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Article

Genomic Insights and Functional Analysis Reveal Plant Growth Promotion Traits of Paenibacillus mucilaginosus G78

by
Dan Wang
1,
Verena Poinsot
2,
Wangxi Li
1,
Yusheng Lu
1,
Chong Liu
1,
Yaying Li
1,
Kaizhi Xie
1,
Lili Sun
1,
Chaohong Shi
1,
Huanlong Peng
1,
Wanling Li
1,
Changmin Zhou
1 and
Wenjie Gu
1,3,*
1
Institute of Agricultural Resources and Environment, Guangdong Academy of Agricultural Sciences, Key Laboratory of Plant Nutrition and Fertilizer in South Region, Ministry of Agriculture, Guangdong Key Laboratory of Nutrient Cycling and Farmland Conservation, Guangdong Engineering Research Center of Soil Microbes and Cultivated Land Conservation, Guangzhou 510640, China
2
Université Toulouse III Paul Sabatier, Inserm, I2MC, Avenue Du Professeur Jean Poulhès, BP 84225, CEDEX 4, 31432 Toulouse, France
3
Maoming Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Maoming 525000, China
*
Author to whom correspondence should be addressed.
Genes 2023, 14(2), 392; https://doi.org/10.3390/genes14020392
Submission received: 9 January 2023 / Revised: 21 January 2023 / Accepted: 26 January 2023 / Published: 2 February 2023
(This article belongs to the Special Issue Molecular Research on Host-Microbe Interactions)
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Abstract

:
Paenibacillus mucilaginosus has widely been reported as a plant growth-promoting rhizobacteria (PGPR). However, the important genomic insights into plant growth promotion in this species remain undescribed. In this study, the genome of P. mucilaginosus G78 was sequenced using Illumina NovaSeq PE150. It contains 8,576,872 bp with a GC content of 58.5%, and was taxonomically characterized. Additionally, a total of 7337 genes with 143 tRNAs, 41 rRNAs, and 5 ncRNAs were identified. This strain can prohibit the growth of the plant pathogen, but also has the capability to form biofilm, solubilize phosphate, and produce IAA. Twenty-six gene clusters encoding secondary metabolites were identified, and the genotypic characterization indirectly proved its resistant ability to ampicillin, bacitracin, polymyxin and chloramphenicol. The putative exopolysaccharide biosynthesis and biofilm formation gene clusters were explored. According to the genetic features, the potential monosaccharides of its exopolysaccharides for P. mucilaginosus G78 may include glucose, mannose, galactose, fucose, that can probably be acetylated and pyruvated. Conservation of the pelADEFG compared with other 40 Paenibacillus species suggests that Pel may be specific biofilm matrix component in P. mucilaginosus. Several genes relevant to plant growth-promoting traits, i.e., IAA production and phosphate solubilization are well conserved compared with other 40 other Paenibacillus strains. The current study can benefit for understanding the plant growth-promoting traits of P. mucilaginosus as well as its potential application in agriculture as PGPR.

1. Introduction

As the world’s population is expected to exceed 9 billion over the next thirty years, an important question to address is how to meet the increasing demands for food [1,2,3]. The application of chemical fertilizers surely helped in increasing crop yields in the last fifty years, but their intensive and continuous use has brought about a lot of environmental problems, such as diverse pollutions and contamination of ecosystems but also soil quality and biodiversity reduction [1]. Plant growth-promoting rhizobacteria (PGPR) are not only able to increase the crop production, but also have the ability to help plants to resist to biotic or abiotic stresses. Thus, the use of PGPR to substitute for part the chemical fertilizers has been considered as an eco-friendly way [4,5].
Most strains from Paenibacillus genus isolated from soil promote plant growth by producing indole-3-acetic acid (IAA) and other auxin phytohormones. Such bacteria can solubilize inaccessible phophorous into forms that can be taken up by plant roots, and some strains can even fix atmospheric nitrogen [6].
P. mucilaginosus was first phylogenetically characterized as Bacillus mucilaginosus in 1967, and it was reclassified to the genus Paenibacillus in 2010 [7,8]. It is widely distributed in the soil or rhizosphere and produces high yield of exopolysaccharides [9,10,11,12,13]. The strains from this species can promote the growth of green gram, trifoliate orange, maize and apple seedling, and it has thus been described as an efficient PGPR [14,15,16,17,18,19].
As many genes are silenced under laboratory conditions, the whole-genome sequencing (WGS) and bioinformatics tools could help biologists investigate more functions and products of PGPR. With the development of genomic era, the next generation sequencing technology makes DNA sequencing faster and cheaper than the first-generation method [20]. Thus, more and more ecologically important metabolites for PGPR have been discovered by the extensive genomic studies [21]. Until now, the genomes of three strains (KNP414, 3016 and K02) from P. mucilaginosus have been sequenced [22,23,24]. However, it is necessary to provide detailed genome-level descriptions of essential features in P. mucilaginosus, including phosphate solubilization, plant hormone production, biofilm formation, and exopolysaccharide biosynthesis.
In this study, we sequenced the genome of a strain P. mucilaginosus G78, and we annotated the genes related to the ability of solubilizing the phosphate, releasing IAA, producing exopolysaccharides, and forming the biofilm. We also compared genomic regions implicated in association with plant hosts among 40 other strains from Paenibacillus genus. This study aimed to provide a foundation for the genetic studies and functions of P. mucilaginosus species and explore the potential ability of plant growth promotion of Paenibacillus genus at the genomic level.

2. Materials and Methods

2.1. Measurement of IAA Production and Phosphate Solubilization

For the measurement of IAA production, P. mucilaginosus G78 was grown in modified ACCC5 medium supplemented with 100 μg/mL Trp (IAA precursor). The modified ACCC5 medium contained sucrose 10 g/L, yeast extracts 0.5 g/L, K2HPO4·3H2O 0.5 g/L, NaCl 0.2 g/L, MgSO4·7H2O 0.2 g/L, CaCO3 1 g/L, at pH 7.2 [22]. The production of IAA was measured by using colorimetric assay, and the modified ACCC5 medium with Trp was used as negative control [25]. For the determination of phosphate solubilization, G78 strain was inoculated into Pikovskaya’s broth containing insoluble tri-calcium phosphate (0.5%) or soybean lecithin (0.02%) and cultured for 72 h, at 30 °C. Water-soluble phosphorus in the supernatant was determined by the chlorostannous-reduced molybdophosphoric acid blue method [26].

2.2. Biofilm Formation Assays

The formation of biofilm was measured applying the crystal violet (CV) method following the experimental procedure as described by Shang [27]. The strain was grown overnight in modified ACCC5 medium, and the N medium was used to develop the bacterial biofilm. The N medium contained maltose 2.5 g/L, MgSO4·7H2O 0.73 g/L, K2HPO4·3H2O 0.4 g/L, NaCl 0.06 g/L, FeCl3 0.6 mg/L, salicylic acid 10 mg/L and CaCO3 1 g/L, at pH 7.2.

2.3. Growth-Promoting Assay

The tomato seeds were surface sterilized by 1% (v/v) NaClO, germinated and transplanted in sterilized vermiculite moistened with Hoagland nutrient solution in Leonard jars, at 25 °C, and placed in a plant growth chamber [28]. The daylight illumination period was 12 h, and the light intensity was 1700 lx. The seedlings were inoculated with 10 mL of bacterial inoculum diluted with 10 mM sterilized MgSO4 solution (1 × 108 cfu/mL) on the 7th, 14th, and 21st days after transplanting. The control seedlings were incorporated with the same volume of 10 mM sterilized MgSO4. Shoot and root lengths, fresh weight were determined at 35 days post inoculation. The root length and scanning version were acquired and analyzed by Root Analysis WINRHIZO System (Regent, CAN). Data obtained were statically analyzed using SPSS software version 25.0 (IBM Corp., Armonk, NY, USA) and were presented in tables as the means ± standard error of mean (SEM). Significant differences between treatment were compared by Independent-samples t test.

2.4. Antagonistic Activity

The antagonistic effects of P. mucilaginosus G78 on the fungus were detected using the dual-culture plate approach by Deng et al. [29], with some modifications. P. mucilaginosus G78 was inoculated and incubated on modified ACCC5 agar medium for 24 h. Fungus inhibition tests were performed by placing the agar plug with Fusarium oxysporum f. sp. Momordicae or F. oxysporum f. sp. Cucumerinum in the center of PDA medium, and three agar plugs with P. mucilaginosus were placed 2.5 cm from the center. The agar plugs with no bacteria were selected as negative control. Plates were incubated, at 28 °C, for 5 days and checked for inhibition.

2.5. Antibiotic Susceptibility Tests

Bacteria was cultured for 24 h, centrifuged, resuspended and diluted 102 times with the modified ACCC5 medium, and then spread onto modified ACCC5 agar medium containing different antibiotics. Bacteria spread onto the medium without any antibiotics was used as a control [30]. The antibiotics used in this study included ampicillin, bacitracin, polymyxin, chloramphenicol, vancomycin, tetracycline, streptomycin and getamicin, with 1 mg/L, 5 mg/L, 10 mg/L, 50 mg/L, 100 mg/L and 150 mg/L, respectively. Plates were incubated, at 28 °C, and checked for inhibition.

2.6. Genome Sequencing and Analysis

The genomic DNA was extracted using a Qiagen Genomic-tip kit and following a modified manufacturer’s protocol as previously described [31]. Sequencing libraries were generated using NEBNext® UltraTM DNA Library Prep Kit for Illumina (Lincoln, NE, USA) following manufacturer’s recommendations, and index codes were added to attribute sequences to the sample. The whole genome of P. mucilaginosus G78 was sequenced using Illumina NovaSeq PE150 at the Beijing Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). The predicted CDSs were annotated from NR (NCBI non-redundant protein sequences; Version 202210, Swiss-Prot (A manually annotated and reviewed protein sequence database; Version 202210), Pfam (Protein family; Version Pfam v35.0), GO (Gene Ontology; Version 20220915), COG (Clusters of Orthologous Groups of proteins; Version 202006), and KEGG (Kyoto Encyclopedia of Genes and Genomes; Version 202210) database using sequence alignment tools such as RPS-BLAST, Diamond and HMMER. Briefly, each set of query proteins were aligned with the databases, and annotations of best-matched subjects (e-value < 10−5) were obtained for gene annotation. Secondary metabolites synthesis clusters were identified using antiSMASH (Version 5.1.2). Antibiotic resistance genes were predicted using CARD (Comprehensive Antibiotic Resistance Database, version 1.1.3). The genomic analyses were also performed using the online platform of Majorbio Cloud Platform (http://cloud.majorbio.com accessed on 1 October 2022) from Shanghai Majorbio Bio-pharm. The GenBank accession number of the sequence for P. mucilaginosus G78 is JAKQYK000000000.

2.7. Comparative Genomic and Phylogenetic Analysis

The core-orthologs from 41 strains were detected by PGAP pipeline-based protein similarity method [32]. The core-orthologs were clustered with at least 50% similarity for protein sequence to each other and 50% overlap with the longest sequence. The total genes within 41 genomes weres defined as the pan genome, and the shared genes among 41 strains was defined as their core genome [26]. Multiple alignment of amino acid sequences was carried out by using ClustalW (version 2.1) [33]. Conserved blocks from multiple alignments of test proteins were selected by using Gblocks [34]. Phylogenetic trees were inferred with 309 sing-copy core genes shared by 41 taxa. Maximum Likelihood (ML) method were inferred with PhyML (version 3.0) using the LG model with 1000 bootstrap replicates to construct the phylogenetic trees [35].

3. Results

3.1. Assessment of Plant Growth-Promoting Traits

Our results indicated significant effect of P. mucilaginosus G78 having the ability to form biofilm, solubilizing the inorganic and organic phosphate, and produce IAA (Table 1). Furthermore, the plant height and fresh weight determined after 35 days of inoculation of P. mucilaginosus G78 showed significant differences (p < 0.05), as presented in Table 2. The inoculation treatment improved the plant height and fresh weight of the tomato plant. The plant height and fresh weight of G78-treated tomato plants increased by 44.1% and 90.0% compared to the control plant, respectively (Table 2, Figure 1a,c), indicating the growth-promoting effect of P. mucilaginosus G78 inoculation.

3.2. Genomic Features

After assembly, the draft genome size of the P. mucilaginosus G78 was 8,576,872 bp with a GC content of 58.5% and 77 scaffolds with the N50 of 250,045 bp. The mean scaffold size was 111,388 bp and the longest scaffold was 778,093 bp. Additionally, a total of 7337 genes with 143 tRNAs, 41 rRNAs, and 5 ncRNAs were identified. The predicted genes included 2274 genes involved in metabolism, 753 genes involved in environmental information processing, and 253 genes in cellular processes. COG function classification showed that 904 genes are involved in carbohydrate transport and metabolism, 701 genes involved in the transcription, 651 genes involved in general function, and 497 genes involved in signal transduction. A total of 423 carbohydrate-active enzyme-encoding genes were identified in G78, including glycosyl hydrolysis (GHs, 58.4%), glycosyl transferases (GTs, 11.8%), carbohydrate esterases (CEs, 31.3%), carbohydrate-binding modules (CBMs, 1.2%), polysaccharide lyases (PLs, 5.7%), and auxiliary activities (AAs, 5%). The circular genome visualization for the P. mucilaginosus G78 was produced by the circular viewer, as shown in Figure 2a.

3.3. Phylogenetic Tree and Comparative Genomic Analysis

A phylogenetic tree based on single-copy core genes was reconstructed using the whole genome sequence (Figure 2b). The information about Paenibacillus strains was shown in Table S1. It was inferred among the 41 Paenibacillus strains that the G78 strain was very closely related to the P. mucilaginosus strain KNP414 and to two other P. mucilaginosus strains: K02 and 3016. The ANI value between P. mucilaginosus G78 and KNP414 equals 99.9%, 98.9% for strain 3016 and 98.49% for K02. It also indicated that the P. mucilaginosus strains grouped closely to P. naphthalenovorans strain 32O-Y.
To visualize the similarity of encoded proteins, the whole-genome alignments of protein coding sequences were conducted for 41 Paenibacillus species strains. Average amino acid identities were calculated using the pair-wise orthologous sets of CDSs. Only 0.13% of the total 234,857 putative protein-coding genes were identified as core genes, which suggests that genetic differentiation and horizontal gene acquisition from other taxa are high. G78 contained a total of 229 strain specific genes, while P. mucilaginosus strain KNP414 has 728 strain specific CDS.

3.4. Secondary Metabolites Production and Antimicrobial Resistance Genes

As shown by the dual-cultural plate, P. mucilaginosus G78 exhibits prohibition of the growth of the plant pathogen, F. oxysporum f. sp. momordicae and F. oxysporum f. sp. cubense (Figure 1b) following incubation for 5 d. Such findings demonstrated the capability of G78 strain to inhibit the growth of F. oxysporum f. sp. momordicae and F. oxysporum f. sp. cubense, with the inhibition rate of 51.2% and 47.3%, respectively. In P. mucilaginosus G78, the genome analysis identified several gene clusters that encode secondary metabolites. The putative natural products include terpene, siderophore, ladderane, flaviolins, polyketides, and NRPS. The NRPS contains some proposed peptide antibiotics, such as icosalide, paenibacterin, tridecaptin, locillomycin. The representative gene clusters encoding putative secondary metabolites were summarized in Table 3. G78 can grow under ampicillin, bacitracin, and polymyxin at a low concentration level, suggesting it contains antimicrobial resistance-related genes (Table 4). These were predicted based on the CARD database (Table 5). G78 was found to contain 429 genes related to the resistance to different antibiotics (Table S2).

3.5. EPS Synthesis Genes

Exopolysaccharides (EPS) play key structural and functional roles in P. mucilaginosus, and were reported to protect the bacteria against the host defense during the plant–microbe interaction. We found an EPS gene cluster in G78 strain, mainly comprising 35 putative genes on a ~39.1 kb DNA fragment, which includes glycosyl transferases, polymerases, enzymes involved in the synthesis of nucleotide precursors and enzymes responsible for sugar modification or the addition of sugar substituents (Table 6, Figure 3). We further blast the putative EPS biosynthetic gene cluster among the sequenced strains from this species, and found P. mucilaginosus KNP414 and K02 have very similar gene structures with strain G78, while strain 3016 showed some of the truncated and non-homologous sequences (Figure S1).

3.6. Biofilm Formation Genes

The key genes involved in the formation of biofilm were investigated using the KAAS database, and 28 genes were explored, including metabolic pathway regulators, diguanylate or adenylate cyclase, matrix protein-coding genes, and putative matrix polysaccharide synthesis genes (Table 7). It was shown that pel-like operon encoded the biofilm polysaccharide in Bacillus cereus [36]. We explored the pel-like genes among 41 Paenibacillus strains, and found that P. mucilaginosus strains had more Pel polysaccharide biosynthetic genes, which indicated that Pel polysaccharide is not a common biofilm matrix component among the genus of Paenibacillus (Figure 4).

3.7. Plant Growth-Promoting Ability Genes

Indole-3-acetic acid (IAA) has been reported as an important phytohormone with the capacity to control plant development, which can be produced by many rhizosphere bacteria [37]. In this study, the indolepyruvate decarboxylase (encoded by ipdC gene) and auxin carrier protein were identified in strain G78 (Figure 5). However, the genes encoding tryptophan monooxygenas or indole-3-acetamide hydrolase were not detected in the G78 strain. Furthermore, the gene ipdC exists among all tested strains, suggesting these bacteria are all capable of IAA production following the indole-3-pyruvic acid pathway, even if auxin carrier proteins are deficient in some strains.
Considering the phosphate-providing ability of strain G78, we found eight phn genes (phnABCDEWXM)and two genes encoding glucose-1-dehydragenase (gcd) and gluconic acid dehydrogenase (gad). We also identified the putative pst operon (pstS, ptsC, pstA, pstB) and PhoP-PhoR system in the genome of G78 (Figure 5). In addition, we screened 20 pathways in total related with the organic acid metabolic pathway, and all above can explain the phosphate solubilization and secretion ability of this strain (Table S2). Additionally, the mineral phosphate solubilizations genes (gcd, gad) and phosphate transport system are present in all 41 sequenced Paenibacillus strains, indicating that all these strains have the ability to promote plant growth on phosphate-limited soil.

4. Discussion

P. mucilaginosus has been widely reported as a plant growth-promoting rhizobacteria (PGPR) [14,15,16,17,18,19]. Until now, only three strains from P. mucilaginosus were sequenced, and the PGPR traits at the genome level has not been described in detail. In this study, we sequenced a PGPR strain, P. mucilaginosus G78, and explored the genes related to microbe-–plant interaction, such as secondary metabolites synthesis, exopolysaccharides biosynthesis, biofilm formation, IAA production, and phosphate-dissolving ability. The genome size of P. mucilaginosus G78 was 8,576,872 bp with a GC content of 58.5%, which is very similar in size with other P. mucilaginosus strains. As shown in Table S1, P. mucilaginosus showed high GC content. Moreover, it presents the second largest genome size than other 37 Paenibacillus strains. Focusing on the influence of several properties including biochemical, genetic flows, selection biases, and the biochemical-energetic properties shaping genome composition, it indicated a trend toward high GC content and larger genomes in free-living organisms, as a result of more complex and varied environments [38,39]. The genes related to the glycoside hydrolase family are much abundant in this strain in comparison to other Paenibacillus, which is consistent with their reported importance for Paenibaicllus survival [40].
Secondary metabolites (SM) produced by plant-associated biocontrol bacteria can directly reduce the pathogen’s ability to cause disease, induce plant defense mechanisms, or promote plant development [41]. P. mucilaginosus G78 showed antifungal activity against phytopathogens such as F. oxysporum, and has the genomic potential to produce a lot of SMs. Recent extensive bacterial genome sequencing and bioinformatic analysis showed that terpene synthases are widely distributed in bacteria [42,43]. The ability to produce or capture siderophores makes the bacteria competitive advantages to colonize plant tissues [44]. Antismash analysis showed that strain G78 has asb operon, which is responsible for petrobactin biosynthesis in Bacillus anthracis [45,46]. Kedarcidin (KED) is an aromatic enediyne that may be produced by strain G78. It was reported to be chromoprotein antitumor antibiotic and was isolated from Streptoalloteichus sp. ATCC 53560 but rarely reported in Paenibacillus or Bacillus genus [47,48]. Bacteriocins are ribosomally synthesized peptides (RSPs) that contain 12~50 amino acid residues which exhibited a broad spectrum of antimicrobial activity. Many of the polyketides (PKs) produced by Bacillus and Paenibacillus species have been described as bioactive natural products that had medical value and can be potentially applied in agriculture for controlling plant pathogens [49,50]. In total, there are 18 NRPS or NRPS-like metabolites proposed gene clusters in strain G78, including 10 unknown ones. The NRPS contains some proposed peptide antibiotics, such as icosalide, paenibacterin, tridecaptin, locillomycin and other new products. However, whether the metabolites mentioned above were produced still needs further determination. In this study, we demonstrated that P. mucilaginosus G78 could grow on the medium supplemented with ampicillin, bacitracin, polymyxin and chloramphenicol. The putative genes which have a role in the resistance to these antibiotics are listed in Table 5. The G78 strain remains susceptible to vancomycin, tetracycline and streptomycin (Table 4), although partial genes participating in these antibiotic resistances were identified by CARD analysis, indicating that the completed operon is necessary for the antibiotic resistance.
Exopolysaccharides secreted by P. mucilaginosus strains was reported to have strong antioxidant abilities [12] and was hypothesized to play an important role during the process of wastewater treatment [13,51]. Studying the genes responsible to EPS synthesis will be helpful to explore its potential functions and structures. Exopolysaccharide biosynthesis for bacteria usually includes the following steps: uptake of substance, nucleotide sugar precursors synthesis, assembling and polymerization, modification, and release [52]. In this study, we explored the potential EPS biosynthesis gene cluster from strain G78. It was shown that the potential monosaccharides include glucose, mannose, galactose, fucose, that can probably be acetylated and pyruvated according to its genetic features. The chemical structure of EPS from this species is strain specific, as different strains from P. mucilaginosus produced various EPS, consistently with the reported biosynthetic genes variations (Figure S1). The reported partial structure of the EPS from P. mucilaginosus SM-01 was mainly composed of β-1, 4-linked-Glc and β-1, 4-linked-Man as the backbone and branched at C-2 position of β-1, - 4-linked-Glc residue by the acetyl esters [13]. The possible structure of polysaccharide from P. mucilaginosus WL412 was identified as [→4)α-Glc(1 → 2)α-Man(1 → 3)β-Glc(1 → 3)α-Man(6-Ac)(1 → 3)β-Gal(1→] [53].
Biofilms are surface-associated microbial communities in which the cells are embedded within an extracellular matrix, and they can help the microorganisms to defend against biotic or abiotic stress, to colonize the plant host and to acquire nutrients or genetic traits [54,55,56]. The major genes for the formation of biofilm include those encoding for important biofilm transcriptional regulators, the matrix structural synthesis (matrix protein, putative matrix polysaccharide), extracellular DNA synthesis and cyclic-di-GMP metabolisms [57,58]. The genome of G78 strain contained several genes that participate in biofilm formation, including transcriptional regulators, matrix structural synthesis genes, eDNA synthesis genes and diguanylate or adenylate cyclase-encoding genes. Pel polysaccharide was reported to play an important role in the biofilm formation of Pseudomonas aeruginosa and Bacillus cereus, and their biosynthesis requires an inner membrane complex comprising of PelD, PelE, PelF, and PelG [34,59,60]. Conservation of the pelADEFG among 41 studied strains from the Paenibacillus genus suggests that Pel may not be a common biofilm matrix component in this genus except for the species of P. mucilaginosus. However, further investigation via a gene deletion approach is required to characterize the function of Pel polysaccharide in P. mucilaginosus G78.
Paenibacillus strains are well known for their beneficial effects of plant growth, including production of IAA and mineral solubilization [6,61,62]. The biosynthesis of indole-3-acetic acid (IAA) is often related to beneficial effects of PGPR on plant development including cell division, elongation, tropism, apical dominance, senescence, flowering, and response to stress [35,63,64,65]. In this study, P. mucilaginosus G78 was demonstrated to produce indole-3-acetic acid (IAA), and to promote the growth of tomato seedlings by increasing the root length, fresh weight, and height of plants. The genes encoding putative indole pyruvate decarboxylase (IpdC) and auxin efflux carrier (AEC) protein are present in the genome of P. mucilaginosus G78. Knocking out the ipdC gene in Bacillus thuringiensis RZ2MS9 resulted in the decreasing production of IAA and significantly reduced its ability to promote maize growth, indicating that IAA biosynthesis by this PGPR is a major mechanism to promote plant growth [66]. Xie et al. found that ipdC homologies are present in all analyzed P. polymyxa genomes, with over 96% amino acid identity between strains across 98% of the sequence [26]. We explored the ipdC and auxin efflux carrier protein-encoding genes among the genomes of 41 Paenibacillus strains, and found that ipdC is present in all analyzed genomes as well. In contrast, not all strains have the auxin efflux carrier protein. This could indicate that their capability of exporting IAA is not common in this genus.
A large proportion of organic and inorganic phosphate is present in the soil, but they cannot be absorbed directly by plants because of the insoluble forms. Phosphate solubilizing bacteria has the ability to convert insoluble phosphates and to make it accessible to the plants [67,68]. It was proved that mineral phosphates solubilization is achieved through gluconic acid production and that the phn genes are responsible for solubilizing organic phosphate [69,70,71]. The glucose-1-dehydrogenase (gcd) and gluconic acid dehydrogenase (gad) are implicated in the production of gluconic acid [68,72,73]. The phosphate transportation is mostly related to the Pst (phosphate-specific transport) system and to the PhoP-PhoR system [24,74,75,76]. The gcd, gad, phnABCDEPWX, pst SCAB and phoPR were all present in the genome of G78, which is consistent with its ability to dissolve both organic or inorganic phosphorus compounds. All the analyzed Paenibacillus strains exhibit the genes for mineral phosphorus solubilization and phosphorus transport, indicating the potential application of Paenibacillus strains as phosphorus activator in the plant rhizosphere. In addition, although G78 strain can grow on the free-nitrogen medium, we did not find the nif genes present in this species (Figure S2), indicating that it could employ an unknown metabolic pathway to survive under nitrogen deficient condition, which needs to be clarified by further investigation.

5. Conclusions

The genome size of the P. mucilaginosus G78 was 8,576,872 bp with a GC content of 58.5%. Additionally, a total of 7337 genes with 143 tRNAs, 41 rRNAs, 5 ncRNAs were identified. It contained 26 gene clusters encoding secondary metabolites and 20 proteins related to the resistance to ampicillin, bacitracin, polymyxin and chloramphenicol, which is in accordance with its antagonist activity and antibiotic resistance ability. According to the genetic features, the potential monosaccharides of its exopolysaccharides for P. mucilaginosus G78 may include glucose, mannose, galactose, fucose, that can probably be acetylated and pyruvated. Conservation of the pelADEFG compared with other 40 Paenibacillus species suggests that Pel may be a specific biofilm matrix component in P. mucilaginosus. The containing genes encoding IAA production and phosphate solubilization associated with the phenotypic analysis highlighted the capability of P. mucilaginosus G78 strain to promote the plant growth. P. mucilaginosus species showed high GC content, and it presents the second largest genome size than other 37 studied Paenibacillus strains. Several genes associated with plant growth-promoting traits, i.e., IAA production and phosphate solubilization, are well conserved among 41 Paenibacillus strains, suggesting their potential uses in agriculture.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/genes14020392/s1, Table S1: The genome information of Paenibacillus strains in this study; Table S2. Putative genes related to phosphate solubilization ability in G78; Predicted exopolysaccharide gene clusters for four P. mucilaginosus strains (3016, K02, KNP414 and G78); Figure S1. Predicted exopolysaccharide gene clusters for four Paenibacillus mucilaginosus strains (3016, K02, KNP414 and G78); Figure S2. Genes involved in nitrogen fixation of 41 Paenibacillus strains.

Author Contributions

D.W., W.G. and V.P. conceived and designed the experiment. D.W. and V.P. wrote the paper. D.W. and W.L. (Wangxi Li) performed the experiment. W.G. supervised the study. Y.L. (Yusheng Lu), C.L., Y.L. (Yaying Li), K.X., L.S., C.S., H.P., W.L. (Wanling Li) and C.Z. contributed in reagents/materials/analysis tools. All authors have read and agreed to the published version of the manuscript.

Funding

This study was financially supported by the National Natural Science Foundation of China (31800102), Guangdong Basic and Applied Basic Research Foundation (2021A1515011331, 2021A1515011211), The Science and Technology Program of Guangdong Province (2021B1212050022), The open competition program of top ten critical priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province (2022SDZG08, 2022SDZG09), Modern Agricultural Industrial Technology System of Guangdong Province (The task of Innovation team-building of key generic technologies in agricultural resources and environment) (2022KJ118, 2022KJ111), High-level Foreign Expert Project of Guangdong Province (2019, 2021), Science and Technology Program of Guangzhou, China (201904010262, 202002020075), Dean project funding of the Guangdong Academy of Agricultural Sciences, China (201934, 201935), Agricultural competitive industry discipline team building project of Guangdong Academy of Agricultural Sciences (202121TD), Low carbon agriculture and carbon neutralization Research Center, GDAAS (XT202220), and Special fund for scientific innovation strategy-construction of high-level Academy of Agriculture Science (R2019PY-QF010, R2020PY-JG012, R2021YJ-YB1003, R2021YJ-QG007, R2022YJ-YB3009).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available from the corresponding author on reasonable request.

Acknowledgments

We thank Muhammad Kamran (College of Pastoral Agriculture Science and Technology, Lanzhou University) for his suggestion about the data analyses and the comments on our manuscript. We acknowledge Aiting Lin (Shanghai Biozeron Biotech Co., Ltd.) for her assistance in bioinformatic analyses.

Conflicts of Interest

The authors declare no conflict of interest.

References

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Figure 1. The plant growth promotion ability and antagonistic activity against pathogenic fungi through the dual-culture test of P. mucilaginosus G78 strain. (a) The effects of G78 strain on the growth of tomato plant. Bar, 2 cm. (b) The growth of F. oxysporum f. sp. momordicae and F. oxysporum f. sp. cubense with or without G78. Fom, F. oxysporum f. sp. momordicae; Foc, F. oxysporum f. sp. cubense; Fom+G78 or Foc+G78, F. oxysporum f. sp. momordicae or F. oxysporum f. sp. cubense with G78. (c) The root scanning image of tomato plants. G78, the treatment which was inoculated by G78; control, the uninoculated treatment.
Figure 1. The plant growth promotion ability and antagonistic activity against pathogenic fungi through the dual-culture test of P. mucilaginosus G78 strain. (a) The effects of G78 strain on the growth of tomato plant. Bar, 2 cm. (b) The growth of F. oxysporum f. sp. momordicae and F. oxysporum f. sp. cubense with or without G78. Fom, F. oxysporum f. sp. momordicae; Foc, F. oxysporum f. sp. cubense; Fom+G78 or Foc+G78, F. oxysporum f. sp. momordicae or F. oxysporum f. sp. cubense with G78. (c) The root scanning image of tomato plants. G78, the treatment which was inoculated by G78; control, the uninoculated treatment.
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Figure 2. Genomic features of P. mucilaginosus G78 and phylogenetic relationship of 41 Paenibacillus strains. (a) Genome map of P. mucilaginosus G78. Circles (from outside to inside) as follows: (1) scale marks (unit, Mb), (2) protein-coding sequences on the forward strand colored by COG category, (3) protein-coding sequences on the reverse strand (same color scheme as the second circle), (4) rRNA genes, (5) tRNA genes, (6) GC content (deviation from average), and (7) positive (green) and negative (orange) GC skew. (b) ML phylogenetic tree was constructed using based on 309 single-copy core proteins shared by 41 genomes.
Figure 2. Genomic features of P. mucilaginosus G78 and phylogenetic relationship of 41 Paenibacillus strains. (a) Genome map of P. mucilaginosus G78. Circles (from outside to inside) as follows: (1) scale marks (unit, Mb), (2) protein-coding sequences on the forward strand colored by COG category, (3) protein-coding sequences on the reverse strand (same color scheme as the second circle), (4) rRNA genes, (5) tRNA genes, (6) GC content (deviation from average), and (7) positive (green) and negative (orange) GC skew. (b) ML phylogenetic tree was constructed using based on 309 single-copy core proteins shared by 41 genomes.
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Figure 3. Predicted exopolysaccharide gene cluster for P. mucilaginosus G78. The predicted functions of each color-coded ORF are indicated at the lower bottom panel.
Figure 3. Predicted exopolysaccharide gene cluster for P. mucilaginosus G78. The predicted functions of each color-coded ORF are indicated at the lower bottom panel.
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Figure 4. pel-like operon of P. mucilaginosus G78 and genes involved in Pel-polysaccharide of 41 Paenibacillus strains. (a) pel-like operon architectures of P. mucilaginosus G78. Arrows are used to denote open reading frames, with the direction of each arrow indicating the direction of transcription. (b) pelAEDFG genes of 41 Paenibacillus strains. Colored box represents the presence of a gene within a genome and white box indicates absence of a gene.
Figure 4. pel-like operon of P. mucilaginosus G78 and genes involved in Pel-polysaccharide of 41 Paenibacillus strains. (a) pel-like operon architectures of P. mucilaginosus G78. Arrows are used to denote open reading frames, with the direction of each arrow indicating the direction of transcription. (b) pelAEDFG genes of 41 Paenibacillus strains. Colored box represents the presence of a gene within a genome and white box indicates absence of a gene.
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Figure 5. Genes involved in IAA production, organic and inorganic phosphate solubilization of 41 Paenibacillus strains. Colored box represents the presence of a gene within a genome and white box indicates absence of a gene.
Figure 5. Genes involved in IAA production, organic and inorganic phosphate solubilization of 41 Paenibacillus strains. Colored box represents the presence of a gene within a genome and white box indicates absence of a gene.
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Table
Table 1. Plant growth-promoting traits of P. mucilaginosus G78 strain.
Table 1. Plant growth-promoting traits of P. mucilaginosus G78 strain.
Soluble P for Inorganic Phosphate
(mg/L)		Soluble P for Organic Phosphate
(mg/L)		IAA Production
(mg/L)		Biofilm Biomass
(OD560)
G7813.6 ± 3.10.48 ± 0.0110.9 ± 0.20.32 ± 0.02
Notice: Values are presented as mean ± SEM of three replications.
Table
Table 2. Effects of P. mucilaginosus G78 strain on the growth of tomato seedlings.
Table 2. Effects of P. mucilaginosus G78 strain on the growth of tomato seedlings.
Root Length *
(cm Plant−1)		Root FW **
(g Plant−1)		Shoot Length **
(cm Plant−1)		Shoot FW **
(g Plant−1)
Control435.3 ± 58.8 a0.22 ± 0.03 a16.1 ± 0.8 a0.90 ± 0.06 a
G781001.2 ± 131.5 b0.78 ± 0.07 b23.2 ± 1.3 b1.71 ± 0.15 b
Notice: *, Values are presented as mean ± SEM of three replications; **, Values are presented as mean ± SEM of five replications. Different letters within the same column indicate significant differences among treatment means according to Independent-samples t test (p < 0.01).
Table
Table 3. The putative gene cluster encoding secondary metabolites in P. mucilaginosus G78.
Table 3. The putative gene cluster encoding secondary metabolites in P. mucilaginosus G78.
Cluster IDTypeSimilar Cluster Similarity (%)MIBiG Accession
1NRPSzwittermicin A7BGC0001059
2NRPS---
3NRPS---
4terpene---
5T3PKS---
6NRPS---
7transAT-PKSdifficidin20BGC0000176
8LAP---
9siderophorepetrobactin83BGC0000942
10bacteriocin---
11NRPSlocillomycin42BGC0001005
12terpenecarotenoid33BGC0000645
13NRPSstigmatellin15BGC0000153
14NRPS---
15ladderanekedarcidin1BGC0000081
16NRPS---
17NRPScyclomarin D8BGC0000333
18NRPSpaenibacterin60BGC0000400
19NRPS---
20NRPS---
21NRPStridecaptin60BGC0000449
22NRPScystothiazole A11BGC0000982
23NRPS---
24NRPS---
25NRPS---
26NRPS-likeicosalide A/icosalide B100BGC0001833
Table
Table 4. Antimicrobial susceptibility test of P. mucilaginosus G78.
Table 4. Antimicrobial susceptibility test of P. mucilaginosus G78.
Antibiotics (μg/mL)0151050100150
Ampicillin++++++-
Bacitracin +++++++
Polymyxin++++---
Chloramphenicol ++-----
Vancomycin+------
Tetracycline+------
Streptomycin+------
Notice: ‘+’ represents the bacteria that can grow on the agar plates but ‘-’ represents not.
Table
Table 5. The putative antibiotic resistance-related genes.
Table 5. The putative antibiotic resistance-related genes.
AntibioticARO NameARO Description
ampicillinampH β-lactamaseampC-like β-lactamase and penicillin-binding protein
ampC1 β-lactamaseβ-lactamase
ampC β-lactamaseβ-lactamase
ampC1 β-lactamaseβ-lactamase
LRA-2β-lactamase
BcIβ-lactamase I
SMB-1hydrolyze a variety of β-lactams
BUT-1cephalosporinase and penicillinase
bacitracin
bcrAABC transporter that confers bacitracin resistance
bacArecycles undecaprenyl pyrophosphate that confers bacitracin resistance
bcrBABC transporter that confers bacitracin resistance
bacArecycles undecaprenyl pyrophosphate that confers bacitracin resistance
polymyxin
arnAmodifies lipid A with 4-amino-4-deoxy-L-arabinose (Ara4N) that confers antimicrobial peptides resistance
ugdsynthesis and transfer of 4-amino-4-deoxy-L-arabinose (Ara4N) to Lipid A that confers antimicrobial peptides resistance
rosAefflux pump/potassium antiporter system that confers resistance to cationic antimicrobial peptides
PmrFrequired for the synthesis and transfer of 4-amino-4-deoxy-L-arabinose (Ara4N) to Lipid A, which confers antimicrobial peptides resistance
chloramphenicol
fexAchloramphenicol exporter
cmlvchloramphenicol phoshotransferase
cmrAchloramphenicol exporter
cmlRchloramphenicol resistance determinant (putative transmembrane protein)
Table
Table 6. Putative exopolysaccharides production-related gene cluster in G78.
Table 6. Putative exopolysaccharides production-related gene cluster in G78.
Predicted Gene ProductsLength
[aa]		Predicted FunctionNR Description
65helix-turn-helix transcriptional regulatorCro/C1-type HTH DNA-binding domain
147Hypothetical protein
254polysaccharide biosynthesis proteinTyrosine-protein phosphatase YwqE
246Polysaccharide biosynthesis proteinChain length determinant protein
226Polysaccharide biosynthesis proteinTyrosine-protein kinase YwqD
galU297Nucleotidyl transferase UTP--glucose-1-phosphate uridylyltransferase GalU
fcl318EpimeraseGDP-L-fucose synthase
gmd331EpimeraseGDP-mannose 4,6-dehydratase
519Flippaseoligosaccharide flippase family protein
378GlycosyltransferaseGlycosyltransferase family 4 protein
379GlycosyltransferaseGlycosyltransferase group 1
407Hypothetical protein
393Galactose transferaseα-galactose transferase
wcaF187acetyltransferasecolanic acid biosynthesis acetyltransferase WcaF
412glycosyltransferaseglycosyltransferase family 4 protein
247Polysaccharide biosynthesis proteinChain length determinant protein
galU297Nucleotidyl transferaseUTP--glucose-1-phosphate uridylyl-transferase GalU
232sugar transferaseGalactosyl transferase
420glycosyltransferaseglycosyltransferase
452O-Antigen ligaseO-antigen ligase family protein
413glycosyltransferaseglycosyltransferase family 4 protein
351glycosyltransferaseGDP-mannose: glycolipid 4-β-D-mannosyltransferase
ugd443EpimeraseUDP-glucose/GDP-mannose dehydrogenase family protein
manC460Nucleotidyl transferaseMannose-6-phosphate isomerase
299glycosyltransferaseglycosyltransferase family 2 protein
463Flippaseoligosaccharide flippase family protein
325Polysaccharide pyruvyl transferasepolysaccharide pyruvyl transferase family protein
181acetyltransferaseO-acetyltransferase
500acetyltransferasemembrane-bound O-acyltransferase
296Hypothetical proteinHypothetical protein
321Actin-binding proteinActin-binding protein
528 right-handed parallel β-helix repeat-containing protein
558 right-handed parallel β-helix repeat-containing protein
362pyruvyl transferasepolysaccharide pyruvyl transferase family protein
490flippaseoligosaccharide flippase family protein
Table
Table 7. Putative biofilm formation-related genes in P. mucilaginosus G78.
Table 7. Putative biofilm formation-related genes in P. mucilaginosus G78.
KEGG OrthologyGenesProtein Product
metabolic pathway regulators
K10914crpCRP/FNR family transcriptional regulator, cyclic AMP receptor protein
K03092rpoNRNA polymerase sigma-54 factor
K03666hfqhost factor-I protein
K02398flgMnegative regulator of flagellin synthesis FlgM
K03563csrAcarbon storage regulator
K02405fliARNA polymerase sigma factor for flagellar operon FliA
diguanylate or adenylate cyclase
K21023mucRdiguanylate cyclase
K01768 adenylate cyclase
K21019sadCdiguanylate cyclase
Matrix protein-encoding genes
K13280sipWSignal peptidase I
Putaitve matrix polysaccharide synthesis genes
K00640cysEserine O-acetyltransferase
K00975glgCglucose-1-phosphate adenylyl-transferase
K02777crrsugar PTS system EIIA component
K05946tagAN-acetylglucosaminyl, diphosphoundecaprenol, N-acetyl-β-D-mannosaminyl-transferase
K00688glgPglycogen phosphorylase
K01657trpEanthranilate synthase
K01791wecBUDP-N-acetylglucosamine 2-epimerase
K00703glgAstarch synthase (glycosyl-transferring)
K21006pelAGlycoside-hydrolase family protein
K21009pelDNAD-dependent epimerase dehydratase
K21010pelEPolysaccharide biosynthesis protein PelE
K21011pelFGlycosyl transferase
K21012pelGPutative exopolysaccharide Exporter
eDNA synthesis genes
K01939purAAdenylosuccinate synthase
K01923purCPhosphoribosyl, aminoimidazole, succinocarboxamide synthase
K23269purLPhosphoribosyl, formylglycinamidine synthase subunit PurL
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Wang, D.; Poinsot, V.; Li, W.; Lu, Y.; Liu, C.; Li, Y.; Xie, K.; Sun, L.; Shi, C.; Peng, H.; et al. Genomic Insights and Functional Analysis Reveal Plant Growth Promotion Traits of Paenibacillus mucilaginosus G78. Genes 2023, 14, 392. https://doi.org/10.3390/genes14020392

AMA Style

Wang D, Poinsot V, Li W, Lu Y, Liu C, Li Y, Xie K, Sun L, Shi C, Peng H, et al. Genomic Insights and Functional Analysis Reveal Plant Growth Promotion Traits of Paenibacillus mucilaginosus G78. Genes. 2023; 14(2):392. https://doi.org/10.3390/genes14020392

Chicago/Turabian Style

Wang, Dan, Verena Poinsot, Wangxi Li, Yusheng Lu, Chong Liu, Yaying Li, Kaizhi Xie, Lili Sun, Chaohong Shi, Huanlong Peng, and et al. 2023. "Genomic Insights and Functional Analysis Reveal Plant Growth Promotion Traits of Paenibacillus mucilaginosus G78" Genes 14, no. 2: 392. https://doi.org/10.3390/genes14020392

APA Style

Wang, D., Poinsot, V., Li, W., Lu, Y., Liu, C., Li, Y., Xie, K., Sun, L., Shi, C., Peng, H., Li, W., Zhou, C., & Gu, W. (2023). Genomic Insights and Functional Analysis Reveal Plant Growth Promotion Traits of Paenibacillus mucilaginosus G78. Genes, 14(2), 392. https://doi.org/10.3390/genes14020392

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