Novel oligonucleotide primers reveal a high diversity of microbes which drive phosphorous turnover in soil
Introduction
Phosphorus (P) is an essential macronutrient for all biota on earth as it is integral for processes of cellular bioenergetics, the formation of lipid bilayers and the genetic backup (Elser, 2012). In most natural ecosystems in addition to nitrogen (N), P is a major growth limiting factor of primary production (Vitousek et al., 2010). Although many soils contain extensive stocks of total P, the bioavailability of soluble orthophosphate, which can be used by most biota is low (Rodriguez and Fraga, 1999). Consequently P is considered as the most inaccessible and unavailable of all soil nutrients (Holford, 1997). Therefore the prominent role of microorganisms for the turnover of soil phosphorus is generally accepted, since they can increase the P availability by different means, which lead to improved P nutrition of plants and other biota (Richardson and Simpson, 2011). Especially the metabolic traits which perform the mineralization of organic-P and the solubilization of inorganic-P are of peculiar interest. Most previous studies mainly focused on the characterization of the respective enzymes using cultivated bacterial and fungal strains (Rodriguez and Fraga, 1999). However only few attempts have been made to directly target genes that drive the turnover of soil P in microbial communities derived from environmental samples without introducing the bias caused by isolation. This might be of great relevance since strains that perform well under controlled conditions might easily be outcompeted in natural environments (Rodriguez and Fraga, 1999). It was not until 2008 that Sakurai et al. (2008) developed PCR primers specifically targeting the alkaline phosphatase phoD gene. Since an amplification bias towards Alphaproteobacteria was recently observed (Tan et al., 2013), Ragot et al. (2015) introduced a new set of primers which increased the covered diversity of phoD genes in soils by the factor of 7. For genes encoding other key processes of the microbial P turnover like the mineralization of organic-P, the solubilization of inorganic-P or phosphorus uptake, primer systems are still missing.
Recently, we performed a metagenomic analysis based on whole genome shotgun (WGS) sequencing in two undistorted beech forest soils to gain insights into the microbial P turnover in soil (Bergkemper et al., 2015). The results highlighted Rhizobiales, Actinomycetales and Acidobacteriales as drivers for P turnover but also rare orders like Solibacterales contributed to the turnover of soil P. Dominating processes were the uptake of P by the phosphate inorganic transporter (Pit) and the phosphate specific transport (Pst) system, the solubilization of inorganic P and the mineralization of organic P by alkaline and acid phosphatases, phosphonatases and phytases. In contrast, Glycerol-3-phosphate transporter or C–P lyases were of minor importance. As the obtained sequencing depth of metagenomic approaches is still far away from allowing quantitative conclusions (Delmont et al., 2011), metagenomic data can serve as a starting point for a targeted primer development to answer questions about spatial and temporal distribution of microbial key players (Schöler et al., 2016). Moreover due to the important role of microorganisms for P turnover we propose that there is a need to investigate the entire set of enzymes (genes) involved in processes of microbial P mineralization, solubilization and uptake to better understand the turnover of this crucial nutrient in soil. Thus it was the aim of this study to develop primer systems suitable for high-throughput amplicon sequencing as well as quantitative real-time PCR approaches. Based on the obtained metagenomic data we chose 7 marker genes, which were highly dominant in the metagenomes of the two forest soils. Overall we covered important steps of the solubilization of inorganic P, the mineralization of organic P and the cellular P uptake. For targeting P solubilization processes, which are mainly attributed to the efflux of protons and organic anions during the oxidation of glucose and other aldose sugars (Goldstein, 1994), we targeted the quinoprotein glucose dehydrogenase (gcd) gene (Cleton-Jansen et al., 1990). With respect to the mineralization of organic-P three different classes of enzymes were investigated: (i) Nonspecific acid phosphohydrolases (NSAPs) and alkaline phosphatases (ALPs) perform the dephosphorylation of phosphoester and — anhydride bonds. Here we focused on the NSAP class A (phoN) (Rossolini et al., 1998) and the ALP PhoD (phoD) (Eder et al., 1996). (ii) The mineralization of more complex myo-Inositol-1,2,3,4,5,6-hexakisphosphates (IP6) is catalyzed by microbial phytases. Especially enzymes which are classified as 6-phytases (appA) (Golovan et al., 2000) were targeted here. (iii) The enzymatic cleavage of relatively stable carbon–phosphorus bonds, which occur in natural and synthetic organophosphonates, is performed by C–P lyases and phosphonoacetaldehyde hydrolases. The latter one (phosphonatase; phnX) (Hsieh and Wanner, 2010) was further investigated in this study. Moreover, microbes also compete for the available P with other biota as they have efficient phosphate uptake systems (Pst, Pit transporter). Therefore, we also targeted the key genes pitA and pstS (Hsieh and Wanner, 2010).
We aimed to cover a broad diversity of distinct microorganisms for the mentioned processes. The primer specificity towards the individual target genes and the diversity of the amplified microbial communities were investigated by Illumina amplicon sequencing of genomic DNA extracted from beech forest soil, which has also been included in the metagenomic analysis described above.
Section snippets
Site description and soil sampling
For this study soil samples were taken from a beech (Fagus sylvatica) dominated German forest site located in the Bavarian Rhoen Mountains near Bad Brueckenau (BBR) (50.352009°N, 9.929028°E). The stand has an average age of 120 years and is part of the International Co-operative Program for the Assessment and Monitoring of Air Pollution Effects on Forests (ICP Level II). The forest site reaches up to 850 m above sea level and the mean annual precipitation and temperature are 1031 mm respectively
Primer specificity
In total seven oligonucleotide primers were developed for genes that code for enzymes which perform major processes of the soil microbial phosphorus (P) turnover. For the entire set of primers a high degree of specificity was determined during amplification of genomic DNA extracted from forest soil. The PCR consistently generated distinct bands of the expected size while unspecific amplification was not observed (Supplementary Fig. S1). Marginal levels of smear around the central band were
Conclusion
In conclusion a new set of oligonucleotide primers was introduced in this study that covers the major processes of the soil microbial phosphorus turnover. The seven primers target genes which code for proteins involved in mineralization of various forms of soil organic-P, solubilization of inorganic-P as well as cellular P uptake. A novel strategy for primer design was applied to allow both the amplification of target genes from a broad diversity of distinct microorganisms and simultaneously
Acknowledgments
Fabian Bergkemper was supported by the German Research Foundation (DFG) (SCHL 446/20-1) in frame of the Priority Program “Ecosystem Nutrition: Forest Strategies for limited Phosphorus Resources” (SPP 1685). The project was further supported by the BMBF funded project “InnoSoilPhos”.
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