Genome sequencing of Bacillus cereus isolate EB-40: a plant growth-promoting bacterium

Abstract

Bacillus sp. isolate EB-40 was characterized in 'Prata Anã' banana (Musa sp.) plants as an endophyte capable of colonizing both the inter- and intracellular spaces of roots, nitrogen fixation, phosphate solubilization, in vitro synthesis of indole-3-acetic acid, and promotion of the development of micropropagated banana seedlings. Here, we report the whole-genome sequence of Bacillus sp. isolate EB-40 and its taxonomic assignment. Its genome is composed of one chromosome and three plasmids. The circular double-stranded DNA chromosome (5,613,235 base pairs (bp)) has a GC content of 35.3% and 5462 genes. The three plasmids have a total length of 237,685 bp with 201 genes. Comparative genomics revealed that its genome shares similarity indices with Bacillus cereus genomes above the thresholds recommended for species assignment.

Article Highlights

B. cereus EB-40 is a novel bacterium with phosphorus-solubilizing activity, nitrogen fixation ability and IAA production ability.

The genome of B. cereus EB-40 consists of a circular double-stranded chromosome and three plasmids.

The complete genome sequence of strain EB-40 provides a genetic basis for multifunctional biofertilizers.

Introduction

Plant growth-promoting bacteria (PGPB) belong to a group of beneficial and heterogeneous microorganisms that can be found in the rhizosphere, on the surface of or associated with the root, and are capable of increasing plant growth and protecting plants from diseases and abiotic stresses [1]. Among the various PGPB, some species of the Bacillus genus stand out [2]. They are considered good options for the formulation of commercial products [3].

Bacillus species can directly or indirectly stimulate plant growth by providing plants with resources or nutrients and modulating growth regulator levels in agriculturally significant crops, and these effects improve the productivity of plants and their content of biologically active substances [4, 5]. Bacillus sp. EB-40, which was isolated by Souza et al. (2013) [6] from 'Prata Anã' banana (Musa spp.) roots, has been studied for over a decade by our group. Our research has demonstrated its ability to colonize both the inter- and intracellular spaces of banana roots [7], fix nitrogen, solubilize phosphate [8], and promote increases in the length and diameter of pseudostems, fresh mass, and dry mass in micropropagated seedlings of 'Prata Anã' banana during the acclimatization period [9]. Furthermore, when used in combination with other bacteria (Bacillus pumilus EB-51 and Lysinibacillus sp. EB-53), Bacillus sp. EB-40 led to a 174% increase in root dry weight and facilitated increased accumulation of macroelements in the aerial parts of micropropagated banana seedlings during the acclimatization period [8]. Although the mechanisms of action of Bacillus sp. EB-40 in promoting plant growth have been extensively studied, its genetic background and taxonomy remain unknown.

The development of sequencing methodologies has revolutionized microbial genomics, and approaches such as hybrid assembly have emerged as solutions for obtaining complete and accurate bacterial genomes [10]. The quality of genome assembly has been a concern in microbial genomics, as it directly impacts the accuracy of gene annotation and can hinder comparative genomic analyses [11]. Among the 7,017 genomes of Bacillus species available in the NCBI Microbial Genomes database (https://www.ncbi.nlm.nih.gov/genome/microbes/; accessed on 2023/03/24), 1,687 are complete genomes.

The active principles of PGPB are mediated by enzymes and secondary metabolites, and genome mining offers the opportunity to scrutinize the whole genome of a PGPB strain for genes encoding beneficial enzymes. Whole-genome analyses combined with biochemical assays offer the advantage that the full arsenal of a PGPR strain can be unveiled and investigated [12]. Here, we report the whole-genome sequence of Bacillus sp. EB-40 and its taxonomic assignment on the basis of its genomic features.

Materials and methods

Genome sequencing, assembly, and annotation

Bacillus sp. EB-40 was previously isolated from the internal tissues of banana roots by Souza et al. (2013) [6]. We multiplied the isolate EB-40 by streaking on a Petri dish containing Luria Bertani medium (2%) and incubating it at 37 °C for 24 h. Then, we selected one isolated colony and cultured it in 10 ml of Luria Bertani medium (2%) for 24 h at 37 °C and 200 RPM. We subsequently extracted the genomic DNA via the Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA) and assessed its quality and quantity via a NanoDrop™ fluorometer and a Qubit™ fluorometer (Thermo Fischer Scientific, Waltham, MA, USA), respectively. A hybrid approach combining Illumina NextSeq 550 short paired reads (2 × 150 nt) and PacBio RSII long reads was used to determine the complete genome of Bacillus sp. EB-40. The quality of the short-read sequencing data was assessed via FASTQC version 0.11.9 (https://github.com/s-andrews/FastQC). Adapter sequences were detected and removed from the sequencing data via the autodetection setting of TrimGalore version 0.6.7 [13]. The paired reads were subsequently trimmed for quality and filtered for length via Trimmomatic version 0.39 [14] with the following parameters: HEADCROP:25, CROP:125, SLIDINGWINDOW:4:20, and MINLEN:100. The long reads were corrected and trimmed by LoRDEC version 0.3 [15] using the processed short reads, and Seqkit filtered the long reads with lengths equal to or greater than 1,000 nt. Unicycler version 0.5.0 [16] was used with both long and short reads to execute the de novo assembly of the genome via the normal method, and BBMap version 38.76 (https://sourceforge.net/projects/bbmap) was used to calculate the sequencing coverage. The NCBI Prokaryotic Genome Annotation Pipeline (PGAP) [17–19] annotated the genome during the processing of its submission to the GenBank database (https://www.ncbi.nlm.nih.gov/genbank), and BUSCO version 5.4.2 [20] was used to assess the completeness of the genome using the bacillales_odb10 database. The Rapid Annotation Subsystem Technology (RAST) server (https://rast.nmpdr.org/) [21] classified the predicted genes into subsystems of the SEED database [22], and the RPSBLAST tool of BLAST version 2.13.0 [23] classified the proteins according to the functional categories of the Clusters of Orthologous Genes (COG) database [24], considering an E-value threshold of 1e⁻¹⁰ to select the significant alignments. In addition, the KEGG Automatic Annotation Server (KASS) [25] was used to classify the proteins according to the KEGG Orthology (KO) system to allow metabolic pathway mapping.

Results and discussion

Genome sequencing

We sequenced the Bacillus sp. EB-40 genome with 745X coverage and assembled it into a single chromosome totaling 5,613,235 bp with an average GC content of 35.3%. The genome also harbors three plasmids with sizes of 215,503, 7,710, and 14,472 bp, each with GC contents of 32.6, 33.8, and 34.2%, respectively (Table 1, Fig. 1). The chromosome has 5,462 genes predicted by the NCBI PGAP, including 5,201 coding DNA sequences (CDSs), 106 tRNA genes, 42 rRNA genes, one tmRNA, four ncRNAs, and 108 pseudogenes that encode nonfunctional proteins. Additionally, the three plasmids possess a set of 185 CDSs and 16 pseudogenes. The BUSCO analysis estimated a completeness of 99.8% for the genome of Bacillus sp. EB-40 among the 450 BUSCO groups expected for the order Bacillales, identifying 444 complete and single-copy BUSCOs, five complete and duplicate BUSCOs, and only one missing BUSCO.

Table 1. List of features associated with the B. cereus isolate EB-40 genome

Item	Features
Project name	Genome sequencing and assembly of Bacillus cereus isolate EB-40
Sample name	EB-40
Taxonomy ID Accession number	1396 [Bacillus cereus species] CP115717
Latitude and longitude	14° 13′ 12.0" S and 42° 46′ 48.0" W
Geographic location	Brazil, Bahia, Guanambi
Collection date	2010
Environment	banana roots (Musa spp.)
Sequencing method	Illumina NextSeq 550 and PacBio RSII
Number of replicons	4
Reference for biomaterial	Not applicable
Isolation and growth condition	Luria Bertani medium at 37 °C for 24 h
Assembly quality	Finished genome
Assembly software	Unicycler version 0.5.0
Number of contigs	4

All features are in accordance with the recommendations of the minimum information about the bacterial and archaeal genome sequences (MIGS-BA) (https://www.gensc.org/pages/standards/checklists.html)

Fig. 1 [Images not available. See PDF.]

Genome map of the Bacillus cereus isolate EB-40. The genome contains one chromosome (ID 1) and three plasmids (IDs 2, 3, and 4). Each representation harbors four circles from outside to inside: I. The annotated genes in the heavy strand and light strand (inside); II. The average GC skew; III. The GC content; IV. The DNA coordinates. The colors indicate the categories of the predicted genes: coding DNA sequences (CDS), tRNA, rRNA, ncRNA, and tmRNA. The Proksee web server was used to generate the map (https://proksee.ca; [22])

The species is within the B. cereus group, which is composed of rod-shaped, aerobic or facultatively anaerobic spore-forming gram-positive bacteria widely distributed in natural environments [26]. Several works have demonstrated the role of several species of Bacillus cereus as bacteria that promote plant growth [26, 27].

The RAST server classified 2,225 CDSs (41.31%) into 27 functional categories of the SEED database, and the most frequent were amino acids and derivatives (257 CDSs); cofactors, vitamins, prosthetic groups, and pigments (202 CDSs); and carbohydrates (191 CDSs). The COG database allowed the classification of 3,429 CDSs (63.67%) into 21 functional categories, and [E] amino transport and metabolism (339 CDSs); [K] transcription (304 CDSs); and [J] translation, ribosomal structure, and metabolism (266 CDSs) were the most frequent among those with known functions. Among the 1,702 CDSs (31.60%) that encode unclassified proteins, 562 CDSs encode hypothetical proteins. Table S1 summarizes the functional classification of the predicted proteins.

Considering the aforementioned phenotypic characteristics of the Bacillus sp. isolate EB-40, we conducted a comprehensive analysis of its genome to identify genes associated with phosphate metabolism, the production of indole-3-acetic acid (IAA or 3-IAA), and volatile organic compounds (VOCs).

Some soil endophytic microorganisms can colonize the rhizosphere of plants and produce extracellular phytase enzymes that can act as plant growth-promoting rhizobacteria by increasing the availability of phytate phosphate [23]. In the genome of Bacillus sp. EB-40, we identified the following enzymes that catabolize phytate into forms assimilable by plants: l-myo-inositol-1,4-monophosphatase (locus PF061_19825), phospholipase C (locus PF061_03420), 1-phosphatidylinositol phosphodiesterase (locus PF061_17290), malonate-semialdehyde dehydrogenase (locus PF061_11600), and cyclo-inositol 2-dehydrogenase (NADP⁺) (locus PF061_17765). The identification of these enzymes corroborates the results obtained in vitro and in vivo by Andrade et al. (2014) [24], who demonstrated phosphate solubilization, and Souza et al. (2017) [5], who investigated the growth of banana seedlings inoculated with Bacillus sp. isolate EB-40 during the acclimatization period in greenhouse experiments.

Bacillus sp. EB-40 also produces auxin in media with and without the addition of tryptophan. Considering tryptophan-dependent pathways, endophytic microorganisms can synthesize indole-3-acetic acid (IAA) via different pathway routes classified based on involved intermediates, such as indole-3-acetamide (IAM), indole-3-pyruvic acid (IPA), indole-3-acetonitrile (IAN), tryptamine (TAM), and tryptophan side-chain oxidase (TSO) pathways [25, 26]. In the genome of the EB-40 isolate, we identified enzymes associated with the TAM, IAM, and TSO pathways. Among the genes involved in tryptophan metabolism, we identified trpA (tryptophan synthase subunit alpha; locus PF061_06305), trpB (tryptophan synthase subunit beta; locus PF061_06300), trpC (indole-3-glycerol phosphate synthase; locus PF061_06290), trpD (anthranilate phosphoribosyltransferase 2; locus PF061_06285), trpE (anthranilate synthase component I; loci PF061_00390 and PF061_06275), trpF (phosphoribosylanthranilate isomerase; loci PF061_06295), and trpG (anthranilate synthase component II; loci PF061_00395 and PF061_06280).

Volatile organic compounds (VOCs) have the potential to control plant pathogens, stimulate plant growth, and induce systemic disease resistance [27]. Acetoin and 2,3-butanediol are VOCs produced by plant growth-promoting bacteria and are synthesized through the condensation of two pyruvate molecules into acetolactate. Acetolactate synthase catalyzes the production of acetolactate, which is decarboxylated into acetoin by acetolactate decarboxylase. Finally, the reduction of acetoin by the enzyme acetoin reductase produces 2,3-butanediol [28]. The genome of Bacillus sp. isolate EB-40 contains all the genes necessary for the production of acetoin and 2,3-butanediol: (R,R)-butanediol dehydrogenase (locus PF061_03410), acetolactate synthase AlsS (locus PF061_04530), alpha-acetolactate decarboxylase (locus PF061_04535), the acetolactate synthase large subunit (loci PF061_07025 and PF061_09285), and the acetolactate synthase small subunit (locus PF061_07030).

Taxonomic assignment

We confirmed the species assignment of Bacillus sp. EB-40 through sequence alignment and genomics analysis. First, we aligned its genome sequence with the representative prokaryotic genomes of the NCBI Refseq database (https://ftp.ncbi.nlm.nih.gov/blast/db/; version 23/12/2023) using the BLASTn tool of BLAST version 2.13.0 [19] and considering an E-value threshold of 1e⁻¹⁰ to select the significant alignments. The JSpeciesWS server (https://jspecies.ribohost.com/jspeciesws/) [29] was subsequently used to compare the Bacillus sp. EB-40 genome with the ten best-ranked genomes among the significant alignments identified via BLASTn and calculate two average nucleotide identity (ANI) indices via BLAST (ANIb) and MUMMER (ANIm). In addition, the Genome-to-Genome Distance Calculator (GGDC) server version 3.0 (https://ggdc.dsmz.de/) [30, 31] was used to calculate the degree of digital DNA‒DNA hybridization (dDDH) via BLAST alignment.

Compared with the prokaryotic representative genomes of the NCBI RefSeq database, the genome of Bacillus sp. EB-40 shares genomic similarity indices that surpass the thresholds recommended for species assignment, which include 95% for ANI and 70% for dDDH [32, 33], with the reference genome of Bacillus cereus (Table 2). These indices were even higher in comparative analyses with other B. cereus isolates available in GenBank, such as the genome of strain DQ01 (accession CP097351), which had values of 99.14% for ANIb, 99.30% for ANIm, and 93.80% for dDDH. Therefore, we classified Bacillus sp. EB-40 as a Bacillus cereus species.

Table 2. Taxonomic assignment of Bacillus sp. EB-40

GenBank ID	Species	dDDH [P(DDH ≥ 70%)]	ANIb [Coverage]	ANIm [Coverage]
NZ_CP017060	Bacillus cereus	70.80 [79.67]	96.30 [87.81]	96.70 [90.77]
NZ_CP049019	Bacillus tropicus	46.30 [10.46]	91.56 [79.43]	92.28 [83.03]
NZ_CP101135	Bacillus paranthracis	45.60 [9.14]	91.29 [77.22]	92.06 [80.60]
NZ_NWUW00000000	Bacillus fungorum	45.40 [8.89]	90.87 [73.53]	92.06 [76.61]
NC_007530	Bacillus anthracis	45.10 [8.33]	91.14 [78.59]	91.94 [81.87]
NZ_CP086328	Bacillus pacificus	45.10 [8.32]	91.05 [76.39]	91.92 [79.90]
NZ_CP032365	Bacillus wiedmannii	44.70 [7.69]	91.05 [79.10]	91.83 [82.59]
NZ_CP064875	Bacillus toyonensis	44.60 [7.60]	91.17 [80.77]	91.82 [84.97]
NZ_CP128152	Bacillus albus	44.40 [7.24]	91.00 [80.29]	91.71 [84.12]
NZ_CP040336	Bacillus luti	43.60 [6.18]	90.57 [76.14]	91.56 [79.42]

The comparative genomic analysis of the isolate EB-40 genome with the reference prokaryotic genomes of the NCBI RefSeq database guided its taxonomic classification by calculating different genomic identity indices. All values are in percentages

Genome sequencing and annotation are the first steps toward complete comprehension of the genetic basis of microbial activities that directly influence the beneficial genetic and physiological responses of plants. Furthermore, transcriptomic and metabolomic studies may be conducted to elucidate how environmental stimuli influence these bacteria.

Conclusion

The genome of Bacillus cereus isolate EB-40 comprises one chromosome and three plasmids. The chromosome is a 5,613,235-bp circular double-stranded DNA with a GC content of 35.3% and 5462 genes, and the three plasmids have sizes of 215,503, 7710, and 14,472 bp with a total of 201 genes. Among the 5,386 CDSs annotated in the genome, we identified genes associated with pathways associated with its previously reported phenotypic characteristics describing it as an endophyte able to promote plant growth. Comparative genomics revealed that its genome shares similarity indices with Bacillus cereus genomes above the thresholds recommended for species assignment. In addition, this isolate has the capacity for effective colonization of the intercellular and intracellular spaces in the banana root system, suggesting that it is a novel isolate of Bacillus cereus.

Acknowledgements

We thank the Núcleo de Análise de Biomoléculas (NuBioMol) of the Universidade Federal de Viçosa (UFV) for providing the facilities for data analysis. NuBioMol is financially supported by the following Brazilian agencies: Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (Finep) and Sistema Nacional de Laboratórios e Nanotecnologias (SisNANO)/Ministério da Ciência, Tecnologia e Informação (MCTI).

Author contributions

Debora Francine Gomes Silva Pereira executed the experiments. Pedro Marcus Pereira Vidigal and Samuel A. Santos executed the genome assembly and analyzed the data. Silvia Nietsche coordinated the research. Debora Francine Gomes Silva Pereira and Pedro Marcus Pereira Vidigal wrote the manuscript. Adelica A. Xavier and Marlon Cristian Toledo Pereira provided funds for the research and commented on the draft manuscript. All the authors reviewed and approved the manuscript.

Funding

This research was funded by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), grant numbers CAG00390-15 and APQ00146-22, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-CAPES, Financing Code 001 and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), grant numbers 427667/2016-0 and 306391/2021-0.

Availability of data and materials

The datasets generated and analyzed during the present research are available from the corresponding author upon reasonable request.

Declarations

Competing interest

The authors declare that they have no conflicts of interest.

Nucleotide sequence accession number

The complete genome sequence of the B. cereus isolate EB-40 was deposited on DDBJ/EMBL/GenBank under accession number CP115717 for the chromosome and numbers CP115718, CP115719, and CP115720 for the plasmids.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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