Screening, cloning and expression patterns of phosphorus-related genes of Burkholderia multivorans WS-FJ9 at different phosphorus levels

As important plant growth promoting rhizobacteria, phosphate-solubilizing bacteria (PSB) fix nitrogen, dissolve potassium, promote growth, improve the soil microenvironment, and enhance soil fertility. A high-efficiency PSB strain from the pine tree rhizosphere, Burkholderia multivorans WS-FJ9, was screened in our laboratory. In this study, we using a Bio Screener fully automatic microbial growth curve meter to determine the growth of the WS-FJ9 strain in phosphate-removing medium, the growth and mineral phosphate solubilization of WS-FJ9 were obtained by Mo-Sb colorimetry and organophosphate-degradation plate assays. Second-generation sequencing technology was used to obtain genomic information and analyze possible phosphorus decomposition genes. The quantitative expression of these genes under different phosphorus levels was determined by real-time PCR. The results showed that WS-FJ9 had strong adaptability and capacity for mineral phosphate solubilization at low phosphorus levels, which is characterized by its low phosphorus induction and high phosphorus inhibition.and the amount of solubilized mineral phosphate could exceed 140 mg/L. The total length of WS-FJ9 was 7,497,552 bp after splicing, and the GC content was 67.37%. Eight phosphate-related genes were selected for further study of their expression patterns at different phosphorus levels. Among them, AP-2, GspE and GspF were only related to organic phosphorus, HlyB was only related to inorganic phosphorus, and PhoR, PhoA, AP-1 and AP-3 were related to both. The strain utilizes multiple pathways for mineral phosphate solubilization, and the degradation processes of different phosphorus sources are interrelated and independent, indicating that WS-FJ9 can adapt to different phosphorus source environments and has good application potential. levels were further analyzed to reveal the mechanism of phosphorus solubilization and plant growth promotion of strain at the molecular level.


Introduction
Phosphorus (P) is an essential nutrient element for plant growth and development. P participates in most plant metabolic processes and is one of the factors that limits crop yield. It has been reported that 74% of the cultivated land in China lacks phosphorus; 95% of the phosphorus in the soil is insoluble, and the phosphorus that can be absorbed and utilized by plants is insufficient to meet plant demands (Chen et al. 2017;Blume et al. 2010). To solve this problem, a large amount of phosphate fertilizer is often used to alleviate the problems caused by phosphorus deficiency in agricultural and forestry production. However, long-term application of phosphate fertilizer not only causes soil hardening, acidification and water pollution but also may harm human health (Chaney 2012). In addition, the raw materials of phosphate fertilizer mainly come from nonrenewable phosphate rock. Therefore, improving the utilization rate of soil insoluble phosphorus has become the primary limitation in agricultural and forestry development.
Phosphate-solubilizing bacteria (PSB) in the rhizosphere have attracted increasing attention because of their advantages, such as environmental protection, low cost, and high efficiency (Khan et al. 2007;Owen et al. 2015). Over the years, many studies have been carried out on the characteristics and mechanisms of PSB (Lin et al. 2015). It is generally believed that PSB can dissolve insoluble inorganic phosphates by secretion of small molecule organic acids, proton exchange, and complexation and degrade organic phosphorus by secretion of degradation enzymes such as phosphatases and proteases (Qin et al. 2019).
At present, research on the phosphorus solubilization genes of PSB is mainly focused on genes related to the degradation of insoluble inorganic phosphorus. For example, Kim and others transferred a phosphorus solubilization gene (PQQ) to Escherichia coli transgenically, which significantly improved the phosphorus solubilization efficiency of the E. coli (Kim et al. 2003). Song et al. cloned the microbial phosphate solubilization gene GabY from the red soil of Guangxi Province and induced its expression in E. coli, and the recombinant E. coli degraded insoluble inorganic phosphorus ). Research on organic phosphorus degradation genes is focused on phosphatase genes, mainly including acid phosphatase, alkaline phosphatase and inositol hexaphosphatase genes. Fraga et al. cloned the acid phosphatase gene napA and transferred it to Burkholderia cepacia IS-16. It was found that the activity of acid phosphatase and the phosphorus dissolving activity of this strain were significantly increased in vitro (Fraga et al. 2001). Due to the wide variety of PSB, there are still relatively few studies on phosphate solubilization pathways and expression patterns of phosphate solubilization genes, which need to be further studied.
The Burkholderia cepacia complex (Bcc) is widely distributed in the soil and is an important component of plant growth promoting rhizobacteria (PGPR) that can promote the growth of wheat, rice, poplar and other plants (Nishiyama et al. 2010;V. Trân Van et al. 2000;Li et al. 2014 (Min et al. 2019) but also produce a variety of antibacterial substances Zhang et al. 2018), inhibit soil-borne diseases, and antagonize a variety of plant pathogens (Ren et al. 2006). At the same time, Bcc, as a bioremediation agent, can decompose herbicides and pesticides that are difficult to degrade (Li et al. 2013). To date, there are 17 genotypes of Bcc, and Burkholderia multivorans belongs to Bcc genotype II. Some of its strains are human pathogens (Varga et al. 2012), while others have antagonistic effects against some plant pathogens (Sijam et al. 2005). At present, the research on this bacterium is mostly focused on its antagonistic substances, but there have been few reports on the molecular mechanisms for phosphate solubilization of this bacterium.
A high-efficiency PSB from the rhizosphere of pine trees, B. multivorans WS-FJ9, was screened in our laboratory. Previous studies have shown that WS-FJ9 has a good ability to promote plant growth, dissolve phosphorus and antagonize a variety of plant pathogenic bacteria (Hou et al. 2012), and preliminary studies have explored its degradation mechanism of inorganic phosphorus by transcriptome analysis (Zeng et al. 2017). However, the ability of this strain to degrade organic phosphorus and the expression levels of phosphorus solubilization genes under different phosphorus levels are not clear. In this study, the growth and mineral phosphate solubilization ability of WS-FJ9 under different phosphorus levels were determined to explore its phosphorus solubilization characteristics when presented with different phosphorus sources. To precisely locate the phosphate solubilization genes and systematically understand the phosphate solubilization pathway of this strain, the second-generation genome of this strain was sequenced to mine genes related to phosphorus solubilization. Furthermore, the expression patterns of these genes under different phosphorus levels were further analyzed to reveal the mechanism of phosphorus solubilization and plant growth promotion of this strain at the molecular level.

Strain and culture conditions
The phosphate-solubilizing bacterium B. multivorans WS-FJ9 was isolated from the rhizosphere soil of a 28-year-old slash pine (Pinus elliotii) in Guangzhuang Forestry Center, Fujian, China (Hou et al. 2012) and deposited in the Chinese Center for Type Culture Collection (Accession No. CCTCCM2011435). The Genomic data uploaded to NCBI (Accession No. JAAGNW000000000). After WS-FJ9 was activated, a single colony was removed and transferred into LB medium and cultured at 28 ℃ for 10 hours at 200 rpm. Then, 1% of the WS-FJ9 strain seed solution was transferred to a phosphate solubilizing medium with different exogenous phosphorus levels. Samples were transferred from bottles into a 100 µL 96-well plate with a liquid pipette, and each sample was repeated 3 times.
The 96-well plate with the bacterial solutions was placed in a Bio Screener automatic microbial growth curve instrument for determination of their OD values.

Sample preparation for genome sequencing
The WS-FJ9 strain was washed 3-4 times with 1*PBS until the supernatant was clear. The samples were quickly frozen in liquid nitrogen and stored at -80 ℃. Three tubes of samples were prepared, each of which was approximately 0.5 g. The samples were sent to a sequencing company (Pasano, Shanghai), and high-quality samples of B. multivorans WS-FJ9 total DNA were extracted and sequenced.

RNA extraction and reverse transcription
A bacterial total RNA extraction kit and reverse transcription kit were used according to the manufacturer's instructions (Vazyme, Nanjing).

Quantitative real-time PCR
To understand the phosphate-solubilization mechanism of the WS-FJ9 strain from multiple angles, the phosphate solubilization genes from different phosphate solubilization pathways were selected to detect their relative expressions by qRT-PCR, including the PhoR gene responsible for sensing the two-component system of external phosphorus sources, which can sense the concentration of soluble phosphorus in the outside world; phosphatase genes AP-1, AP-2, AP-3, which encode the acid phosphatase gene and are important enzymes regulating phosphorus metabolism; organic acid genes, such as PhoA, which encode alkaline phosphatase, which can mineralize the activity of organic acids; HlyB, GspE, and GspF, are related to the secretion system responsible for secreting organic acids and enzymes into the environment. Primer 5.0 software was used to design specific primers for quantitative real-time PCR. The specific primers were designed as follows:  CA, USA) were used for qRT-PCR. The kit instructions were followed, and the reaction mixture was prepared on ice.

Statistical analyses
Statistical analyses were carried out using Excel 2010 (Microsoft Corporation, Redmond, WA, USA) and SPSS software (ver. 23.0 IBM Corp., Armonk, NY, USA). Comparisons among treatments were analyzed for significance using Duncan's new multiple range test.

Growth of Burkholderia multivorans WS-FJ9 in phosphorus-solubilizing medium with different exogenous phosphorus concentrations
The growth of B. multivorans WS-FJ9 in phosphorus-solubilizing medium with different concentrations of exogenous phosphorus was examined to better understand the phosphate solubilizing ability of WS-FJ9 and determine its phosphate solubilization characteristics. The WS-FJ9 reached the logarithmic phase preferentially under low phosphorus conditions, and the sequence was as follows: 0 mmol/L≈1 mmol/L > 5 mmol/L > 10 mmol/L > 20 mmol/L (Fig. 1) . It is possible that high phosphorus concentrations may hinder the early growth of WS-FJ9. However, whether or not the phosphorus source is sufficient restricts the total number of viable bacteria in the later stage. The number of colonies in the later stage of logarithmic growth was directly proportional to the phosphorus content, that is, 20 mmol/L> 10 mmol/L> 5 mmol/L> 1 mmol/L > 0 mmol/L.

Detection of phosphate-solubilizing capacity of Burkholderia multivorans WS-FJ9
The phosphate degradation by strain WS-FJ9 on a phosphate solubilizing plate was shown in Fig

Genome assembly and annotation of Burkholderia multivorans WS-FJ9
To explore its ability to solubilize phosphate, the genome of strain WS-FJ9 was detected. The original reads of the WS-FJ9 strain obtained by sequencing were subjected to quality control, quality evaluation and assembly. The assembled genome characteristics and genome structural prediction are shown in Table 2 and Table 3

Functional annotation of protein-encoding genes of Burkholderia multivorans WS-FJ9
The functional annotation of protein-encoding genes is at the core content of whole genome analysis of microorganisms, and it can reveal the biological activities of a species at the molecular level.
According to the functional annotation results of the genomic protein coding genes of WS-FJ9 (Table   4), 6271 protein-encoding genes were compared in the NR database, and 106 protein-encoding genes were compared in the KEGG database. The differences were mainly related to the volume and focus of the databases.

Phosphate-related genes and metabolic pathways of Burkholderia multivorans WS-FJ9
The genomic data of strain WS-FJ9 revealed many types of phosphate-related genes, which mainly include genes involved in organic acid synthesis and secretion, phosphatase synthesis and secretion and the sensing of external phosphorus sources, related to the regulatory system (Table 5). According to KEGG analysis of this strain, a total of 106 genes of the WS-FJ9 genome were annotated, enriched in 32 metabolic pathways, and can be divided into 9 types. Among them, the pathways with the most genes were mainly signaling and cellular processes (29), genetic information processing (24), metabolism (21), carbohydrate metabolism (18), and metabolism of cofactors and vitamins (13).
There were more than ten pathways related to phosphorus degradation: the two-component system ( Fig. 4), bacterial secretion system (Fig. 5), phosphonate and phosphinate metabolism, inositol phosphate metabolism, pentose phosphate pathway, glycerol phospholipid metabolism, oxidative phosphorylation, ABC transport system, phosphotransferase system (PTS), phosphatidylinositol signal system, phospholipase D signal pathway, etc. Therefore, the WS-FJ9 strain has the same traditional phosphorus degradation pathway as most of the PSB and has additional phosphorus solubilization pathways.

FJ9 strains under different exogenous phosphorus conditions
The eight phosphate solubilization genes from different phosphate solubilization pathways were detected their relative expressions by qRT-PCR. The result showed that the expression patterns of the eight genes can be roughly divided into 4 categories: (1) PhoR was sensitive to the phosphorus concentration and had the same expression pattern in both organic and inorganic phosphorus media (Fig. 6 B). The expression level of the gene was high under phosphorus-free conditions (approximately 1.0 times), but it was low after the addition of exogenous phosphorus, and there was no significant difference in expression (approximately 0.2 mol / 0.4 times).
(2) PhoA (Fig. 6A), AP-1 (Fig. 6C), and AP-3 (Fig. 6E) were sensitive to the phosphorus concentration but had different expression patterns in both organic and inorganic phosphorus media. In the organic phosphorus medium with a low exogenous phosphorus concentration (0-1 mmol / L), the three genes were upregulated 2.2 times, 1.1 times, and 1.5 times, respectively. When the concentration of exogenous phosphorus was high, these genes were downregulated 0.4 times, 0.5 times, and 0.5 times, respectively. In the inorganic phosphorus medium, the expression level of PhoA was slightly higher than in the absence of phosphorus and under high phosphorus and slightly lower at other levels; the difference, at approximately 0.5 times, was not significant. With the increase in exogenous phosphorus concentration, the expression of AP-1 and AP-3 decreased, but the differences were not significant. The above three genes had slightly larger responses to the exogenous phosphorus content in the organic phosphorus medium.
(3) AP-2 (Fig. 6D), GspE (Fig. 6G), and GspF (Fig. 6H) were sensitive to the phosphorus concentration only in organic phosphorus medium. There was no difference in the expression of these genes in the inorganic phosphorus medium (all approximately 1.0 times). In the organic phosphorus medium, AP-2 had a low expression (1-1.3 times) when the exogenous phosphorus content was low (0-5 mmol / L) and a high expression (2.7 times) when the exogenous phosphorus content was high. The expression patterns of GspE and GspF were as follows: when the exogenous phosphorus concentration was low (0-1 mmol / L), and the level of gene expression was high, up to 1.7 times and 2.4 times, respectively; when the exogenous phosphorus concentration was high, and the one of gene expression was low, approximately 0.8 and 0.5 times, respectively.
(4) HlyB was only sensitive to the phosphorus concentration in inorganic phosphorus medium (Fig.   6F). In the organic phosphorus medium, that gene showed no differences To explore the phosphorus solubilization mechanism of the strain, the whole-genome shotgun strategy was used along with second-generation sequencing technology (Next-Generation Sequencing, NGS). The total length of the assembled genome was 7,497,552 bp, and the GC content was 67.37%, which is relatively high, so the gene density is relatively high, and the ability of this strain to resist high temperatures and an alkaline environment is also strong (Zhou et al. 2014).
Through KEGG pathway analysis, multiple pathways related to dephosphorization were found in this strain, including a two-component system, bacterial secretion system, phosphonate and phosphinate metabolism, inositol phosphate metabolism, pentose phosphate pathway, glycerol phospholipid metabolism, oxidative phosphorylation, ABC transport system, phosphotransferase system (PTS), phosphatidylinositol signal system, phospholipase D signal pathway, etc. The existence of a twocomponent system and bacterial secretion system strongly explained the phosphate solubilizing abilities of the strains that exhibited "low phosphorus induction and high phosphorus inhibition" in previous research, as well as the secretion of organic acids and phosphatase to degrade insoluble inorganic and organic phosphorus.
In the previous studies on the phosphate solubilization pathways of PSB mainly focused on the analysis and discussion of the pathways related to organic acid and phosphatase synthesis (Yin et al. 2011 (Zeng et al. 2017;Geng et al. 2019). On the basis of the above, this study has increased our understanding of two phosphate solubilization systems that "communicate" between the strain and the outside environment, namely, the two-component system and the bacterial secretion system.
Alexander found that there were significant differences in the expression of the PhoA, PhoC,  other in the degradation of organic phosphorus and inorganic phosphorus but also support each other, which enables strain WS-FJ9 to adapt to different phosphorus source environments. Therefore, the strain has good application prospects. In the future, we can examine phosphate-sensing genes or secretion system genes to gain a more comprehensive and in-depth understanding of the phosphorus solubilization mechanism of this strain. In addition, the genomic data obtained from strain WS-FJ9 can be used to accurately locate relevant genes, which provides a good basis for future research on energy and material metabolism pathways and the related regulatory mechanisms.

Acknowledgments
We are grateful to Dr. Long-Jiao Hu for her suggesting in manuscripts .

Authors' contributions
YQL performed and studied most of the experiments in manuscripts and analyzed experimental data and drafted linked content of the manuscript. XQW as research supervisor of YQL was involved in planning of research work; analysis and interpretation of data; WLK participated in the grammar and experimental planning of the manuscript; YHW and WHL and XLX were involved in the planning and execution of the research work; analysis and interpretation of the data; All the authors agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors read and approved the final manuscript.