APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2011, p. 6295–6300 Vol. 77, No. 170099-2240/11/$12.00 doi:10.1128/AEM.05005-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Global Biogeography and Quantitative Seasonal Dynamics ofGemmatimonadetes in Soil�

Jennifer M. DeBruyn, Lauren T. Nixon, Mariam N. Fawaz, Amy M. Johnson, and Mark Radosevich*Department of Biosystems Engineering & Soil Science, The University of Tennessee, Knoxville, Tennessee

Received 4 April 2011/Accepted 7 July 2011

Bacteria belonging to phylum Gemmatimonadetes comprise approximately 2% of soil bacterial communities.However, little is known of their ecology due to a lack of cultured representation. Here we present evidence frombiogeographical analyses and seasonal quantification of Gemmatimonadetes in soils, which suggests an adap-tation to low soil moisture.

Bacteria belonging to phylum Gemmatimonadetes are fre-quently detected in environmental 16S rRNA gene librariesand have been identified as one of the top nine phyla found insoils, comprising ca. 2% of soil bacterial communities (23).More recent high-throughput sequencing approaches haveconfirmed these estimates: Gemmatimonadetes relative abun-dances in large libraries (�500 sequences) from soils rangefrom 0.2% to 6.5%, with a mean of 2.2% (Fig. 1).

Despite their frequency and persistent abundance in soils,only one representative from this phylum has been isolated andcharacterized, Gemmatimonas aurantiaca strain T-27, apolyphosphate-accumulating isolate from wastewater (51).The isolation of four other Gemmatimonadetes strains hasbeen reported (10, 25); however, none of these strains havebeen characterized to date. The highest proportions of Gem-matimonadetes were found in arid soils (Fig. 1, left), suggestingan adaption to low-moisture environments. A limitation ofthese studies is that they are restricted to a singular time ortreatment.

To gain further insight into Gemmatimonadetes ecology, wetested the hypothesis that Gemmatimonadetes are more prev-alent in drier soils. A 2-fold approach was employed: first, abiogeographic analysis of Gemmatimonadetes sequences de-posited in public databases was performed to determine pat-terns of environmental distribution. Second, seasonal Gemma-timonadetes abundances were quantified in five landmanagement systems at a long-term ecological research(LTER) site in Michigan to determine relationships to edaphicfactors.

Biogeography of Gemmatimonadetes. Gemmatimonadetes16S rRNA gene sequences (�1,200 bp in length) deposited inthe RDP were used to analyze biogeographical patterns. Aneighbor-joining phylogenetic tree of 456 sequences was con-structed in MEGA v4 (44) with Fibrobacter succinogenes (ac-cession no. AJ496032) as an outgroup (Fig. 2). NCBI GenBankmetadata were retrieved to classify soils by dominant vegeta-tion and land use. The resulting tree (Fig. 2) confirmed acosmopolitan distribution of the phylum, which was not unex-

pected as it has been observed for other prokaryotic phyla (40).The sequences were grouped into three clades, here arbitrarilydesignated G1, G2, and G3. The greatest number of Gemma-timonadetes phylotypes was from soils, including grassland/prairie/pasture soil (26.4% of sequences), agricultural soil(13.1%), forest soil (11.1%), and contaminated soil (20.6%),confirming its place as one of the nine dominant soil phyla(23). Gemmatimonadetes phylotypes have also been recoveredfrom sediments (4, 19, 38), and other nonsoil locations (16, 20,24, 33, 35, 46, 49). Their presence in environments with a widerange of nutrient concentrations (e.g., eutrophic lake sedi-ments to alpine soils) and redox states (anoxic sediments orinner soil aggregates to airborne dust) suggests versatile me-tabolisms which have contributed to their cosmopolitan suc-cess.

The phylogenetic tree shown in Fig. 2 was statistically ana-lyzed in UniFrac (31) to determine possible biogeographicpatterns. A principal components analysis followed by clusteranalysis with jackknife resampling (100 permutations) revealedtwo significant (P � 0.01) environmental clusters (Fig. 3). Lin-eage-specific analysis using the G test corrected for multiplecomparisons (31) was used to determine if certain clades weresignificantly enriched in particular environments. G1, G2, andG3 have environmental patterns that are significantly nonran-dom (P � 0.001). G1 has an overrepresentation of sequencesfrom grassland and prairie soils; G2 has an overrepresentationof agricultural soils and organically contaminated soils. At afiner phylogenetic resolution (family and genus levels), mostsmall clades are randomly distributed and include members ofthe Gemmatimonadetes from a variety of environments andlocations, suggesting a generalist ecological strategy and adap-tation to a variety of environments.

Seasonal quantification of Gemmatimonadetes in KBS soils.A quantitative PCR assay targeting Gemmatimonadetes wasused to quantify this phylum in soils at the Kellogg BiologicalStation (KBS) LTER in Michigan. Samples were collectedover the 2008 season from replicate plots under five differentland treatments. Plots included two agriculture types: (i) con-ventional till and chemical input with a corn-soybean-wheatrotation (replicate plots T1R1, T1R4, and T1R5) and (ii) or-ganically managed plots with a corn-soybean-wheat rotationand a vetch winter cover crop (T4R2, T4R3, T4R5). Otherplots included early succession fields maintained by annual

* Corresponding author. Mailing address: Department of Biosys-tems Engineering & Soil Science, 2506 E. J. Chapman Drive, TheUniversity of Tennessee, Knoxville, TN 37996. Phone: (865) 974-7266.Fax: (865) 974-4514. E-mail: mrad@utk.edu.

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burning (T7R1, T7R3, T7R5) and annual mowing (T8R1,T8R2, T8R3) and a mid-succession forest plot ca. 50 yearspostagricultural abandonment (SF2). For each date, compositesoil samples (15 cores) were collected from the upper 15 cm ofeach plot and sieved through 2-mm mesh. DNA was extractedusing a PowerSoil DNA extraction kit (MoBio) according tothe manufacturer’s protocol and quantified using a NanoDropspectrophotometer.

To quantify Gemmatimonadetes seasonal abundances, aquantitative PCR (qPCR) assay targeting their 16S rRNAgenes was developed. A primer to target phylum Gemmati-monadetes had been previously designed in silico (34); how-ever, a search of this primer against the RDP databaserevealed matches to other phyla, especially Actinobacteriaand Firmicutes. For our study, primer G1G3-673F (5�-GAATGCGTAGAGATCC) was designed in Primrose (2) to tar-get clades G1 and G3. In silico, primer G1G3-673F targeted55.6% of all full-length sequences classified as Gemmati-monadetes in RDP (2,312 sequences). The primer had aperfect match to 95.6%, 0%, and 100% of sequences inclades G1, G2, and G3, respectively. G1G3-673F matchedonly 11 of 7,446 type strains from other phyla and was thusconsidered to be Gemmatimonadetes specific. Primer speci-ficity was confirmed by melt curve analysis after each run

and by sequencing PCR products (reactions described be-low) from three randomly selected samples: PCR products(ca. 243 bp) were cloned into the pGEM-T Easy vector(Promega) and sequenced on an ABI 3730 DNA analyzer(Applied Biosystems) at the Molecular Biology ResourceFacility at the University of Tennessee. Of the 27 sequencesobtained, 70% were classified as Gemmatimonadetes withRDP Classifier (�80% similarity) and represented a diver-sity comparable to that of clades G1 and G3 (�78% se-quence identity). Quantitation standards for qPCR werecreated by cloning the 16S rRNA gene from strain KBS708,a Gemmatimonadetes isolate from KBS soil (J. M. DeBruynet al., unpublished data). The 16S rRNA gene was PCRamplified using universal bacterial primers 8F and 1392Rand cloned and sequenced as described above (GenBankaccession no. HM154525). Plasmids were diluted to createan 8-point standard curve (101 to 108 copies per reaction).PCRs were done in triplicate and consisted of 12.5 �l SYBRPremix II Taq (Takara), 400 nM G1G3-637F primer, 400nM universal 16S rRNA primer 907R, and 2.5 ng templateDNA extracted from soil. Dilutions of template DNA (1:10and 1:100) were used to determine a 93% PCR efficiencywith a lower detection limit of 100 gene copies per reaction.Template-free negative controls were run in parallel. Reac-tions were performed on a Bio-Rad CFX96 thermocycler asfollows: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s,53°C for 20 s, and 72°C for 20 s. Gemmatimonadetes abun-dances were expressed as a proportion of total 16S rRNAgenes, quantified using universal bacterial primers 1055Fand 1392R as described previously (18).

Gemmatimonadetes 16S rRNA gene copies were detected inall KBS plots, indicating that they are a persistent member ofthese communities (Fig. 4). Mean relative abundances of G1and G3 ranged from 0.09% to 5.31% of total 16S rRNA genecopies. These percentages are in agreement with results fromother soil studies (Fig. 1). A nonparametric analysis of variance(ANOVA) (Kruskal-Wallis; F) revealed no differences by landtreatment. However, there were significant differences by date(F � 19.32; P � 0.0001); relative abundances in October andNovember were significantly different from those of the otherdates (post hoc multiple comparison Z test). The only signifi-cant difference between the five land treatments was observedin September, when the relative abundance of Gemmatimon-adetes was higher in agriculture and field plots (T1, T4, T7, T8)than in forest plots (SF2) (Mann-Whitney U test; P � 0.001).The relative increase in Gemmatimonadetes abundance in Sep-tember is largely driven by a decrease in total bacterial abun-dance. There were no significant changes in other parameters,and field logs do not indicate activity around this time, so it isunclear what may have driven this change. Other studies haveindicated that forest and agriculture soil communities haveseasonal dynamics (22) and that agricultural (crop and pas-ture) soils are enriched with Gemmatimonadetes compared toforest soils (22, 29).

Gemmatimonadetes abundances were compared toedaphic parameters. Percent soil moisture (�) was deter-mined gravimetrically after oven drying. Soil pH was deter-mined by combining 10 g soil with 10 ml deionized H2O.Nitrogen and carbon content were determined by using drycombustion on a Carlo-Erba C/N analyzer, and phosphorus

FIG. 1. Relative abundances of Gemmatimonadetes (as a propor-tion of total bacterial community) in soils and sediments reported inpublished 16S rRNA gene libraries. Studies are grouped by librarysize as follows: �5,000 sequences (pyrosequencing libraries) (blacksquares), 500 to 2000 (small pyrosequencing libraries and largeclone libraries) (gray squares), and �500 sequences (clone librar-ies) (white squares). Studies referenced are as follows: Antarctic drysoils (6, 14), alpine hyper arid soils (9), hot desert (8, 39, 47), alpinetundra (37), Antarctic peninsula/coastal (6, 45), semiarid soils (1,26, 32), prairies/grasslands (12, 41), pasture (22), crop agriculture(7, 22, 29, 41, 50), temperate forest (17, 22, 29, 30, 41), tropicalforest (13), moist acidic tundra (5), and freshwater sediments(27, 48).

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was measured by the molybdate-blue colorimetric proce-dure after Mehlich 3 extraction (43). Plant diversity data(Shannon H index), determined by noncrop biomass andrichness at harvest, were obtained from the KBS LTERwebsite (http://lter.kbs.msu.edu/) and are used with permis-sion. Pearson’s correlation analysis revealed that total bac-

terial 16S rRNA gene copies were significantly correlatedwith several edaphic parameters (Table 1). In contrast, theonly parameter that significantly correlated with Gemmati-monadetes abundances was moisture, which ranged from8.7% to 61.7% during this study. The highest relative Gem-matimonadetes abundances were observed during periods

FIG. 2. Neighbor-joining phylogenetic tree of 456 Gemmatimonadetes 16S rRNA gene sequences deposited in RDP (�1,200 bp in length).Fibrobacter succinogenes was used as an outgroup. Sequences are listed by GenBank accession number and colored according to the sample typeas follows: arid soils and deserts (tan), forest soil (dark green), prairie and grassland soil (light green), agricultural crop soil (yellow), alpine andtundra soil (brown), rock surfaces (black), soil contaminated with organics and hydrocarbons (pink), soil contaminated with radioactive waste (red),wastewater and activated sludge (light blue), freshwater sediments and saturated soils (medium blue), and marine sediments (navy blue). The threemajor clade divisions are labeled G1, G2, and G3.

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with the lowest soil moisture (Table 1; Fig. 5A). (Marchsamples were excluded from the analysis because much ofthe moisture was ice.) Gemmatimonadetes proportions werebest predicted by the following linear regression model: log

(%G1G3) � �1.957 � 1.363 � � (r2 � 0.113; F � 9.538;P � 0.0028), where � is percent soil moisture and %G1G3is the percentage of Gemmatimonadetes in the bacterialcommunity. Multivariate regression was attempted; how-ever, the fit of the univariate models could not be improvedby addition of the other parameters measured in this study(data not shown). Many microbial activities are known toincrease with soil moisture (15, 21, 42); however, microbialcommunity structure responses to moisture are less clear.Some studies have found shifts in soil microbial communitycomposition under different moisture regimens (11, 15),while others have reported no relationship with soil mois-ture changes (3, 29). In this study, Gemmatimonadetes rel-ative abundances were inversely correlated to moisture.There were also many Gemmatimonadetes phylotypes in li-braries from semiarid and arid soils and deserts (1, 6, 8, 9,26, 32) and higher relative abundances in these environ-ments (Fig. 1 and 2). In combination, this evidence suggestseither an adaptation to drier soils or, alternatively, thatGemmatimonadetes may be outcompeted when moisture isavailable. In microbial communities associated with soil mi-croaggregates, a much higher percentage of Gemmatimon-adetes (10 to 32% of the community) was found in the innermicroaggregates than in the whole aggregates (36). This maybe indicative of an adaptation to low-moisture (or low-oxygen) conditions typical of inner aggregates. Desiccationtolerance may have contributed to high dispersal rates, lead-ing to the observed cosmopolitan biogeography of this phy-lum.

Other studies have reported higher relative abundances ofGemmatimonadetes in soils near neutral pH than in acidic soils(28, 32, 48). We observed a slight (but not significant) rela-tionship to soil pH, which ranged from 3.7 to 6.3 over thecourse of the study (Fig. 5B). The highest relative abundanceswere observed near neutral pH, while low abundances wereobserved across all pHs, suggesting that after moisture, pHmay act as a secondary constraint on Gemmatimonadetes inthese soils.

Gemmatimonadetes bacteria have a cosmopolitan distributionin terrestrial systems, and their consistent abundance implicatethem as persistent and important members of soil communities.

FIG. 3. Principal components analysis of sequences of the phylumGemmatimonadetes shown in Fig. 2 (n � 456) by environment type.Soil types are as follows: crop agriculture (SoilAg); alpine (Soil-Alpine); arid and desert (SoilArid); grassland, prairie, and pasture (Soil-Grass); and forest (SoilForest). Also included are soils contaminated withorganics (SoilContamOrg) and radioactive wastes (SoilContamRadio),freshwater sediments and saturated soils (SedimentsFW), marine sedi-ments (SedimentsMar), rock surfaces (Rock), and wastewater and acti-vated sludge (WWAS). Significant clusters are circled.

FIG. 4. Relative abundance of Gemmatimonadetes bacteria (cladesG1 and G3) in five soil types at the Kellogg Biological Station overtime. Quantities are expressed as a percentage of the total bacterial16S rRNA genes. Land treatments include conventional agriculture(T1), organic agriculture (T4), early succession meadows (T7 and T8),and mid-succession forest (SF2) (see text for descriptions). Each pointrepresents the mean and standard deviation of 3 replicate land plots.

TABLE 1. Correlation coefficients between log transformed genequantities and soil parameters

Parametera

Pearson coefficient (r) forb:

Total bacterial 16SrRNA gene copies %G1G3

%G1G3 �0.676Moisture 0.409 �0.336pH �0.239 0.181N 0.307 �0.020C 0.295 �0.038C/N 0.049 �0.144PO4 �0.015 0.032PlantH 0.357 �0.082

a Gemmatimonadetes 16S rRNA gene quantities (%G1G3) are normalized tototal 16S quantities. C/N, carbon-to-nitrogen ratio; PlantH, Shannon diversity ofnoncrop plants.

b The significance of each correlation is indicated by italics (P � 0.05) and bold(P � 0.01) (n � 77).

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Spatial and temporal abundances of Gemmatimonadetes bacteriaacross five land treatments at KBS has demonstrated that theirrelative abundances were not related to land management butwere inversely correlated to soil moisture, suggesting an adapta-tion to drier soils.

Field samples were acquired from the Kellogg Biological StationLTER with the help of K. Roy, D. Ghosh, and K. Sides.

This project was funded by National Research Initiative CompetitiveGrant no. 2007-35319-18432 from the USDA Cooperative State Re-search, Education, and Extension Service and the University of Ten-nessee M-CERV program.

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