone (fengycin 40%), lassopeptide (paeninodin 100%), terpene, 2 RiPP-like

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