Introduction

Phosphorus (P) is a critical macronutrient, essential for various physiological processes in plants including photosynthesis, energy conversion, and reproduction (Shen et al. 2011). Despite its abundance in soil, the bioavailable form of P is limited, leading to extensive use of phosphate fertilizers to enhance crop yields (Lekberg et al. 2021; Peñuelas et al. 2013). However, the inefficiency of these fertilizers, due to a substantial portion of P being bound in an insoluble form, poses economic and environmental challenges (Qiao et al. 2013; Peñuelas et al. 2020). These challenges include soil acidification, eutrophication, and depletion of high-quality P resources, accentuating the need for sustainable alternatives (Harvey et al. 2009). The role of phosphate-solubilizing bacteria (PSB) in converting insoluble P into forms accessible to plants offers a promising solution (Khan et al. 2009; Sackett et al. 1908). PSBs facilitate the solubilization process through the secretion of organic anions such as acetate, lactate, and gluconate, and by the activity of enzymes like acid phosphatases and phytases (Liu et al. 2022), targeting both inorganic and organic P compounds for solubilization and mineralization (Kour et al. 2021; Yu et al. 2022).

Central to the mechanism of P solubilization is the production of gluconic acid (GA) from glucose, a process significantly influenced by the gene pqq encoding the cofactor pyrroloquinoline quinone (PQQ) (Bhanja et al. 2021). PQQ plays a pivotal role in the direct oxidation pathway of glucose to GA, particularly under aerobic conditions or when substrate availability is high (Wagh et al. 2016). This metabolic pathway not only contributes to P solubilization but also provides a competitive advantage by limiting glucose availability to other microorganisms (An and Moe 2016; Cheng et al. 2023). Besides, PQQ serves as a cofactor for glucose dehydrogenase (GDH), which catalyzes the oxidation of glucose to gluconic acid (Wagh et al. 2016). This reaction is crucial for phosphate solubilization, as gluconic acid lowers the pH and chelates cations bound to phosphate, thereby increasing its availability to plants. Despite the absence of a direct linkage between pqq gene presence and P solubilization capabilities, PQQ is recognized as a key gene in the inorganic P solubilization pathway (Joshi et al. 2023). The complexity of the pqq gene family, including variations in gene clusters and synteny among different bacterial species, underscores the intricacies and difficulties involved in accurately predicting a bacterium’s P-solubilization potential based solely on genome analysis (Wu et al. 2022).

The exploration of PSB mechanisms through genomics has been a focal point of recent studies (Li et al. 2023; Wu et al. 2022; Zeng et al. 2022), yet the identification of key genes associated with efficient P solubilization remains a challenge. This is attributed to the vast array of genes involved, especially within the pqq gene family, which complicates the rapid assessment of a newly isolated strain’s potential for effective P solubilization. Our study aims to bridge this gap by establishing a correlation between the presence of specific genes and the ability to solubilize inorganic P efficiently by studying them at once. By analyzing the genomes of 76 known PSB strains (reported in Zheng et al. 2018) and conducting a comparative genomic study on a highly efficient PSB strain, Burkholderia cepacia 51-Y1415, we seek to develop a generalized approach to predict a strain’s P-solubilizing capacity based on its pqq gene profile. This approach not only contributes to the understanding of microbial P solubilization mechanisms but also enhances the potential for utilizing PSBs in sustainable agriculture practices.

Materials and methods

Biochemical characterization and gene identification

The 76 phosphate-solubilizing bacteria (PSBs) were initially isolated from agricultural field soils near Hailun in Heilongjiang Province (47′ 26″ N, 126′ 38″ E) and Yingtan in Jiangxi Province (28′ 14″ N, 116′ 54″ E), China (Zheng et al. 2018). To evaluate the phosphate-solubilizing capabilities of the 76 soil bacterial isolates, the pH and soluble phosphate concentration were measured post-incubation. Each isolate was cultivated in 50 mL of the modified PVK medium at 30 °C for 72 h. After cultivation, supernatants were harvested via centrifugation at 4200 g for 10 min. The pH was assessed using an XL60 pH meter (Fisher Scientific, USA), and phosphate concentrations were quantified using the molybdate-blue method (Olsen et al. 1954). The presence of various pqq gene clusters within these isolates was also examined through quantitative PCR (qPCR), details of which are provided in the Supplementary Materials and Methods.

Universal primers targeting five distinct pqq gene clusters were used (Table S1, referenced from An and Moe 2016; Meyer et al. 2011; Zheng et al. 2017). qPCR assays were prepared with SYBR premix Ex Taq, BSA, and respective primers, and conducted on a LightCycler 480 System (Roche, Basel, Switzerland) following a protocol of initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s. The specificity of reactions was confirmed via melting-curve analysis. Although the primers were designed based on conserved regions across species, it is important to note that not all species may possess the targeted gene fragments, and as such, not all might be detectable by qPCR. The presence of pqq genes in this study was determined based on a threshold of CT < 30, to ensure a more tolerant detection rate for qPCR assays.

The PSB strains were isolated from agricultural field soils with varying phosphate levels in Hailun, Heilongjiang, and Yingtan, Jiangxi, China. Strains were selected based on their phosphate-solubilizing activity, determined by the molybdate-blue method, and their ability to decrease the pH of the medium. While these criteria aimed to capture a diverse range of phosphate-solubilizing capabilities, they may introduce biases by favoring strains adapted to specific soil conditions.

Genome sequencing and annotation of Burkholderia cepacian 51-Y1415

Among the 73 strains assessed for phosphate-solubilizing capabilities, Burkholderia cepacia 51-Y1415 (CCTCC AB 2017151) was identified as one of the strains exhibiting a high phosphate-solubilizing capacity. Significantly, similar to classical P-solubilizing Pseudomonas strains (Meyer et al. 2011), it possesses a complete set of five pqq gene clusters. This feature makes Burkholderia cepacia 51-Y1415 an ideal candidate for detailed genomic analysis to explore the genetic basis of its phosphate-solubilizing ability. Therefore, this strain was subjected to whole-genome sequencing and comparative genomic analysis, serving as a representative example to investigate the role of pqq genes in the phosphate-solubilization process.

Detailed protocols for DNA extraction, sequencing, and assembly, as well as comparative genomic analyses, are available in the Supplementary Materials and Methods. Briefly, the genome of Burkholderia cepacia 51-Y1415 was sequenced to provide insights into its phosphate-solubilizing genetic mechanisms. Following 24-h incubation in PVK medium at 30 °C, genomic DNA was extracted and sequenced using the Illumina HiSeq 2000 platform. Assembly was performed with the SOAPdenovo v2.01 software, producing 233 contigs across 229 scaffolds. Gene prediction was conducted using GeneMarkS v 4.28, with coding DNA sequences (CDSs) annotated through submission to various databases including GenBank and KEGG for functional gene analysis. The assembled genome sequence and associated data were deposited in DDBJ/ENA/GenBank and the Sequence Read Archive, respectively. For the comparative genomic analysis, Burkholderia cepacia 1178_BCEN, B. ubonensis MSMB1138, B. ambifaria FDAARGOS_419, and other 7 Burkholderia strains were selected based on phylogenetic proximity and known P-solubilizing capabilities (Deng et al. 2016; Riera et al. 2017). Besides, digital DNA–DNA hybridization (dDDH) values were estimated using the Genome–Genome Distance Calculator 2.1 (GGDC) to assess genomic relatedness among the strains (Meier-Kolthoff et al. 2013, 2014). Orthologous genes were identified using OrthoFinder, which facilitated the identification of shared and unique genes related to P solubilization (Emms and Kelly 2015).

Statistical analyses

Statistical analyses were conducted to establish correlations between genetic profiles and phosphate-solubilization capabilities of the studied bacterial strains. Sequence alignments and phylogenetic trees were generated using Clustal X 2.0 (Larkin et al. 2007) and MEGA 6.0 (Tamura et al. 2013). The statistical significance of differences in gene presence and phosphate solubilization efficiencies were determined through variance analyses (ANOVAs) performed with IBM SPSS Statistics 21. The specificity of PCR products was verified by melting-curve analysis, and changes in pqq gene expression were quantified using the 2−∆∆CT method, with LB medium conditions serving as the control (Livak and Schmittgen 2001). Each experiment was replicated thrice to ensure the reliability of the results.

Results

The relationship between pqq genes and bacterial phosphate-solubilizing capabilities

Analysis of 76 phosphate-solubilizing bacteria (PSB) strains demonstrated a clear link between phosphate solubilization abilities and the presence of pqq gene clusters, which correlated with decreased medium pH levels (Table 1). Strains such as Bacillus megaterium exhibited high phosphate-solubilizing capacities (96.32 ± 27.05 ug mL−1) with medium pH values ranging from 4.2 to 5.2. These strains almost invariably possessed a comprehensive set of pqqABCD genes. In contrast, strains with lower solubilization capabilities, including Arthrobacter oxydans 08-OY02 and Rhodanobacter sp. 21-Y7, displayed incomplete pqq gene clusters, which coincided with their higher medium pH levels. This pattern underscores the potential role of the pqq gene cluster in enhancing the biochemical processes that reduce pH and increase phosphate solubilization.

Table 1 Biochemical characterization and pqq gene identification of 76 PSB strains
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Genetic insights from Burkholderia Cepacia 51-Y1415

Further investigation into the genus Burkholderia revealed that, similar to classical Pseudomonas strains, Burkholderia spp. also harbored complete pqqABCDE gene clusters. A prime example is Burkholderia cepacia 51-Y1415, which demonstrated high phosphate-solubilizing capability (92.03 ug mL−1) and a medium pH of 5.00. This strain was selected for whole-genome sequencing, providing comprehensive insights into the genetic basis of its solubilization capacity (comprehensive details on the whole-genome sequencing, analysis, functional annotation, and exploration of pqq genes for strain 51-Y1415 providing in-depth insights into the genetic basis of its solubilization capacity refer to the Supplementary Materials and Methods and Supplementary Results, Table S2–S4, Figures S1–S5). The presence of pqq gene clusters was identified and compared with Pseudomonas kilonensis and ten other Burkholderia strains (Figure S6) to analyze the conserved nature of pqq genes across the genus (Fig. 1). Comparisons of pqq gene clusters across eleven Burkholderia strains highlighted the conservation of these genes within the genus. However, it was noted that the presence of complete pqq gene clusters is not universally characteristic across all strains, as evidenced by strains like B. phytofirmans 56-OY3 (P release of 3.85 ug mL−1, Table 1), which lacked a complete pqq gene cluster and showed low P-solubilization activity.

Fig. 1
figure 1

The genetic structures of thepqqoperon inBurkholderia cepacia51-Y1415 and other strains, showing the conserved nature of these genes across different species.Genetic structures of pqq operon of Burkholderia cepacian 51-Y1415, B. ubonensis, B. ambifaria, B. anthina, B. territorii, B. contaminans and B. latens with a Pesudomonas species as reference. The location and polarity of genes are showed with arrows

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Strain 51-Y1415 distinguishes itself by possessing a complete series of enzymes essential for the direct oxidative pathway of glucose, allowing for efficient conversion to gluconate and gluconate-6-phosphate (Figures S7 and S8). During the 144-h cultivation period, strain 51-Y1415 demonstrated the crucial role of 2-keto-d-gluconic acid in phosphate solubilization. The concentration of this metabolite increased alongside enhanced phosphate release and was significantly correlated with the abundance and expression levels of the pqqC gene and the entire pqqABCDE cluster (Fig. 2). Despite the presence of other various organic acids (lactic acid, acetic acid, succinic acid, oxalic acid and citric acid), only 2-keto-d-gluconic acid demonstrated a significant positive correlation with P release in the medium (P < 0.05, Table 2). The secretion of 2-keto-d-gluconic acid was also significantly correlated with the expression levels of the entire pqqABCDE gene cluster (P < 0.01, Table 2), confirming the indispensability of pqq genes in the solubilization mechanism.

Fig. 2
figure 2

Biochemical and genetic analysis of strain 51-Y1415 cultivated in PVK medium over 144 h. a Average values of triplicate measurements with standard deviations. The line graph illustrates P release into the medium, while the bar chart quantifies the concentrations of various organic acids produced. Significant differences between groups indicated by different letters were determined by ANOVA (P < 0.05). b Radar charts depicting the relative expression levels (fold change) of the pqq gene cluster in strain 51-Y1415, with expression normalized to conditions in LB medium over 144 h

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Table 2 Pearson’s correlation analysis between P release and organic acid produced by strain 51-Y1415 and its pqq gene abundance based on qPCR results
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Discussions

To elucidate the role of pqq gene clusters in bacterial phosphate-solubilizing capabilities, our examination reveals that these clusters are pivotal, not merely for their functional attributes but also for the regulatory mechanisms they impose on environmental pH modifications. Central to this discussion, the correlation between the integrity of pqq gene clusters and phosphate solubilization efficiency has been substantiated by comparing strains such as Bacillus megaterium and Arthrobacter oxydans. Notably, Bacillus megaterium strains, characterized by a robust assembly of pqqABCD genes, exhibit significantly enhanced phosphate solubilization, typically accompanied by acidification of their milieu (An and Moe 2016; Miller et al. 2010). Contrastingly, strains like Arthrobacter oxydans, which possess fragmented pqq gene clusters, show a concomitant rise in medium pH, underscoring a diminished solubilizing activity (Magnusson et al. 2004). This dichotomy not only highlights the functional imperative of these genes but also emphasizes their role in ecological adaptations, mediating microbial interactions with their abiotic environment (Cheng et al. 2023; Zheng et al. 2019).

Further stratification within the genus Burkholderia illustrates this concept robustly. For instance, Burkholderia cepacia, distinguished by its complete pqqABCDE gene assembly, showcases significant phosphate-solubilization capacities. This phenotype is mirrored across strains within the genus that share a similar genetic architecture, suggesting a conserved evolutionary strategy aimed at optimizing nutrient acquisition (Rodrı́guez and Fraga 1999; Sashidhar and Podile 2010). The synthesis and regulatory functions of the PQQ (pyrroloquinoline quinone) cofactor, facilitated by the orchestration of pqqD/pqqE, pqqB, and pqqC, underline a complex biochemical pathway critical for effective phosphate solubilization (Fig. 2, Latham et al. 2015; Magnusson et al. 2004; Velterop et al. 1995). These findings not only refine our understanding of the biochemical pathways involved but also highlight the ecological significance of these gene clusters in microbial population dynamics and nutrient cycling.

This nuanced comprehension extends beyond the operational mechanics to suggest that the presence and configuration of pqq genes are indicative of a broader ecological strategy, enabling certain microbial taxa to thrive under nutrient-limited conditions. The ecological significance of pqq gene clusters extends beyond individual strains, influencing microbial community dynamics and nutrient cycling. The disparate presence of these genes across different strains further illustrates the evolutionary plasticity and ecological ramifications of phosphate solubilization capabilities within microbial communities. These insights, supported by comprehensive genomic data available in the Supplementary materials, offer profound implications for microbial ecology and the management of biogeochemical cycles.

The pivotal role of the pqq gene cluster in enhancing phosphate solubilization efficiency emerges distinctly in our findings, particularly illustrated by strain 51-Y1415. The presence of a complete pqqABCDE gene cluster underpins the biosynthesis of the cofactor PQQ, which is crucial for the activity of PQQ-dependent glucose dehydrogenase (PQQ-GDH) (Fig. 3). This enzyme facilitates the transformation of glucose into gluconic acid, a key step in the direct oxidative pathway of glucose metabolism leading to enhanced phosphate solubilization. This pathway, predominant in strain 51-Y1415 and more effective than the alternative Entner-Doudoroff pathway, exemplifies the biochemical precision with which these bacteria adapt to phosphorus-limited environments (Holden et al. 2004; Nierman et al. 2004).

Fig. 3
figure 3

The schematic diagram of the proposed pyrroloquinoline quinone (PQQ) biosynthetic pathway and glucose to gluconic acid conversion via PQQ-GDH (PQQ-dependent glucose dehydrogenase). (Adapted from Martins et al. 2019; Mi et al. 2020). Depicts the biosynthesis of PQQ from the peptide PqqA through four conserved enzymes: PqqE (radical SAM enzyme), PqqD (peptide chaperone), PqqB (dual hydroxylase), and PqqC (eight-electron, eight-proton oxidase), alongside an alternative pathway involving PqqF/G. This process underscores PQQ’s role in the conversion of glucose to gluconic acid

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The role of 2-keto-d-gluconic acid during a 144-h cultivation period further accentuates the efficacy of this metabolic pathway. This acid, significantly correlated with phosphate release, marks a critical phase in P solubilization, overshadowing the contribution of other organic acids detected. The robust expression of the pqqABCDE cluster, particularly pqqC, during this period underscores the genetic basis for this enhanced solubilization capacity. Notably, pqqC’s function as an oxidase in the redox reactions vital for effective phosphate solubilization highlights its indispensability in this biochemical process (An and Moe 2016; Zheng et al. 2018, 2019).

Our analysis also reveals a significant variability in the presence of complete pqq gene clusters among different strains, reflecting a sophisticated regulatory mechanism that correlates gene expression with phosphate solubilization efficiency. The distinct expression dynamics of pqqC, directly associated with solubilization capacity, underscores its potential as a valuable marker for identifying efficient PSB. This gene’s expression is not only stimulated by glucose and phosphorus availability but also appears to be upregulated in response to phosphate release, suggesting a feedback mechanism that enhances its solubilization potential (Magnusson et al. 2004).

Moreover, our findings elucidate that despite variations in the biosynthetic pathways for PQQ, the essential redox reactions mediated by pqqC are critical for phosphate solubilization across diverse ecological niches. This realization expands our understanding of microbial solubilization capabilities and highlights the potential of genomic insights to inform bioaugmentation strategies aimed at improving soil fertility and plant phosphorus uptake. As such, the comprehensive genetic framework provided by the pqqC gene offers a strategic target for enhancing microbial interventions in nutrient cycling, emphasizing the need for continued exploration into the regulatory networks that govern pqq gene expression and activity.

This research advances our understanding of microbial contributions to soil phosphorus cycling and underscores the potential of leveraging genetic insights to optimize microbial capabilities for sustainable agricultural practices. Future studies should focus on the precise regulatory networks governing pqq gene expression and activity, particularly the unique contributions of pqqC, to harness these insights for enhancing soil health and crop productivity. Besides, our study’s primary limitation lies in its focus on the pqq gene cluster and the use of whole-genome sequencing, which, while precise, does not capture the full complexity of microbial community interactions. Additionally, the selection criteria for PSB strains may introduce biases, affecting the study’s conclusions. Future research should incorporate metagenomic and metatranscriptomic analyses to explore functional interactions within microbial communities and conduct field trials to assess the practical applications of our findings in diverse agricultural settings.