Performance of the introduced primer design pipeline

Prior to application of the newly designed oligonucleotide primers in quantitative real-time PCR approaches, the specificity towards the respective target genes was verified (M3). In fact, the occurrence of unspecific amplicons would cause an overestimation of the gene abundance due to false-positive signals (Sundquist, 2005). As revealed by Illumina amplicon sequencing of genomic DNA from the organic layer of site BBR, the primers preferably amplified DNA from the respective target genes, since the specificity generally reached ninety-eight percent or more. Sole exception in this regard was the acid phosphatase class A (phoN), where a marginal fraction of unspecific amplicons was detected (13%). Unfortunately the metalloenzyme Enolase (EC 4.2.1.11), which is involved in the glycolysis, comprises several conserved amino acids within the signature sequence motif of class A acid phosphatases (G-S-Y-P-S-G-H-T) (Rossolini et al., 1998). Still, the latter conserved region was targeted by the phoN primers, since alternative putative primer binding sites were lacking in the alignment.

Regarding the phoD primers, three distinct amplicons were detected after PCR that differed by approximately 60 bp, but nonetheless uniformly represented the target gene. The group of Ragot et al. (2015) did not report the occurrence of distinct amplicon sizes, while their primer system targeted a different section of the phoD gene. Thus, the variable region is likely to be situated in the upstream region of the PhoD conserved domain (W-D-D-H-E), which is involved in the coordination of metal ions in the PhoD active site of Bacillus subtilis (Rodrı ́guez et al., 2014). The length variation in the PhoD polypeptide chain likewise comes into view by visual inspection of the “Clusters of Orthologous Groups” number 3540 within the Conserved Domain Database (Marchler-Bauer et al., 2015). While the corresponding PhoD protein of Caulobacter crescentus comprises 564 amino acids, the respective enzyme of Corynebacterium glutamicum is significantly shorter (516 amino acids). In this context, the three different amplicon sizes were likewise affiliated to distinct phoD harboring microbial communities (M3). After all, the introduced phoD primers amplified the target gene from fifteen distinct bacterial and fungal phyla, which is comparable to the results described by Ragot et al.

(2015). While the authors detected a slightly lower diversity of thirteen bacterial phyla using their primer system on six different grassland soils, the sequencing depth was limited on the other hand.

In comparison, the primer system by Sakurai et al. (2008) merely amplified phoD genes from six bacterial phyla using the same soil samples as Ragot and colleagues (2015). In accordance with Tan et al. (2013), the latter primer system produced an amplification bias leading to an overrepresentation of Alphaproteobacteria in the datasets (Ragot et al., 2015). Obviously the amplified microbial diversity was strongly affected by the sample type per se. Recently, Ragot et al. (2017) performed phoD amplicon sequencing of thirty different soil samples, comprising forest and grassland ecosystems with distinct soil types, vegetation and climatic conditions. This time the authors were

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able to amplify the target gene from sixteen microbial phyla, including one archaeal and two fungal phyla. Although archaeal sequences were lacking in the underlying amplicon datasets of this study (M3), this does not necessarily constrain the performance of the newly introduced phoD primers.

Taking into account, that merely one single soil sample was applied for amplicon sequencing, the covered microbial diversity is likely to increase when several samples from distinct ecosystems are compared.

To sum up, the amplicon sequencing approach (M3) basically reproduced the results obtained by SGS (M1) to the greatest extent. The introduced oligonucleotide primers reliably covered the designated microbial key players of the individual processes, while the relative abundance of specific taxa varied slightly between both approaches. Most probably this referred to the investigated sample type (organic layer or mineral topsoil of beech forest soil), since microorganisms are known to be stratified vertically along the different soil horizons (Baldrian et al., 2012). Beyond that, the diversity analysis using the newly designed oligonucleotide primers (M3) revealed a tremendous contribution of various low-abundant taxa to the processes of soil P turnover, which were not detected during SGS (M1). Consequently, the constraints of SGS were overruled by targeted, deep amplicon sequencing.

Noteworthy, the covered microbial diversity varied substantially among the investigated genes (M3).

While phoD and pitA were amplified from fifteen, respectively, thirteen microbial phyla, the lower limit of two amplified bacterial phyla was observed for the phosphonatase (phnX) and the histidine acid phosphatase (appA). Regarding the latter one, the low diversity was barely influenced by the primer system itself, but rather by the applied database and the low number of curated sequences.

According to Jorquera et al. (2008) database entries of corresponding proteins are restricted to members of Alpha-, Delta-, Gammaproteobacteria and Acidobacteria, which were already covered by the appA primers. Although the introduced oligonucleotide primers represent a valuable tool for an in depth diversity analysis the accurate annotation of datasets depends on reliable databases, which still appears as the bottleneck of data evaluation (Hugenholtz, 2002; Nilsson et al., 2006). Thereby the validity of sequencing approaches is constrained to some extent.

In contrast to the histidine acid phosphatase, the reduced diversity of amplified phnX genes was actually induced by the primer system itself. The corresponding phosphonatase (PhnX) is a member of the Haloacid Dehalogenase (HAD) superfamily of aspartate-nucleophile hydrolases. Yet, all known phosphonatases comprise a modified version of the HAD conserved catalytic motif, which comprises four amino acids (D-12, A-14, T-16 and D-186) (Morais et al., 2000). Besides this catalytic motif, however, the individual members of the HAD superfamily show very weak sequence similarity (Morais et al., 2000). In consequence, the number and especially the diversity of the aligned sequences had to be reduced during primer design, thereby providing the opportunity to play off the

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major strength of the introduced pipeline. As revealed by metagenomics (M1), phnX was exclusively harbored by members of Proteobacteria and Firmicutes in the investigated soils. Consequently, merely sequences of the latter phyla were included in the alignment to reduce the degeneracy and ensure primer specificity (M3).

In conclusion, solely the primer systems of Sakurai et al. (2008) and Ragot and colleagues (2015) may benchmark the performance of the introduced pipeline, since further primers targeting processes of the microbial soil P turnover are lacking in literature. The former primer system was clearly outperformed regarding the amplified microbial diversity in soil samples, while the latter was on a par with the newly introduced primers. Ragot et al. (2017) recently developed another primer system for amplification of the alkaline phosphatase PhoX (phoX) from environmental samples. Since phoX was not detected during SGS (M1), the gene was obviously low abundant if not absent in the two forest soils and consequently was omitted from primer design.

Once the specificity as well as the covered microbial diversity was verified (M3), the primers were used for downstream application in qPCR approaches (M5). In this respect, the quantification of the phoN gene abundance succeeded readily in samples that were derived from the organic layer and the mineral topsoil of the two forest sites BBR and LUE. Following this, the prospective high-throughput implementation of the entire primer set enables the quantification of microbial potentials (based on DNA) and activities (based on mRNA) on the field-scale, thereby addressing questions of spatial and temporal heterogeneity regarding the microbial turnover of soil P.

In summary, the introduced pipeline represents an effective and straightforward strategy for the development of oligonucleotide primer systems that enable the specific amplification of target genes from a broad diversity of distinct microorganisms in environmental samples (M3). The pipeline is more than helpful in case of poorly conserved proteins, since it specifies which taxa to focus on whereby the degeneracy is reduced. The two-phasic approach ensures that the target genes of all microbial key players that are actually present in an investigated sample are amplified. In principle, the entire set of primers was designed for application in the two forest soils that were also used for SGS (M1). As shown by Ragot et al. (2015; 2017) degenerated primers might generally be suitable for a range of comparable ecosystems. However, it has to be considered, that predominantly target genes of ubiquitous, dominant microorganisms are amplified, while other taxa that are specific to a certain environment might remain undiscovered.

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2. Microbial community structure in soils of beech forest ecosystems along a