Introduction

Phage auxiliary metabolic genes (AMGs) were originally from the genomes of cellular microorganisms but have been detected in several phage genomic sequences1. The sequences of phage AMGs are often homologous to their host genes, but phylogenetic analysis can separate AMGs from hosts2. Therefore, several AMGs can be used as biomarker genes to study phage diversity in natural environments3. These AMGs include the photosynthetic genes psbA and psbD, which encode the photosystem D1 and D2 proteins4,5,6; the mazG gene, which encodes pyrophosphatase7; and the phoH gene, which is relates to phosphate metabolism8.

PhoH has some advantages as a biomarker gene over other biomarker genes for studying phage diversity, such as g239, g2010, DNA pol11 and psbA4, since those genes are restricted to specific morphological phages. The phoH gene was detected in various morphological types of phages (including siphophages, myophages and podophages) and in a wide host range (including autotrophic and heterotrophic bacteria) and even in the viruses of autotrophic eukaryotes8. By using this gene, viral phoH sequences in the Sargasso Sea and worldwide oceans were clustered into six novel groups (Groups 1~6). In addition, the distribution patterns of viral phoH assemblages in the Sargasso Sea differed with water depth and sampling time12,13. Although the phoH gene was detected in 40% of cultured marine phages and in only 4% of cultured nonmarine phages8, it appears that this gene is not suitable for assessing the diversity of phages in terrestrial environments. However, more than 400 phage phoH sequences were recently obtained from several paddy floodwaters in northeast China, and 4 specific groups and 7 subgroups of phage phoH were detected in paddy waters14. These findings suggest that this biomarker gene can be used to investigate the diversity of phages in both marine and terrestrial environments.

Wetlands are ecotones between uplands and water bodies characterized by shallow water that is permanently or periodically close to the soil surface, and they establish specific wetland plant communities15,16. Wetlands have a functional role in several key biogeochemical processes, such as pollutant degradation, nitrification, denitrification, methanogenesis, methanotrophy, and iron and sulfate reduction17,18. Thus, wetlands are usually considered the “kidney” of the earth. Since these biogeochemical processes are mostly mediated by microbes19, wetlands are regarded as the hotspots for studying microbial ecology18; however, assessments of virus or phage diversity in wetlands are limited20,21. In this study, by targeting the biomarker gene phoH, we surveyed phage diversity and distribution in several wetland sediments in northeast China using culture-independent PCR, cloning and Sanger sequencing methods. The objectives were (i) to test whether the phage phoH gene can be obtained from natural wetlands; (ii) to compare the phage phoH sequence diversity and novelty with known sequences; and (iii) to compare the difference or similarity of phage phoH assemblages in natural wetlands with that in marine and paddy fields.

Results

PCR amplification and the closest relatives of phoH clones

Using degenerate primers vPhoHf and vPhoHr, PCR products were generated from wetland sediments obtained from two coastal wetlands of Liaohekou (LHK) and Yalujiangkou (YLJK), a lake wetland of Xingkaihu (XKH), and a swamp wetland of Honghe (HH), respectively. Electrophoresis revealed several faint bands of PCR products were showed on the gel (Supplementary Fig. S1). Only bands with a target length of approximately 420 bp were excised, purified and cloned for sequencing.

In total, 219 positive clones were submitted for sequencing, but only 102 clones were confirmed as phoH sequences. A BLASTp search at the amino acid level indicated that 44 clones had the highest identities ranging from 78% to 96% to bacterial strains and environmental bacterial clones, with exception of the clone LHK-phoH-7 and clone HH-phoH-3, which had 51% identity to Spirochaetes bacterium GWB1_48_622 and Methylobacterium extorquens AM123, respectively (Supplementary Table S1). The remaining 58 clones had the highest identities ranging from 48% to 100% to environmental phage clones obtained from paddy water14 and sea waters8, as well as marine cultured Synechococcus phages24,25,26 (Supplementary Table S2).

Phylogenetic analysis of phoH sequences

A neighbor-joining phylogenetic tree was constructed from the phoH sequences isolated in this study and the phoH sequences from the closest relatives identified in Blastp (Fig. 1). The tree showed that all clones in Supplementary Table S1 formed a large cluster with a 100% bootstrap support value with bacterial phoH sequences, with the exception of two clones, HH-phoH-3 and LHK-phoH-7. Thus, these clone sequences were considered phoH sequences obtained from wetland bacteria. The clones in Supplementary Table S2 formed two clusters with bootstrap support values of 99% and 48% (data not shown in the Figure), respectively, with environmental viral clones or phages; therefore, those clones were thought to be obtained from wetland viruses. The two clones HH-phoH-3 and LHK-phoH-7 had 51% identity to the phoH sequences of bacteria (Supplementary Table S1) but formed several low bootstrap-supported branches with phoH sequences of bacteria and phages. Thus, the origin of clones HH-phoH-3 and LHK-phoH-7 could not be determined.

Figure 1
figure 1

Neighbor-joining phylogenetic tree constructed with phoH sequences obtained in wetland sediments and their closest relatives retrieved from GenBank at the amino acid level. Numbers in parentheses are the accession numbers of phoH sequences in the NCBI website. The phoH sequences from bacteria and viruses (phages) are shown in normal gray letters and bold black letters, respectively. The bootstrap support values < 50 are not shown. The scale bar represents a 10% difference in amino acid sequence.

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Through the above exclusion analysis, 58 viral phoH sequences were obtained from wetland sediments. Among them, 41, 2, 1 and 14 sequences were from sediments of YLJK, LHK, HH and XKH, respectively. The identity among 58 viral phoH sequences at amino acid level ranged from 22% to 99%, which indicates that the diversity of viral phoH gene in wetland sediments is high. To determine the phylogenetic position of viral phoH sequences in wetlands, 58 viral phoH sequences obtained in this study were used to build a phylogenetic tree with phoH sequences from cultured phages of Synechococcus, Prochlorococcus and heterotrophic bacteria, as well as the phoH sequences from autotrophic and heterotrophic bacteria (Fig. 2). All sequences were divided into three clusters. Cluster I contained 50 clones obtained in this study with phages of autotrophic bacteria, and Cluster II included 8 clones obtained in this study with phages of heterotrophic bacteria. None of the phage phoH sequences in this study fell into Cluster III. Cluster III comprised the viruses infecting eukaryotes, phages infecting several heterotrophic bacteria, a phage Ma-LMM01 of Microcystis, and some bacterial phoH sequences. Thus, all 58 viral phoH sequences obtained in this study were considered to come from phages.

Figure 2
figure 2

Neighbor-joining phylogenetic tree constructed with phage phoH sequences obtained in wetland sediments and the phoH sequences of cultured bacteria, phages and eukaryotic viruses at the amino acid level. The phoH sequences from cultured phages of Synechococcus, Prochlorococcus and heterotrophic bacteria are shown in bold black letters, while the phoH sequences from cultured bacteria are shown in normal black letters. Numbers in parentheses are the accession numbers of phoH sequences in the NCBI website. Bootstrap support values < 50 are not shown. The scale bar represents a 10% difference in amino acid sequence.

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To test whether novel groups of phage phoH sequences exist in wetlands, a phylogenetic tree at the amino acid level was constructed with the phage phoH sequences obtained in this study, marine waters8, paddy waters14, cultured phages of autotrophic and heterotrophic bacteria, and cultured viruses of autotrophic eukaryotes (Fig. 3). Based on the grouping standard reported previously8,14, 35, 1, 8 and 14 clones from wetlands fell into Groups 2, 3, 4 and 6, respectively. Within Groups 2 and 4, two new subgroups, Group 2c and Group 4c, which contained 21 and 4 clones exclusive to this study, were identified. The proportional distributions of phage phoH clones across different groups in wetland sediments, marine waters and paddy waters are summarized in Table 1, which indicates that the distribution of this biomarker gene differed among the three environments.

Figure 3
figure 3

Unrooted phylogenetic tree showed the relationships of phage phoH amino acid sequences obtained from environmental clones of wetlands in this study, marine waters (Goldsmith et al. 2011) and paddy waters (Wang et al. 2016), and cultured phages and cultured eukaryotic viruses. The size of the circles at the end of the branches is proportional to the number of clones/phages, and the small, medium and large circles represent one, four and eight clones/phages, respectively. The bootstrap support values < 50 are not shown. The scale bar represents a 10% difference in amino acid sequence.

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Table 1 Number and proportional distribution of phage phoH clones in phylogenetic groups obtained from marine waters, paddy waters and wetland soils.
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Biogeography of phage phoH sequence assemblage

To characterize the relationships of phage communities in different environments, the phage phoH sequence assemblages from two wetland sediments of this study, 10 marine waters reported previously8, and four paddy waters of northeast China14 were evaluated with nonmetric multidimensional scaling (NMDS) analysis (Supplementary Table S3). Of note, since only two phage phoH clones from LHK and one clone from HH were obtained in this study, these two wetland samples were not used for NMDS analysis. Overall, the phoH assemblages from different environments can be divided into two major groups: samples from YLJK and marine waters fell into one group, while samples from XKH and paddy waters fell into the other group (Fig. 4).

Figure 4
figure 4

A nonmetric multidimensional scaling plot showing the distribution pattern of the phage phoH assemblages obtained from different environments. Samples located close to each other on the plot are grouped by dashed circles.

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Discussion

The degenerate primers vPhoHf and vPhoHr were first designed based on alignment of the full-length phoH gene from Synechococcus phage S-PM2, Prochlorococcus phages P-SSM2 and P-SSM4, as well as Vibrio phage KVP408. Using this primer set, Goldsmith et al.8,12 successfully obtained many phage phoH sequences from various marine waters. Following that, our research group further isolated 424 sequences of phage phoH from four paddy floodwaters in northeast China, indicating that this primer set was applicable for studying phage diversity in both marine and paddy waters14. It should be noted that the water samples used for the above studies excluded host DNA using the ultracentrifuge or Millipore filtering method; we do not know whether this primer set can be applied to studies of nonwater environments. In this study, we extracted microbial DNA directly from wetland sediments as previously conducted for studying the diversity of the g23 gene of T4-type phage27. The extracted DNAs were used as a template for PCR amplification, and the results showed that 44 sequences had the highest identity to bacterial strains (Supplementary Table S1). Phylogenetic analysis also separated these sequences from phage or virus groups (Fig. 1), suggesting that these sequences originated from bacteria. This finding conflicted with Goldsmith et al.12, whom stated that this primer set does not amplify known bacterial phoH genes. To the best of our knowledge, this is the first report to reveal that host phoH genes can also be amplified with the primers vPhoHf and vPhoHr. This finding underscores the importance of excluding host phoH sequences when investigating viral or phage phoH genes in natural environments.

It should be noted that both clones LHK-phoH-7 and HH-phoH-3 had 51% identity to phoH sequences of known bacterial strains (Supplementary Table S1), and HH-phoH-3 had 53% identity to Yersinia phage phiR1-37 (YP_004934283) (Supplementary Table S2); however, both clones fell into two branches with bacteria, although the bootstrap supporting value was lower than 50% (Fig. 1). Based on this finding, the origins of clones LHK-phoH-7 and HH-phoH-3 remained unclear. The fact that 58 clones of phage phoH genes were obtained in wetland sediments after excluding potential host phoH sequences indicated that this primer set can be used to assess phage ecology in soil ecosystems.

Although AMGs of phages were derived from their host counterparts, many of these genes, such as psbA28, psbD29 and mazG7, are evolving differently from their hosts, resulting in phages and hosts residing in different phylogenetic branches or clusters. Likewise, Goldsmith et al.8 demonstrated that phoH sequences coming from phages could be separated from those coming from hosts by constructing a phylogenetic tree, and phages of cyanobacteria segregated into different evolutionary branches than phages of heterotrophic bacteria or viruses of eukaryotes. In this study, we found that the majority (86%) of the phage phoH sequences from wetlands formed a high bootstrap supporting (99%) group of Cluster I with several cyanophages of Synechococcus and Prochlorococcus. None of the phoH sequences from heterotrophic bacterial phages, eukaryotic viruses, or host-derived phoH gene sequences fell into Cluster I (Fig. 2). Thus, we deduce that the phoH sequences from this study in Cluster I are likely derived from phages of autotrophic bacteria, i.e., the phage of cyanobacteria. The remaining 14% of wetland phage phoH clones fell into Cluster II along with several phoH sequences of the heterotrophic bacterial phages; thus, the origins of these phoH sequences were considered as coming from phages of heterotrophic bacteria. Similar to our previous finding14, the number of clones belonging to cyanophages containing phoH genes was higher than that of heterotrophic bacterial phages in wetlands; thus, the AMG of phoH was suspected to be mainly carried by cyanophages rather than heterotrophic bacterial phages.

The phage phoH sequences from marine waters were clustered into six groups (Groups 1–6)8; however, only Group 2 contained reference phoH sequences of cyanophages infecting Synechococcus and Prochlorococcus8. Subsequently, our research group surveyed the genetic diversity of phage phoH in paddy waters in northeast China, and we found that the sequences from paddy waters fell into Groups 2, 3, 4, 6 and four newly designed groups (Groups α, β, γ and δ). In addition, 7 novel subgroups (Groups 2b, 3d, 3e, 6a, 6b, 6c and 6d) were established within individual groups. In this study, 58 phage phoH clones were obtained from the four wetland sediments, although the number was not comparable between samples. All sequences were clustered into Groups 2, 3, 4 and 6, and two new subgroups were detected within Groups 2 and 4 (Group 2c and Group 4c) (Fig. 3). None of the phage phoH sequences from wetland sediments were distributed into Groups α~δ and Group 5 (Table 1), which suggested that the distribution of phage phoH genes in wetlands differed from those in marine waters8 and paddy waters14.

The compositions of phage phoH sequences were determined to be different among six worldwide oceans8, among four paddy waters and between ocean and paddy environments14. In this study, we further revealed that the composition of phage phoH sequences in wetland sediments was distinctly different from that in marine waters and paddy waters (Table 1). This finding to some extent was in accord with the results of another study using the biomarker gene g23 of T4-type phages in wetlands20, which suggested that the distribution of phage biomarker genes in wetlands differed from that in other environments.

Our previous study revealed that the phage phoH sequence assemblages were grouped into marine water group and paddy water group, irrespective of where those samples were obtained14. These two groups were also confirmed in this study, and the two wetland samples were distributed into the two groups separately, that is the samples from YLJK and XKH fell into the marine water group and paddy water group, respectively (Fig. 4). This finding suggested that the phage communities evaluated by phoH sequence assemblage in coastal wetland of YLJK were similar to those in oceans, while in lake wetland of XKH, the phage communities were similar to those in paddy waters. This finding was somewhat unsurprising because the coastal wetland of YLJK was frequently influenced by tidal water of the Bohai Sea, and the lake wetland of XKH was surrounded by paddy fields. This finding supported the common opinion that “everything is everywhere, but the environment selects”30,31, which suggests that the selection effect is not only reflected in the bacterial community but also reflected in AMGs of phage genomes8.

In conclusion, this paper first demonstrated that the primers vPhoHf and vPhoHr were not highly specific to phage phoH genes. Through the exclusion analysis, 58 phage phoH clones were obtained from wetland sediments. We found that all clones were clustered into formerly known Groups 2, 3, 4 and 6. The finding of two new subclusters (Group 2c and Group 4c) containing clones exclusively obtained from wetlands in this study suggested that novel subgroups of phages inhabit wetlands. In addition, the finding that phage phoH assemblages of YLJK and XKH were similar to those in marine waters and paddy waters, respectively, suggested that the distribution patterns of phage phoH sequences were distinctly different between coastal wetland and lake wetland.

Materials and Methods

Wetland sediment sampling

Four wetland sediment samples, including two coastal wetlands of Liaohekou (LHK) and Yalujiangkou (YLJK), a lake wetland of Xingkaihu (XKH) and a swamp wetland of Honghe (HH) were collected across northeast China during July and August 2016. Briefly, approximately 2 kg of sediments within a soil depth of 0–10 cm were randomly collected from 5 sites in each wetland; the mixed samples were deposited into a polyethylene bag and transported to the laboratory at 4 °C. Then, some samples were loaded into 50 mL centrifuge tubes and stored at −80 °C for DNA extraction. The remaining sediment samples were air-dried for determination of physicochemical properties. The locations of sampling sites are shown in Supplementary Fig. S2 and some soil physiochemical properties of sediments are shown in Table 2.

Table 2 Locations of wetlands and some sediment soil properties.
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DNA extraction and PCR amplification

Soil DNA was extracted from 0.5 g of fresh sediment sample with a Fast DNA SPIN Kit for Soil (MP Biomedicals, Santa Ana, CA, USA) according to the manufacturer’s instructions. The phoH gene was amplified with the degenerate primers vPhoHf (5′-TGC RGG WAC AGG TAA RAC AT-3′) and vPhoHr (5′-TCR CCR CAG AAA AYM ATT TT-3′)8. PCR reactions were performed in a 50 μL mixture volume as reported previously (Wang et al., 2017). The PCR program parameters included initial denaturation at 95 °C for 5 min, followed by 35 cycles of 95 °C for 1 min, 53 °C for 1 min (annealing), and 72 °C for 1 min (extension), with a final extension at 72 °C for 10 min8.

Cloning and sequencing

The PCR product with a target length of 420 bp was excised from a 2% agarose gel and purified using the QIAExII Gel Extraction Kit (QIAGEN, Shanghai, China). The purified DNA was cloned into the pMD 18-T vector (TaKaRa, Dalian, China) and transformed into competent cells of Escherichia coli (E. coli) DH 5α. White clones were randomly selected, and positive clones were confirmed by PCR amplification. The plasmid DNA of the positive clone was obtained through overnight culture of E. coli DH 5α and sequenced by a commercial company (BGI, Shenzhen, China). The phoH nucleotide sequences obtained in this study were deposited in GenBank under accession numbers MH479451-MH479530 and MH479532-MH479553.

Sequence analysis

The clone sequences were translated to deduced amino acid sequences using the EMBOSS Transeq program on the European Bioinformatics Institute (EBI) website (https://www.ebi.ac.uk/Tools/st/emboss_transeq/). The closest relatives of the respective phoH clones were examined at the amino acid level by the Basic Local Alignment Search Tool (BLAST) search program within the National Center for Biotechnology Information (NCBI) website (https://blast.ncbi.nlm.nih.gov/). References of phoH sequences from cultured viruses and bacteria, as well as environmental viral clones, were retrieved from GenBank. The amino acid sequences of phoH genes were aligned using ClustalX1.8132, and the neighbor-joining phylogenetic tree was constructed by MEGA 6.0 with 1000-fold bootstrap support33. The phage phoH sequences obtained from the different biomes were defined by the number of operational taxonomic units (OTUs) at sequence divergences of 3%. The NMDS analysis was performed in R-3.4.3 with the ‘vegan’ package. This NMDS analysis employed all published environmental phage phoH sequences available at present. The sequence information for this analysis is provided in Supplementary Table S3.