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Draft Genome Sequence of Bacillus subtilis SB-14, an Antimicrobially Active Isolate from Namibian Social Spiders ( Stegodyphus dumicola )

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Abstract

We present the high-quality draft genome sequence of Bacillus subtilis SB-14, isolated from the Namibian social spider Stegodyphus dumicola . In accordance with its antimicrobial activity, both known and potentially novel antimicrobial biosynthetic gene clusters were identified in the genome of SB-14.
Draft Genome Sequence of Bacillus subtilis SB-14, an
Antimicrobially Active Isolate from Namibian Social Spiders
(Stegodyphus dumicola)
Stine Sofie Frank Nielsen,
a
Simone Weiss,
a
Seven Nazipi,
b
Ian P. G. Marshall,
b
Trine Bilde,
c
Andreas Schramm
b
a
Department of Clinical Medicine, Aarhus University, Aarhus, Denmark
b
Department of Bioscience, Section for Microbiology, Aarhus University, Aarhus, Denmark
c
Department of Bioscience, Section for Genetics, Ecology and Evolution, Aarhus University, Aarhus, Denmark
ABSTRACT We present the high-quality draft genome sequence of Bacillus subtilis
SB-14, isolated from the Namibian social spider Stegodyphus dumicola. In accordance
with its antimicrobial activity, both known and potentially novel antimicrobial bio-
synthetic gene clusters were identified in the genome of SB-14.
Increasing antibiotic consumption has accelerated resistance development, prompt-
ing the need for novel antibiotics (1, 2). Stegodyphus dumicola spiders are social
spiders that live in communal nests with a high degree of inbreeding and low genetic
variation, making them potentially vulnerable for pathogen attack. We therefore sug-
gest an intricate symbiotic relationship with a well-developed protective microbiome.
This microbiome may present an unexplored source of antibiotics (3, 4).
Bacillus subtilis SB-14 was isolated from the body surface of a Stegodyphus dumicola
spider, as follows: a spider was immersed in nutrient broth (Sigma-Aldrich), which was
incubated at 30°C overnight, plated onto nutrient broth agar plates, and grown aerobically
at 30°C. Single colonies from serial dilutions were restreaked until a pure culture was
established. Colonies were screened by colony PCR and 16S rRNA gene sequencing (5). The
antimicrobial activity of SB-14 against Staphylococcus epidermidis BMC-HMP0060 was de-
tected using the Kirby-Bauer disk diffusion susceptibility test protocol (6), with the modi-
fication that SB-14 was point inoculated onto a lawn of S. epidermidis.
Genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen) and
prepared for sequencing with a Nextera XT DNA library prep kit (Illumina). Sequencing was
performed utilizing an Illumina MiSeq platform with a paired-end 300-bp read MiSeq
reagent kit, yielding 5.5 million sequencing reads in total (1.11 Gbp), with approxi-
mately 260coverage. Read quality was analyzed by FastQC version 0.11.7 (https://www
.bioinformatics.babraham.ac.uk/projects/fastqc/), and reads were trimmed by Trimmomatic
version 0.36 (7) using length-based trimming with the following parameters: headcrop, 20;
crop, 290; a minimum average quality score of 20; and a 4-bp sliding window. Finally, the
genome was assembled using SPAdes version 3.11.1 (8), resulting in 37 scaffolds, of which
31 were 200 bp when applying the parameters – careful -k 21, 33, 55, 77, 99, 127.
CheckM version 1.0.9 (9) estimated 98.28% completeness and 0.00% contamination
of the SB-14 genome, using the gene marker set for the domain Bacteria; the corre-
sponding values using the gene marker set for Bacillus subtilis were 97.29% and 2.63%,
respectively.
NCBI BLAST (10) analysis of the 16S rRNA gene revealed 99% identity to the 16S rRNA
genes of multiple Bacillus subtilis strains. This genus and species identity was confirmed by
an average amino acid identity of 98% (11), average nucleotide identity of 98% (11),
and 94.18% digital DNA-DNA hybridization value (http://ggdc.dsmz.de/), compared to
Bacillus subtilis subsp. inaquosorum (NCBI RefSeq accession no. NZ_CP013984).
Citation Nielsen SSF, Weiss S, Nazipi S, Marshall
IPG, Bilde T, Schramm A. 2019. Draft genome
sequence of Bacillus subtilis SB-14, an
antimicrobially active isolate from Namibian
social spiders (Stegodyphus dumicola).
Microbiol Resour Announc 8:e00156-19.
https://doi.org/10.1128/MRA.00156-19.
Editor Christina A. Cuomo, Broad Institute
Copyright © 2019 Nielsen et al. This is an
open-access article distributed under the terms
of the Creative Commons Attribution 4.0
International license.
Address correspondence to Andreas Schramm,
andreas.schramm@bios.au.dk.
S.S.F.N. and S.W. contributed equally to this
article.
Received 12 February 2019
Accepted 27 May 2019
Published 20 June 2019
GENOME SEQUENCES
crossm
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The draft genome of Bacillus subtilis SB-14 contains 4,262,181 bp on 31 scaffolds of
200 bp, with an average GC content of 44.02% and an N
50
value of 781,251 bp. Prokka
version 1.12 (12) analysis found 81 tRNA, 10 rRNA, and 4,210 protein-coding sequences.
Upon antiSMASH version 3.0 (13) analysis, 30 secondary metabolite biosynthetic gene
clusters were identified. Five of these were 100% identical to known clusters encoding the
biosynthesis of teichuronic acid (14), bacillibactin (15), the antifungal lipopeptide fengycin
(16), and the antibiotics bacillaene (17) and subtilosin A (18). Three other clusters had 82%,
40%, and 18% identity to clusters known for the biosynthesis of the antimicrobial metab-
olites surfactin (19), bacillomycin (20), and zwittermicin A (21), respectively; these three may
therefore represent novel variations of these metabolites.
Data availability. The isolate Bacillus subtilis SB-14 has been deposited at the DSMZ
(https://www.dsmz.de/) under accession no. DSM 109343. This whole-genome shotgun
project has been deposited in DDBJ/ENA/GenBank under BioProject accession no.
PRJNA438195. The raw data can be found under accession no. SRR8560781, and the
assembled genome has GenBank accession no. PXUR00000000. The version described
in this paper is the first version, PXUR01000000.
ACKNOWLEDGMENTS
We thank Virginia Settepani for collecting and handling social spiders, Britta Poulsen
and Susanne Nielsen for help with Illumina MiSeq sequencing, and Anne Stentebjerg
for handling the bacterial culture/DSMZ submission.
This study was funded by the Novo Nordisk Foundation, the European Research
Council (grant ERC StG-2011_282163 to T.B.), and The Danish Council for Independent
Research, Natural Sciences.
REFERENCES
1. Ventola CL. 2015. The antibiotic resistance crisis: part 1: causes and
threats. P T 40:277–283.
2. Munita JM, Arias CA. 2016. Mechanisms of antibiotic resistance. Micro-
biol Spectr 4:VMBF-0016-2015. https://doi.org/10.1128/microbiolspec
.VMBF-0016-2015.
3. Bilde T, Coates KS, Birkhofer K, Bird T, Maklakov AA, Lubin Y, Avilés L.
2007. Survival benefits select for group living in a social spider despite
reproductive costs. J Evol Biol 20:2412–2426. https://doi.org/10.1111/j
.1420-9101.2007.01407.x.
4. Johannesen J, Lubin Y, Smith DR, Bilde T, Schneider JM. 2007. The age
and evolution of sociality in Stegodyphus spiders: a molecular phyloge-
netic perspective. Proc Biol Sci 274:231–237. https://doi.org/10.1098/
rspb.2006.3699.
5. Foesel BU, Gössner AS, Drake HL, Schramm A. 2007. Geminicoccus roseus
gen. nov., sp. nov., an aerobic phototrophic alphaproteobacterium iso-
lated from a marine aquaculture biofilter. Syst Appl Microbiol 30:
581–586. https://doi.org/10.1016/j.syapm.2007.05.005.
6. Hudzicki J. 2009. Kirby-Bauer disk diffusion susceptibility test proto-
col. American Society for Microbiology, Washington, DC. http://www
.asmscience.org/content/education/protocol/protocol.3189.
7. Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for
Illumina sequence data. Bioinformatics 30:2114 –2120. https://doi.org/10
.1093/bioinformatics/btu170.
8. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS,
Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV,
Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new
genome assembly algorithm and its applications to single-cell sequenc-
ing. J Comput Biol 19:455– 477. https://doi.org/10.1089/cmb.2012.0021.
9. Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. 2015.
CheckM: assessing the quality of microbial genomes recovered from
isolates, single cells, and metagenomes. Genome Res 8:1043–1055.
https://doi.org/10.1101/gr.186072.114.
10. Johnson M, Zaretskaya I, Raytselis Y, Merezhuk Y, McGinnis S, Madden
TL. 2008. NCBI BLAST: a better Web interface. Nucleic Acids Res 36:
W5–W9. https://doi.org/10.1093/nar/gkn201.
11. Rodriguez-R LM, Konstantinidis KT. 2016. The enveomics collection: a
toolbox for specialized analyses of microbial genomes and metag-
enomes. PeerJ Preprints 4:e1900v1.
12. Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioin-
formatics 30:2068 –2069. https://doi.org/10.1093/bioinformatics/btu153.
13. Weber T, Blin K, Duddela S, Krug D, Kim HU, Bruccoleri R, Lee SY,
Fischbach MA, Müller R, Wohlleben W, Breitling R, Takano E, Medema
MH. 2015. antiSMASH 3.0 —a comprehensive resource for the genome
mining of biosynthetic gene clusters. Nucleic Acids Res 43:W237–W243.
https://doi.org/10.1093/nar/gkv437.
14. Janczura E, Perkins HR, Rogers HJ. 1961. Teichuronic acid: a mucopoly-
saccharide present in wall preparations from vegetative cells of Bacillus
subtilis. Biochem J 80:82–93. https://doi.org/10.1042/bj0800082.
15. May JJ, Wendrich TM, Marahiel MA. 2001. The dhb operon of Bacillus
subtilis encodes the biosynthetic template for the catecholic siderophore
2,3-dihydroxybenzoate-glycine-threonine trimeric ester bacillibactin. J
Biol Chem 276:7209 –7217. https://doi.org/10.1074/jbc.M009140200.
16. Vanittanakom N, Loeffler W, Koch U, Jung G. 1986. Fengycin—a novel
antifungal lipopeptide antibiotic produced by Bacillus subtilis F-29-3. J
Antibiot (Tokyo) 39:888 –901. https://doi.org/10.7164/antibiotics.39.888.
17. Patel PS, Huang S, Fisher S, Pirnik D, Aklonis C, Dean L, Meyers E, Fernandes
P, Mayerl F. 1995. Bacillaene, a novel inhibitor of procaryotic protein syn-
thesis produced by Bacillus subtilis: production, taxonomy, isolation,
physico-chemical characterization and biological activity. J Antibiot (Tokyo)
48:997–1003. https://doi.org/10.7164/antibiotics.48.997.
18. Babasaki K, Takao T, Shimonishi Y, Kurahashi K. 1985. Subtilosin A, a new
antibiotic peptide produced by Bacillus subtilis 168: isolation, structural
analysis, and biogenesis. J Biochem 98:585– 603. https://doi.org/10
.1093/oxfordjournals.jbchem.a135315.
19. Vollenbroich D, Pauli G, Ozel M, Vater J. 1997. Antimycoplasma proper-
ties and application in cell culture of surfactin, a lipopeptide antibiotic
from Bacillus subtilis. Appl Environ Microbiol 63:44 –49.
20. Landy M, Warren GH, Rosenman SB, Colio LG. 1948. Bacillomycin: an
antibiotic from Bacillus subtilis active against pathogenic fungi. Proc Soc
Exp Biol Med 67:539 –541. https://doi.org/10.3181/00379727-67-16367.
21. Silo-Suh LA, Stabb EV, Raffel SJ, Handelsman J. 1998. Target range of
zwittermicin A, an aminopolyol antibiotic from Bacillus cereus. Curr
Microbiol 37:6 –11.
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