# Draft Genome Sequence of Bacillus velezensis Strain Marseille-Q1230, Isolated from a Stool Sample from a Severely Malnourished Child

Salimata Konaté, Rita Zgheib, Aminata Camara, Ogobara Doumbo, Abdoulaye Djimdé, Abdoulaye Kassoum Koné, Mahamadou Ali Théra, Pierre-Edouard Fournier, Maryam Tidjani Alou, Didier Raoult, et al.

<figure><img src="image_1.png" /></figure>

To cite this version:

Salimata Konaté, Rita Zgheib, Aminata Camara, Ogobara Doumbo, Abdoulaye Djimdé, et al. Draft Genome Sequence of Bacillus velezensis Strain Marseille-Q1230, Isolated from a Stool Sample from a Severely Malnourished Child. Microbiology Resource Announcements, 2021, 10 (33), 10.1128/mra.00514-21. hal-03429416

HAL Id: hal-03429416

https://amu.hal.science/hal-03429416v1

Submitted on 6 Nov 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_3.png" /></figure>

# Draft Genome Sequence of *Bacillus velezensis* Strain Marseille-Q1230, Isolated from a Stool Sample from a Severely Malnourished Child

Salimata Konaté,a,b Rita Zgheib,b,c Aminata Camara,a,b Ogobara Doumbo,d Abdoulaye Djimdé,d Abdoulaye Kassoum Koné,d Mahamadou Ali Théra,d Pierre-Edouard Fournier,c Maryam Tidjani Alou,a Didier Raoult,a,b Matthieu Milliona,b

**ABSTRACT** *Bacillus velezensis*, a species first described in 2005, has been mostly associated with plants and the environment. To date, there is no genome available for this species from human samples. In this announcement, we present the genome of *Bacillus velezensis* strain Marseille-Q1230, which was isolated from a stool sample from a child suffering from severe acute malnutrition. The genome assembled into 15 contigs and had a size of 3,861,152 bp, with a GC content of 46.6%. A total of 3,716 protein-coding genes, including 3 antibiotic resistance genes and 92 RNAs, were predicted.

The genus *Bacillus*, a member of the phylum *Firmicutes*, includes more than 200 val-
idly published species (https://lpsn.dsmz.de/genus/bacillus) (1). *Bacillus velezensis*
was first isolated by Ruiz-García et al. from the Vélez River in Malaga, Spain (2), and
possesses bactericidal (3, 4) and fungicidal (5) abilities, thus promoting plant growth
and controlling wheat diseases (6). No previous studies have reported its presence in
the human microbiome, as this species has been isolated only from plants or environ-
mental samples (7, 8). Here, we present the genome sequence of a *B. velezensis* strain
that was isolated from a human sample collected as part of a childhood malnutrition
study in Mali; the study was approved by the Malian ethical committee (approval num-
ber 2014/46/CE/FMPOS). More specifically, *B. velezensis* strain Marseille-Q1230 was iso-
lated from the stool of a severely malnourished Malian child <5 years of age using the
culturomics approach, which is a high-throughput approach consisting of diversifica-
tion and multiplication of culture conditions followed by rapid identification by matrix-
assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)
(9, 10).

Genomic DNA of strain Marseille-Q1230 was extracted using the EZ1 biorobot (Qiagen) with the EZ1 DNA tissue kit before sequencing using the MiSeq platform (Illumina Inc., San Diego, CA, USA) with the paired-end strategy. The library was prepared following the workflow of the Nextera XT DNA library preparation kit (Illumina) (11). Automated cluster generation and paired-end sequencing with dual index reads were performed in a single 39-hour run (2 × 250 bp); 5.00 Gb of data was obtained from a 521-K/mm² cluster density, with 94.11% of clusters passing the quality control filters. Within this run, the index representation for strain Marseille-Q1230 was assessed as 5.09%. Finally, 10,125,033 paired-end reads were filtered according to read quality using FastQC v0.11.8 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc).

The resulting forward (113,107,188 bases) and reverse (113,148,534 bases) reads were assembled using SPAdes v3.14.0 (12). FastQC and SPAdes were used with default parameters. Strain Marseille-Q1230 was identified after the 16S rRNA gene

**Citation** Konaté S, Zgheib R, Camara A, Doumbo O, Djimdé A, Koné AK, Théra MA, Fournier P-E, Tidjani Alou M, Raoult D, Million M. 2021. Draft genome sequence of *Bacillus velezensis* strain Marseille-Q1230, isolated from a stool sample from a severely malnourished child. Microbiol Resour Announc 10:e00514-21. https://doi.org/10.1128/MRA.00514-21.

**Editor** Steven R. Gill, University of Rochester School of Medicine and Dentistry

**Copyright** © 2021 Konaté et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

Address correspondence to Matthieu Million,
matthieumillion@gmail.com.

**Received** 18 May 2021

**Accepted** 28 July 2021

**Published** 19 August 2021

<footnote>aAix-Marseille Université, IRD, AP-HM, MEPHI, Marseille, France</footnote>

<footnote>bInstitut Hospitalo-Universitaire Méditerranée Infection, Marseille, France</footnote>

<footnote>cAix-Marseille Université, IRD, AP-HM, SSA, VITROME, Marseille, France</footnote>

<footnote>dMalaria Research and Training Center, Faculty of Medicine, Pharmacy, and Odontostomatology, University of Bamako, Bamako, Mali</footnote>
<table><caption>TABLE 1 Pairwise OrthoANI values and *recQ* gene identities between strain Marseille-Q1230 and other type strains from the *Bacillus subtilis* species complex</caption><thead><tr><th rowspan="2">Strain<sup>a</sup></th><th colspan="10">OrthoANI value (%) or recQ gene identity (%)<sup>b</sup></th></tr><tr><th>Strain 1</th><th>Strain 2</th><th>Strain 3</th><th>Strain 4</th><th>Strain 5</th><th>Strain 6</th><th>Strain 7</th><th>Strain 8</th><th>Strain 9</th><th>Strain 10</th></tr></thead><tbody><tr><td>1</td><td>100.00</td><td>77.33</td><td>70.24</td><td>97.82</td><td>94.47</td><td>77.04</td><td>86.42</td><td>94.07</td><td>99.95</td><td>77.26</td></tr><tr><td>2</td><td>75.64</td><td>100.00</td><td>71.06</td><td>77.09</td><td>77.41</td><td>87.31</td><td>77.83</td><td>77.51</td><td>77.39</td><td>80.57</td></tr><tr><td>3</td><td>66.29</td><td>67.18</td><td>100.00</td><td>70.43</td><td>70.37</td><td>70.96</td><td>70.54</td><td>70.48</td><td>70.29</td><td>70.75</td></tr><tr><td>4</td><td>98.54</td><td>75.36</td><td>66.29</td><td>100.00</td><td>94.35</td><td>77.00</td><td>86.47</td><td>94.05</td><td>97.84</td><td>77.08</td></tr><tr><td>5</td><td>95.28</td><td>74.56</td><td>66.05</td><td>95.17</td><td>100.00</td><td>77.00</td><td>86.11</td><td>93.98</td><td>94.44</td><td>77.45</td></tr><tr><td>6</td><td>75.99</td><td>84.84</td><td>67.92</td><td>75.75</td><td>74.70</td><td>100.00</td><td>77.46</td><td>77.07</td><td>77.06</td><td>79.56</td></tr><tr><td>7</td><td>85.69</td><td>75.42</td><td>67.10</td><td>85.35</td><td>85.13</td><td>76.91</td><td>100.00</td><td>86.82</td><td>86.59</td><td>78.39</td></tr><tr><td>8</td><td>94.94</td><td>75.87</td><td>65.77</td><td>94.77</td><td>94.44</td><td>75.54</td><td>85.69</td><td>100.00</td><td>94.10</td><td>77.32</td></tr><tr><td>9</td><td>100.00</td><td>75.64</td><td>66.29</td><td>98.54</td><td>95.28</td><td>75.99</td><td>85.69</td><td>94.94</td><td>100.00</td><td>77.37</td></tr><tr><td>10</td><td>77.02</td><td>79.77</td><td>67.39</td><td>76.80</td><td>76.37</td><td>77.82</td><td>78.37</td><td>77.19</td><td>77.02</td><td>100.00</td></tr></tbody></table>

sequence was extracted from the genome and matched against the GenBank database using BLASTn (accessed 6 August 2020) (13). Strain Marseille-Q1230 exhibited 16S rRNA gene sequence similarity of >99% to multiple species from the *Bacillus subtilis* species complex. Because the 16S rRNA gene sequence failed to discriminate within the aforementioned species complex, we also used OrthoANI v0.93.1 software (14), core genome-based and *rpoB*-based phylogeny (15), and *recQ* sequence identities (determined by local BLASTn v2.9.0) (16). Both *rpoB* and *recQ* genes were extracted from the annotated genomes. All of these methods revealed a close relationship between strain Marseille-Q1230 and *B. velezensis*, as both *recQ* sequence identities and OrthoANI values were higher than the species-delineating thresholds (96.6% for *recQ* and 95% to 96% for OrthoANI) (Table 1 and Fig. 1) (14, 16). Therefore, phylogenetic and genomic analyses identified strain Marseille-Q1230 as *B. velezensis*.

Strain Marseille-Q1230 had a genome size of 3,861,152 bp (N<sub>50</sub> value of 583,987 bp) assembled into 15 contigs, with a GC content of 46.6% and 29.3× coverage. Annotation with Prokka v1.13 software (17) predicted 3,716 protein-coding genes and 92 RNA genes

<figure><img src="image_4.png" /><figcaption>FIG 1 (a) Phylogenetic tree inferred from a comparison of the *rpoB* gene sequences using the maximum likelihood method with 1,000 bootstrap replicates and the Kimura-2 parameter with MEGA X software (19). (b) Core genome-based phylogenetic tree generated from the alignment of 208 core genes for all of the compared strains, including strain Marseille-Q1230 (red). The core gene alignment was generated using Roary v3.13.0 with 80% identity (20). The tree was inferred with FastTree v2.1.10 (21). Genome accession numbers are indicated in parentheses. Type strains are indicated with a superscript T. Numbers at the nodes are bootstrap values. The scale bars indicate 10% sequence divergence.</figcaption></figure>

<footnote><sup>a</sup> Strain 1, *Bacillus velezensis* KCTC 13012<sup>T</sup>; strain 2, *Bacillus halotolerans* FJAT-2398<sup>T</sup>; strain 3, *Bacillus xiamenensis* HYC-10<sup>T</sup>; strain 4, *Bacillus velezensis* Marseille-Q1230; strain 5, *Bacillus siamensis* KCTC 13613<sup>T</sup>; strain 6, *Bacillus subtilis* NCIB 3610<sup>T</sup>; strain 7, *Bacillus nakamurai* NRRL B-41091<sup>T</sup>; strain 8, *Bacillus amyloliquefaciens* DSM 7<sup>T</sup>; strain 9, *Bacillus velezensis* NRRL B-41580<sup>T</sup>; strain 10, *Bacillus atrophaeus* NRRL NRS 213<sup>T</sup>.</footnote>

<footnote><sup>b</sup> Pairwise OrthoANI values are shown in the upper half of the table and *recQ* gene identity values in the lower half.</footnote>
<figure><img src="image_5.png" /></figure>

(10 rRNA genes, 81 tRNA genes, and 1 transfer-messenger RNA gene). Compared to NCBI Prokaryotic Genome Annotation Pipeline (PGAP) annotation, a few differences were obtained using the Prokka software, probably due to different parameters and databases used by the two tools. Using ABRicate with the NCBI resistance database and an identity cutoff value of 80%, three resistance genes, namely, *rphC* (rifamycin-inactivating phosphotransferase), *tet(L* (tetracycline efflux major facilitator superfamily [MFS] transporter), and *satA* (streptothricin *N*-acetyltransferase), were detected (18).

**Data availability.** The raw reads and genome sequence of *Bacillus velezensis* strain Marseille-Q1230 have been deposited in GenBank under accession numbers SRR14270290 and CACRZA000000000, respectively.

## ACKNOWLEDGMENTS

The study was financed by the Méditerranée Infection Foundation, through the National Research Agency under the Investissement d'Avenir program (reference Méditerranée Infection 10-IAHU-03).

S.K. and A.C. collected the sample and isolated the strain. R.Z. performed the bioinformatic analysis. S.K., R.Z., M.T.A., and M.M. wrote the manuscript. A.D., A.K.K., P.-E.F., M.T.A., and M.M. supervised the study. O.D., M.A.T., and D.R. designed and supervised the study.

## REFERENCES

1. Parte AC, Sardà Carbasse J, Meier-Kolthoff JP, Reimer LC, Göker M. 2020. List of Prokaryotic names with Standing in Nomenclature (LPSN) moves to the DSMZ. Int J Syst Evol Microbiol 70:5607–5612. https://doi.org/10.1099/ijsem.0.004332.

2. Ruiz-García C, Béjar V, Martínez-Checa F, Llamas I, Quesada E. 2005. *Bacillus velezensis* sp. nov., a surfactant-producing bacterium isolated from the river Vélez in Málaga, southern Spain. Int J Syst Evol Microbiol 55: 191–195. https://doi.org/10.1099/ijs.0.63310-0.

3. Fan B, Wang C, Song X, Ding X, Wu L, Wu H, Gao X, Borriss R. 2018. *Bacillus velezensis* FZB42 in 2018: the Gram-positive model strain for plant growth promotion and biocontrol. Front Microbiol 9:2491. https://doi.org/10.3389/fmicb.2018.02491.

4. Yoo Y, Seo D-H, Lee H, Cho E-S, Song N-E, Nam TG, Nam Y-D, Seo M-J. 2019. Inhibitory effect of *Bacillus velezensis* on biofilm formation by *Streptococcus mutans*. J Biotechnol 298:57–63. https://doi.org/10.1016/j.jbiotec.2019.04.009.

5. Wambacq E, Audenaert K, Höfte M, De Saeger S, Haesaert G. 2018. *Bacillus velezensis* as antagonist towards *Penicillium roqueforti* s.l. in silage: in vitro and in vivo evaluation. J Appl Microbiol 125:986–996. https://doi.org/10.1111/jam.13944.

6. Kang X, Zhang W, Cai X, Zhu T, Xue Y, Liu C. 2018. *Bacillus velezensis* CC09: a potential "vaccine" for controlling wheat diseases. Mol Plant Microbe Interact 31:623–632. https://doi.org/10.1094/MPMI-09-17-0227-R.

7. Pajcin I, Vlajkov V, Frohme M, Grebinyk S, Grahovac M, Mojićević M, Grahovac J. 2020. Pepper bacterial spot control by *Bacillus velezensis*: bioprocess solution. Microorganisms 8:1463. https://doi.org/10.3390/microorganisms8101463.

8. Kim SY, Lee SY, Weon H-Y, Sang MK, Song J. 2017. Complete genome sequence of *Bacillus velezensis* M75, a biocontrol agent against fungal plant pathogens, isolated from cotton waste. J Biotechnol 241:112–115. https://doi.org/10.1016/j.jbiotec.2016.11.023.

9. Lagier J-C, Armougom F, Million M, Hugon P, Pagnier I, Robert C, Bittar F, Fournous G, Gimenez G, Maraninchi M, Trape J-F, Koonin EV, La Scola B, Raoult D. 2012. Microbial culturomics: paradigm shift in the human gut microbiome study. Clin Microbiol Infect 18:1185–1193. https://doi.org/10.1111/1469-0691.12023.

10. Lagier J-C, Dubourg G, Million M, Cadoret F, Bilen M, Fenollar F, Levasseur A, Rolain J-M, Fournier P-E, Raoult D. 2018. Culturing the human microbiota and culturomics. Nat Rev Microbiol 16:540–550. https://doi.org/10.1038/s41579-018-0041-0.

11. Anani H, Raoult D, Fournier P-E. 2019. Whole-genome sequence of *Haloimpatiens linggiaoenensis* strain P8956. Microbiol Resour Announc 8:e00699-19. https://doi.org/10.1128/MRA.00699-19.

12. 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 sequencing. J Comput Biol 19:455–477. https://doi.org/10.1089/cmb.2012.0021.

13. Zhang Z, Schwartz S, Wagner L, Miller W. 2000. A greedy algorithm for aligning DNA sequences. J Comput Biol 7:203–214. https://doi.org/10.1089/10665270050081478.

14. Lee I, Ouk Kim Y, Park S-C, Chun J. 2016. OrthoANI: an improved algorithm and software for calculating average nucleotide identity. Int J Syst Evol Microbiol 66:1100–1103. https://doi.org/10.1099/ijsem.0.000760.

15. Rooney AP, Price NPJ, Ehrhardt C, Swezey JL, Bannan JD. 2009. Phylogeny and molecular taxonomy of the *Bacillus subtilis* species complex and description of *Bacillus subtilis* subsp. *inaquosorum* subsp. nov. Int J Syst Evol Microbiol 59:2429–2436. https://doi.org/10.1099/ijs.0.009126-0.

16. Heo J, Kim J-S, Hong S-B, Park B-Y, Kim S-J, Kwon S-W. 2019. Genetic marker gene, *recQ*, differentiating *Bacillus subtilis* and the closely related *Bacillus* species. FEMS Microbiol Lett 366:fnz172. https://doi.org/10.1093/femsle/fnz172.

17. Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068–2069. https://doi.org/10.1093 bioinformatics/btu153.

18. Feldgarden M, Brover V, Haft DH, Prasad AB, Slotta DJ, Tolstoy I, Tyson GH, Zhao S, Hsu CH, McDermott PF, Tadesse DA, Morales C, Simmons M, Tillman G, Wasilenko J, Folster JP, Klimke W. 2019. Validating the AMRFinder tool and Resistance Gene Database by using antimicrobial resistance genotype-phenotype correlations in a collection of isolates. Antimicrob Agents Chemother 63:e00483-19. https://doi.org/10.1128/AAC.00483-19.

19. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Mol Biol Evol 35:1547–1549. https://doi.org/10.1093/molbev/msy096.

20. Page AJ, Cummins CA, Hunt M, Wong VK, Reuter S, Holden MTG, Fookes M, Falush D, Keane JA, Parkhill J. 2015. Roary: rapid large-scale prokaryote pan genome analysis. Bioinformatics 31:3691–3693. https://doi.org/10.1093/bioinformatics/btv421.

21. Price MN, Dehal PS, Arkin AP. 2010. FastTree 2—approximately maximum-likelihood trees for large alignments. PLoS One 5:e9490. https://doi.org/10.1371/journal.pone.0009490.