Abstract
Many Gram-positive spore-forming rhizobacteria of the genus Bacillus show potential as biocontrol biopesticides that promise improved sustainability and ecological safety in agriculture. Here, we present a draft-quality genome sequence for Bacillus velezensis EU07, which shows growth-promotion in tomato plants and biocontrol against Fusarium head blight. We found that the genome of EU07 is almost identical to that of the commercially used strain QST713, but identified 46 single-nucleotide differences that distinguish these strains from each other. The availability of this genome sequence will facilitate future efforts to unravel the genetic and molecular basis for EU07's beneficial properties.
Keywords: Bacillus velezensis, biological control, genome sequence, plant-growth promoting
Data Summary
In this study, we generated genome sequence data, which has been deposited in public databases:
National Center for Biotechnology Information (NCBI) BioProject accession number PRJNA743875 – https://www.ncbi.nlm.nih.gov/bioproject/743875
Assembly NCBI GenBank accession number GCA_019997305.2 – https://www.ncbi.nlm.nih.gov/nuccore/JAIFZJ000000000
NCBI RefSeq accession number GCF_019997305.2
NCBI Sequence Read Archive (SRA) accession number SRR27184279.
Introduction
Many Gram-positive spore-forming rhizobacteria of the genus Bacillus show potential as biocontrol biopesticides that promise improved sustainability and ecological safety in agriculture [1,3]. Here, we present genomic sequencing data for Bacillus strain Egem-Utku 07, hereafter known as EU07. This strain was previously isolated from the rhizosphere of diseased tomato plants [4] in an effort to collect strains that could inhibit the soilborne pathogen Fusarium oxysporum f. sp. radicis-lycopersici [4], which causes crown rot in tomato. We demonstrated that EU07 inhibits this pathogen in vitro [4]. Furthermore, EU07 promotes growth and inhibits fusarium head blight in planta [5]. We previously established that EU07 is a member of the genus Bacillus, but its precise species identity was ambiguous. Furthermore, in the absence of sequence data, little was known about the potential molecular mechanisms for its beneficial properties. Here, we present a draft-quality genome sequence assembly and genomic sequence reads from strain EU07. This dataset will help in better understanding EU07’s phylogeny and taxonomy, and provide a resource to assist elucidation of the molecular mechanisms of EU07’s beneficial traits.
Methods
Bacterial strain and isolation of genomic DNA
We isolated genomic DNA from bacterial strain EU07 from fresh liquid culture grown for 24 h in nutrient broth pH 7.2. We note that this medium provides a laboratory environment quite different from the bacterium’s normal soil environment. The liquid culture was inoculated from a single colony and, therefore, was assumed to be clonal. We used the ISOLATE II genomic DNA kit (Bioline), following the manufacturer’s instructions. The quality and concentration of the genomic DNA were assessed using a NanoDrop 2000c spectrophotometer (ThermoFisher Scientific).
DNA sequencing
Genomic DNA was sent to the University of Exeter’s Sequencing Facility (https://biosciences.exeter.ac.uk/sequencing/) for Illumina Nextera XT library preparation and sequencing on the Illumina MiSeq platform to generate 748 528 pairs of 300 bp reads with a mean insert size of approximately 400 bp.
Genome sequence assembly
We performed adapter trimming and quality filtering on the MiSeq reads using Trim Galore version 0.6.7 [6], which incorporates Cutadapt version 3.5 [7]. The -q parameter was set to 30 and we used the --paired option. The resulting cleaned read-pairs served as input for de novo assembly using SPAdes version 3.13.1 [8] with the --careful option. The resulting scaffolds and contigs were re-ordered against the reference genome of strain FZB42 with the Mauve Contig Mover [9]. Annotation was added by the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline version 6.6 [10] after submission of the genome assembly. The command lines are documented in GitHub at https://github.com/davidjstudholme/bacillus_EU07/tree/main/assembly and in the Zenodo repository (https://doi.org/10.5281/zenodo.10968102) [11].
Assessment of genome-assembly quality
We calculated assembly statistics using quast version 5.2.0 [12]. We checked read coverage of the genome assembly by aligning the EU07 reads against the EU07 assembly and calculating alignment statistics with Qualimap version 2.3 [13]. The alignment was performed using bwa-mem version 0.7.17 [14]; then, we reformatted and sorted the output using SAMtools version 1.13 [15]. The full details of the command lines are documented at https://github.com/davidjstudholme/bacillus_EU07/blob/main/assemblyQC/README.md and in the Zenodo repository [11].
Average nucleotide identity (ANI)
We used fastANI [16] to calculate ANI between the genome of EU07 and each of the Bacillus amyloliquefaciens group (taxonomy ID: 1938374) genome assemblies retrieved from GenBank [17,18]. The exact command lines are documented in GitHub at https://github.com/davidjstudholme/bacillus_EU07/ and in the Zenodo repository [11].
Phylogenomics
To generate a maximum-likelihood phylogenetic tree based on genome-wide SNPs, we used PhaME [19] with FastTree [20]. The exact command lines used are documented at https://github.com/davidjstudholme/bacillus_EU07/ and in the Zenodo repository [11]. The resulting tree was rendered using the Interactive Tree of Life (iTOL) 6.8.1 [21].
Whole-genome alignment
Genome sequences were aligned using progressiveMauve version 2.4.0 [22] after first re-ordering the contigs against the reference genome of strain KNU-28 [23] with the Mauve Contig Mover [9]. The resulting alignment was visualized using Mauve snapshot_2015-02-25 [24]. The exact command lines used are documented at https://github.com/davidjstudholme/bacillus_EU07/ and in the Zenodo repository [11].
Further whole-genome analyses
We used the Proksee web server [25] to perform several analyses of the assembled EU07 genome. This included blastn searches against 888 related genomes, annotation of horizontally acquired genomic regions with Alien Hunter [26], and identification of bacteriophage sequences using VirSorter [27,28] and Phigaro [29]. Variant-calling was performed using the Parsnp tool in Harvest [30].
Results and discussion
Genome sequencing and assembly
We generated 748 528 pairs of 300 bp Illumina MiSeq sequencing reads from EU07 genomic DNA. This represents approximately 100× coverage of the 4.2 Mbp genome. Trimming and filtering with Trim Galore left 715 442 pairs of reads, with lengths ranging from 20 to 300 bp. De novo assembly with SPAdes yielded 266 contigs with a total length of 4.2 Mbp and N50 length of 52.8 kb. This was deposited in GenBank via the NCBI Submission Portal under accession number GCA_019997305.2. The NCBI’s contamination filtering removed 5 contigs, leaving 261. The NCBI PGAP annotation system predicted 4 273 genes, of which 4 081 encode putative proteins. The results of NCBI’s quality check with CheckM v1.2.2 [31,32] revealed a completeness of 98.16 % (85th percentile) and 0.47 % contamination.
Alignment of sequencing reads against the genome assembly and analysis with Qualimap revealed a mean coverage of 93.25× and standard deviation of 89.87. Almost all of the genome assembly (99.96 %) had at least 1× coverage, and 97.59 % of the assembly had at least 10× coverage. The full Qualimap report and output files are available in the Zenodo repository (https://doi.org/10.5281/zenodo.10968102) [11], allowing users of this data to take coverage into account when performing analyses. We note that the contig with least coverage is JAIFZJ020000237.1, having only 1.04× coverage. Nevertheless, blast searches reveal that this contig shows very high levels of sequence similarity to genomes of other Bacillus velezensis strains, increasing confidence in its validity.
EU07 belongs to the species B. velezensis
Previously, the phylogenetic and taxonomic position of strain EU07 had been ambiguous and we previously referred to it as ‘B. sp.’ and ‘B. subtilis’ [4,5]. To identify the species to which strain EU07 belongs, we uploaded the genome assembly to the Type Strain Genome Server (TYGS) [33]. This classified EU07 to the species B. amyloliquefaciens. Among the sequenced type strains in TYGS, the most similar to EU07 was FZB42 [34], which is the type strain of B. amyloliquefaciens subsp. plantarum [35]. However, this taxon is now considered to be synonymous with B. velezensis and distinct from B. amyloliquefaciens [36]. Hereafter, we refer to our strain as B. velezensis EU07.
EU07 belongs to a clade of plant-associated strains of B. velezensis
To identify previously sequenced similar genomes, we calculated ANI between B. velezensis EU07 and all 888 genome assemblies available in GenBank for the B. amyloliquefaciens group. This revealed that EU07 shares more than 99.9 % ANI with 13 previously sequenced genomes. Table 1 lists the genomes showing the highest levels of ANI to that of B. velezensis EU07. This includes strains that previously have been classified variously as B. amyloliquefaciens or B. velezensis. However, they all fall within the B. velezensis clade [36,38] and should be considered as belonging to that species. To further elucidate the evolutionary relationships of EU07, we generated a phylogenomic tree including these closely related strains and the relevant type strains; this is presented in Fig. 1. Consistent with the ANI results, strain EU07 falls within a clade that includes the same 13 strains that showed greatest ANI with EU07. Alignment of these genomes with Mauve (Fig. 2) reveals extensive conservation and co-linearity of the chromosome sequence among these strains. Comparison of the EU07 chromosome versus the genome sequences of related strains, as shown in Fig. 3, revealed that most of the presence–absence polymorphism was associated with loci predicted to originate from bacteriophage genomes.
Table 1. Genomes that share more than 99 % ANI with B. velezensis EU07.
GenBank accession no. | Reference | Strain | ANI (%) |
GCA_004421045.1 | [47] | ‘B. amyloliquefaciens’ FS1092 | 99.99 |
GCA_021228895.1 | [48] | B. velezensis A4P130 | 99.99 |
GCA_003986895.1 | – | B. velezensis BE2 | 99.99 |
GCA_007678125.1 | [49] | B. velezensis DE0189 | 99.99 |
GCA_003073255.1 | [37] | B. velezensis QST713 | 99.99 |
GCA_026156445.1 | [50] | B. velezensis CHBv2 | 99.98 |
GCA_001709055.1 | – | B. velezensis CFSAN034339 | 99.98 |
GCA_019093835.1 | – | ‘B. amyloliquefaciens’ BK | 99.98 |
GCA_014791945.1 | – | ‘B. amyloliquefaciens’ INH2-4b | 99.98 |
GCA_028609625.1 | [42] | B. velezensis DMW1 | 99.98 |
GCA_003149795.1 | [40] | ‘B. amyloliquefaciens’ ALB79 | 99.95 |
GCA_024300805.1 | [23] | ‘B. amyloliquefaciens’ KNU-28 | 99.95 |
GCA_001278635.1 | [39] | ‘B. amyloliquefaciens’ BS006 | 99.94 |
GCA_024134605.1 | – | B. velezensis 2987tsa1 | 99.12 |
GCA_000817575.1 | [51] | ‘B. amyloliquefaciens’ TF28 | 99.10 |
GCA_034060585.1 | – | B. velezensis Y-4 | 99.07 |
GCA_010671715.1 | [52] | B. velezensis HU-91 | 99.07 |
GCA_009193045.1 | [53] | B. velezensis BPC6 | 99.07 |
GCA_034061945.1 | – | B. velezensis YN-2A | 99.05 |
GCA_026786545.1 | – | B. velezensis NRRL B-59289 | 99.04 |
GCA_024138555.1 | [54] | ‘B. amyloliquefaciens’ TPS17 | 99.04 |
GCA_029866505.1 | [55] | ‘B. amyloliquefaciens’ MN-13 | 99.03 |
GCA_000341875.1 | [56] | B. velezensis UCMB5036 | 99.02 |
GCA_009789615.1 | [57] | B. velezensis BA-26 | 99.02 |
GCA_029910295.1 | – | B. velezensis PT4 | 99.01 |
GCA_009738165.1 | [58] | B. velezensis HN-Q-8 | 99.01 |
GCA_021559715.1 | [59] | B. velezensis CF57 | 99.01 |
GCA_012647845.1 | [60] | B. velezensis UCMB5140 | 99.01 |
Among the strains closely related to EU07 are several that previously have been described as having growth-promoting and/or pathogen-inhibitory properties. For example, strain BS006 was isolated from roots of Physalis peruviana in Colombia and promotes growth in banana [39]. Strain KNU-28 was isolated from peach leaves in Korea [23]. Strain ALB79 was isolated from grapes in northern California and shown to inhibit the growth of Listeria monocytogenes in vitro [40], while strain QST713 is used commercially (Serenade; Bayer) to protect mushroom crops against green mould disease and promotes growth in banana [37,41], among other applications. The endophytic Bacillus strain DMW1 was isolated from the inner tissues of potato tubers and exhibited strong biocontrol activity [42]. The near-identity of these genome sequences, independently isolated from plants in diverse geographical locations, suggests that EU07 is a member of a widely disseminated lineage of B. velezensis with biocontrol and growth-promoting properties. The molecular mechanisms and genetic determinants of these properties have been extensively reviewed elsewhere [43,45], and include gene clusters for secondary metabolites such as bacilysin, fengycin and macrolactin, which are conserved in the B. velezensis lineage that includes BS006 and EU07 [38].
Since our previous phenotypic comparisons between strains EU07 and QST713 revealed differences in their abilities to suppress fungal growth, we compared their genome sequences to identify possible genetic determinants of the observed differences. Their genomes are almost identical, with no detectable differences in their gene contents. However, we identified 46 single-nucleotide differences, which are listed in Table 2. These differences appear to be non-uniformly distributed across the genome. For example, 20 of the 46 SNPs occur within a single gene that encodes the beta subunit of a class-1b ribonucleoside-diphosphate reductase [46] (RefSeq WP_108702400.1; locus tag BVQ_RS09140). This suggests that these differences might be explained by recombination events associated with horizontal genetic transfer rather than point mutations. We also identified some sequence differences between EU07 and QST713 in the intergenic regions between several tRNA genes (GenBank accession no. JAIFZJ010000168.1). These genetic differences may explain the previously observed differences observed between the DNA fingerprints of these two strains when previously assayed using RAPDs [4].
Table 2. Forty-six SNPs between B. velezensis strains EU07 and QST713.
Position in CP025079.1 | Nucleotide in QST713 | Nucleotide in EU07 | Amino acid change | Predicted gene product |
21 222 | A | G | K→E | BVQ_RS00080: serine-tRNA ligase |
230 096 | A | C | E→A | BVQ_RS21890: non-ribosomal peptide synthetase |
230 098 | A | C | K→Q | BVQ_RS21890: non-ribosomal peptide synthetase |
230 111 | C | A | A→E | BVQ_RS21890: non-ribosomal peptide synthetase |
530 737 | T | G | Y→STOP | BVQ_RS02595: hypothetical protein |
530 789 | T | G | L→V | BVQ_RS02595: hypothetical protein |
530 811 | T | G | I→>S | BVQ_RS02595: hypothetical protein |
531 288 | T | G | I→S | BVQ_RS02595: hypothetical protein |
705 298 | A | C | F→V | BVQ_RS03655: GNAT family N-acetyltransferase |
855 165 | A | C | Non-coding | |
1 168 486 | A | C | Non-coding | |
1 215 136 | A | C | F→C | BVQ_RS06330: contact-dependent growth inhibition system immunity protein |
1 851 920 | T | G | F→L | BVQ_RS09140: class 1b ribonucleoside-diphosphate reductase subunit beta |
1 851 923 | A | T | G→G (synonymous) | BVQ_RS09140: class 1b ribonucleoside-diphosphate reductase subunit beta |
1 851 925 | C | A | T→K | BVQ_RS09140: class 1b ribonucleoside-diphosphate reductase subunit beta |
1 851 929 | G | T | K→N | BVQ_RS09140: class 1b ribonucleoside-diphosphate reductase subunit beta |
1 851 932 | A | G | E→E (synonymous) | BVQ_RS09140: class 1b ribonucleoside-diphosphate reductase subunit beta |
1 851 935 | A | G | Q→Q (synonymous) | BVQ_RS09140: class 1b ribonucleoside-diphosphate reductase subunit beta |
1 851 938 | C | T | D→D (synonymous) | BVQ_RS09140: class 1b ribonucleoside-diphosphate reductase subunit beta |
1 851 941 | T | G | T→T (synonymous) | BVQ_RS09140: class 1b ribonucleoside-diphosphate reductase subunit beta |
1 851 944 | T | C | Y→Y (synonymous) | BVQ_RS09140: class 1b ribonucleoside-diphosphate reductase subunit beta |
1 851 950 | A | G | K→K (synonymous) | BVQ_RS09140: class 1b ribonucleoside-diphosphate reductase subunit beta |
1 851 953 | T | G | V→V (synonymous) | BVQ_RS09140: class 1b ribonucleoside-diphosphate reductase subunit beta |
1 851 954 | T | C | L→L (synonymous) | BVQ_RS09140: class 1b ribonucleoside-diphosphate reductase subunit beta |
1 851 956 | A | C | L→F | BVQ_RS09140: class 1b ribonucleoside-diphosphate reductase subunit beta |
1 851 959 | T | C | A→A (synonymous) | BVQ_RS09140: class 1b ribonucleoside-diphosphate reductase subunit beta |
1 851 962 | A | C | G→G (synonymous) | BVQ_RS09140: class 1b ribonucleoside-diphosphate reductase subunit beta |
1 851 965 | T | G | L→L (synonymous) | BVQ_RS09140: class 1b ribonucleoside-diphosphate reductase subunit beta |
1 851 969 | T | C | L→L (synonymous) | BVQ_RS09140: class 1b ribonucleoside-diphosphate reductase subunit beta |
1 851 971 | A | G | L→L (synonymous) | BVQ_RS09140: class 1b ribonucleoside-diphosphate reductase subunit beta |
1 851 972 | T | C | L→L (synonymous) | BVQ_RS09140: class 1b ribonucleoside-diphosphate reductase subunit beta |
1 851 974 | G | T | L→F | BVQ_RS09140: class 1b ribonucleoside-diphosphate reductase subunit beta |
1 878 004 | T | G | Non-coding | |
2 191 740 | T | C | D→G | BVQ_RS10680: cysteine hydrolase family protein |
2 415 378 | C | A | Non-coding | |
2 415 381 | C | A | Non-coding | |
2 415 440 | C | A | Non-coding | |
2 722 225 | G | T | Non-coding | |
2 722 243 | T | G | Non-coding | |
3 268 938 | G | T | A→E | BVQ_RS16510: class 1 isoprenoid biosynthesis enzyme |
3 269 022 | T | G | N→T | BVQ_RS16510: class 1 isoprenoid biosynthesis enzyme |
3 467 035 | A | C | Non-coding | |
3 489 562 | A | G | F→F (synonymous) | BVQ_RS17685: lantibiotic immunity ABC transporter MutG family permease subunit |
3 490 697 | T | A | I→I (synonymous) | BVQ_RS17690: lantibiotic immunity ABC transporter MutE/EpiE family permease subunit |
3 573 178 | T | A | Non-coding | |
4 000 822 | T | G | Non-coding |
Table 3. Genome sequences included in the phylogenomic analysis.
GenBank accession no. | Taxon | Reference |
GCA_003149795.1 | ‘B. amyloliquefaciens’ ALB79 | [40] |
GCA_019093835.1 | ‘B. amyloliquefaciens’ BK | – |
GCA_001278635.1 | ‘B. amyloliquefaciens’ BS006 | [39] |
GCA_000196735.1 | B. amyloliquefaciens DSM7T | [34] |
GCA_004421045.1 | ‘B. amyloliquefaciens’ FS1092 | [47] |
GCA_014791945.1 | ‘B. amyloliquefaciens’ INH2-4b | – |
GCA_024300805.1 | ‘B. amyloliquefaciens’ KNU-28 | [23] |
GCA_029866505.1 | ‘B. amyloliquefaciens’ MN-13 | [55] |
GCA_000817575.1 | ‘B. amyloliquefaciens’ TF28 | [51] |
GCA_024138555.1 | ‘B. amyloliquefaciens’ TPS17 | [54] |
GCA_000262045.1 | B. siamensis KCTC 13613T | [61] |
GCA_024134605.1 | B. velezensis 2987tsa1 | – |
GCA_021228895.1 | B. velezensis A4P130 | [48] |
GCA_001647965.1 | B. velezensis AP194 | [62] |
GCA_009789615.1 | B. velezensis BA-26 | [57] |
GCA_003986895.1 | B. velezensis BE2 | – |
GCA_009193045.1 | B. velezensis BPC6 | [53] |
GCA_003431885.1 | B. velezensis (B. methylotrophicus) CBMB205T | [63] |
GCA_021559715.1 | B. velezensis CF57 | [59] |
GCA_001709055.1 | B. velezensis CFSAN034339 | – |
GCA_026156445.1 | B. velezensis CHBv2 | [50] |
GCA_007678125.1 | B. velezensis DE0189 | [49] |
GCA_028609625.1 | B. velezensis DMW1 | [42] |
GCA_000015785.2 | B. velezensis (B. amyloliquefaciens subsp. plantarum) FZB42T | [34] |
GCA_009738165.1 | B. velezensis HN-Q-8 | [58] |
GCA_010671715.1 | B. velezensis HU-91 | [52] |
GCA_001461835.1 | B. velezensis (='B. oryzicola') KACC 18228T | [64] |
GCA_001267695.1 | B. velezensis KCTC 13012 | [65] |
GCA_001461825.1 | B. velezensis NRRL B-41580T | [36] |
GCA_026786545.1 | B. velezensis NRRL B-59289 | – |
GCA_026787705.1 | B. velezensis NRRL BD-154 | – |
GCA_029910295.1 | B. velezensis PT4 | – |
GCA_003073255.1 | B. velezensis QST713 | [37] |
GCA_000341875.1 | B. velezensis UCMB5036 | [56] |
GCA_012647845.1 | B. velezensis UCMB5140 | [60] |
GCA_034060585.1 | B. velezensis Y-4 | – |
GCA_034061945.1 | B. velezensis YN-2A | – |
GCA_019997305.1 | B. velezensis EU07 | This study |
Conclusion
Genome sequencing of potential biocontrol strain EU07 revealed that it belongs to the species B. velezensis, a species often closely associated with plant roots, and well known for promoting plant growth and biocontrol. The EU07 strain is genetically almost identical to the commercially used strain QST713 (Serenade) and several other previously sequenced and characterized strains; however, we identified several genes containing single-nucleotide differences that can distinguish between EU07 and QST713. Strain EU07 is more distantly related to the commercially used B. velezensis strain FZB24 (TAEGRO), previously known as the type-strain of B. amyloliquefaciens subsp. plantarum. The availability of this genome sequence will facilitate future efforts to unravel the genetic and molecular basis for the strains beneficial properties.
Acknowledgements
We acknowledge the University of Exeter’s DNA Sequencing Facility for performing library preparation and sequencing, and the University of Exeter’s Research IT team for provision of advanced research computing infrastructure used for sequence analysis.
Abbreviations
- ANI
average nucleotide identity
- NCBI
National Center for Biotechnology Information
Footnotes
Funding: Support for C. Jimenez-Quiros from the University of Worcester is gratefully acknowledged. This project utilized equipment funded by the Wellcome Trust Institutional Strategic Support Fund (WT097835MF), a Wellcome Trust Multi-User Equipment Award (WT101650MA) and a Biotechnology and Biological Sciences Research Council (BBSRC) LOLA award (BB/K003240/1).
Accession No: The genome sequence data generated in this work have been deposited in public databases: BioProject accession number PRJNA743875, https://www.ncbi.nlm.nih.gov/bioproject/743875; GenBank
assembly accession number GCA_019997305.2, https://www.ncbi.nlm.nih.gov/nuccore/JAIFZJ000000000; NCBI RefSeq accession number GCF_019997305.2; Sequence Read Archive (SRA) accession number SRX22864526.
Author contributions: Conceptualization: O.B. and M.T. Data curation: all authors. Formal analysis: all authors. Investigation: all authors. Writing – original draft: O.B., D.J.S. and M.T. Writing – review and editing: all authors.
Contributor Information
David J. Studholme, Email: d.j.studholme@exeter.ac.uk.
Catherine Jimenez-Quiros, Email: catherinejq@gmail.com.
Mahmut Tör, Email: m.tor@worc.ac.uk.
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