The draft genome of RZ3MS14 consisted of 28 scaffolds in a total size of 5,493,110 bp (G + C content of 34.9%), N50 and L50 values of 397,909 bp and 3, respectively (Table 1). Genome completeness scored 97.7% in Checkm analysis and 99.8% using BUSCOs. Metrics and PGAP prediction are summarized in Table 1.
Table 1
Genome metrics of Bacillus paramycoides RZ3MS14
Assessment | Value |
Genome size (bp) | 5,493,110 |
Scaffolds | 28 |
Contigs | 28 |
Largest contig (bp) | 1,521,286 |
G + C content (%) | 34.9 |
Quality assessment | |
Completeness (%) | 97.7 |
Contamination (%) | 2.3 |
Complete BUSCOs (nº of core genes detected, %) | 449, 99.8 |
Missing BUSCOs (nº of core genes detected, %) | 1, 0.2 |
PGAP annotation | |
Genes (total) | 5,744 |
CDSs (total) | 5,637 |
Genes (coding) | 5,339 |
Genes (RNA) | 107 |
rRNAs (5S, 16S, 23S) | 5, 4, 1 |
Complete rRNAs (5S, 23S) | 4, 1 |
Partial rRNAs (5S, 16S) | 1, 4 |
tRNAs | 92 |
ncRNAs | 5 |
Pseudo genes (total) | 298 |
Pseudo genes (ambiguous residues) | 0 of 298 |
Pseudo genes (frameshifted) | 99 of 298 |
Pseudo genes (incomplete) | 227 of 298 |
Pseudo genes (internal stop) | 75 of 298 |
Pseudo genes (multiple problems) | 89 of 298 |
RZ3MS14 clustered in the Bacillus paramycoides clade, represented by the type strain B. paramycoides NH24A2 (Fig. 1). Within this cluster, RZ3MS14 presented a separate branch (length 0.03350), followed by B. paramycoides DE0103 (length 0.02653). The OrthoANI value between the genomes of RZ3MS14 and B. paramycoides strains (97.48 to 97.63%) provided initial support for its inclusion in this species (Fig. 1). Moreover, dDDH metrics ranged from 73-80.6% when compared to strain type NH24A2. Both measurements were higher than ANI (95 ~ 96%) and dDDH (70%) thresholds, widely used and supported for bacterial species delineation based on genomic analyses (Chun et al. 2018). Therefore, this study refers to this strain as B. paramycoides RZ3MS14.
The RAST provided an overview of the biological features of RZ3MS14, categorizing 2.408 (44%) genes (including both protein- and RNA-coding genes) into 466 SEED subsystems (Fig. 2A). The draft genome contained genes primarily involved in the metabolism of carbohydrates (498), proteins (359), amino acids, and derivatives (580), as well as in the cofactors, vitamins, prosthetic groups, and pigments category (274). Metabolism of phosphorus (102), sulfur (43), nitrogen (34), and iron (19) were other notable categories, as these are essential nutrients, and PGPR are directly tied to their recycling in the soil. The highest number of protein-coding genes (84.24%) was annotated using COG database. According to the distribution in COG classes, 1683 of these genes (30.48%) were associated with metabolic processes, predominantly within the amino acid transport and metabolism subclass, 932 genes (16.88%) in Information storage and processing, and 775 (14.03%) for Cellular processes and signaling (Fig. 2B). With KEGG, 1.46% of protein-coding genes were mapped in 210 metabolic pathways, mainly in carbohydrate (427) and amino acid (361) metabolism, alongside protein families related to genetic information processing (633) and cellular processes and signaling (683) (Fig. 2C).
The presence of genes involved in ammonia assimilation (glnR, glnA, and gltB) and in aerobic denitrification (narG, narH, narJ, and narI) revealed the ability of RZ3MS14 to regulate nitrogen (N) in the rhizosphere (Table S1). Furthermore, this rhizobacterium exhibited genetic potential in the cycling of other nutrients essential for plant growth and development. A repertoire of genes associated with inorganic phosphorus (Pi) solubilization through the synthesis of organic acids such as citric, formic, glycolic, glyoxylic, malic, oxalacetic, 2-oxoglutaric, pyruvic, and succinic acids, were found in RZ3MS14 genome; along with genes encoding the Pi transport system (pstSCAB), the two-component PhoP/PhoR system, and its regulon (Table S1). Regarding the release of P from organic sources for plant uptake, three genes with phosphatase activity were identified, among others related to phosphonoacetate and 2-aminoethylphosphonic acid (ciliatin) mineralization (Table S1). The RZ3MS14 genome also harbors a biosynthetic cluster of catecholate-type bacillabactin siderophore (dhbFBECA). However, it might be able to internalize iron (Fe) bound to other hydroxamate-type siderophores or by direct Fe2+ (Feo genes) iron transport system (Table S2).
Auxin production is another important mechanism to increase plant growth, uptake of nutrients, and even alleviate environmental stresses. We detected the indole-3-pyruvate (IPA) pathway tryptophan-dependent composed of the key genes ipdC (encoding for indole-3-pyruvate decarboxylase) and aldA (encoding an aldehyde dehydrogenase). These results would uncover the genes responsible for the in vitro IAA production reported previously for strain RZ3MS14 (Batista et al. 2018). The tryptophan biosynthesis genes trpA, trpB, trpC, trpF, trpD, trpG and trpE, were also found.
Several protective mechanisms for plant health were widely distributed in the genome of the Amazonian isolate RZ3MS14; among them, genes responsible for production of hydrogen sulfide (Table S3). This gas-signaling molecule plays an emerging role in regulating plant senescence and maturation, as well as providing resistance against fungal pathogens and protection against drought, extreme temperatures, toxic metals, and salinity (Aroca et al. 2018; Corpas and Palma 2020; Liu et al. 2021). The genome also contains genes involved in synthesizing, transporting, and utilizing polyamines putrescine and spermidine (Table S3). Various roles have been ascribed to bacterial polyamines, including induction of positive physiological changes in plants and protection against various abiotic stresses and pathogen attacks. In particular, spermidine has been linked to biofilm formation in Bacillus subtilis (Hobley et al. 2017). Additionally, genes associated with the formation of γ-aminobutyrate (GABA), butanoic acid, acetoin, 2,3-butanediol, and 2,3-butanedione (Table S3) evidence the potential of the strain to produce volatile organic compounds (VOCs), that elicit induced systemic resistance in plants and responses against environmental stresses (Dias et al. 2021; Yi et al. 2016).
By AntiSMASH v6.1.1 were predicted six biosynthetic gene clusters for relevant natural products: three biosynthetic clusters of antibiotics micrococcin, cerecidin, and paeninodin; a cluster for the root elongation promoter trehangelin; and a biosynthetic cluster of antifungal lipopeptide fengycin (Table S4). This analysis also detected one last cluster for NRPS siderophore bacillibactin, previously described.
The bioinformatics analysis of the draft genome of B. paramycoides strain RZ3MS14 unveils several mechanisms contributing to the nutrient dynamics in the rhizosphere and plant growth-promoting multitraits. RZ3MS14 also harbors genes related with antifungal activities and responses to environmental stress through VOCs among a wide range of metabolites. Overall, this study provides new insights into the species and its potential as microbial bioinoculants.