Microarray Analysis of Bacterial Diversity and Distribution in Aggregates from a Desert Agricultural Soil

Biol Fertil Soils (2008) 44:1003–1011 DOI 10.1007/s00374-008-0291-5

ORIGINAL PAPER

Microarray analysis of bacterial diversity and distribution in aggregates from a desert agricultural soil

Jong-Shik Kim & Robert S. Dungan & David Crowley

Received: 1 February 2008 /Revised: 2 April 2008 /Accepted: 8 April 2008 /Published online: 7 May 2008 # Springer-Verlag 2008

Abstract Previous research has shown that soil structure distribution and organization of bacterial populations at can influence the distribution of bacteria in aggregates and, small scales (van Gestel et al. 1996; Ranjard et al. 2000; thereby, influence microbiological processes and diversity Mummey and Stahl 2004; Schutter and Dick 2002). Early at small spatial scales. Here, we studied the microbial research using cultivation-based methods demonstrated that community structure of inner and outer fractions of micro- Gram-positive bacteria were predominant in the outer aggregates of a desert agricultural soil from the Imperial fractions of microaggregates, whereas Gram-negative bac- Valley of Southern California. To study the distribution of teria occupied the inner fractions (Hattori and Hattori soil bacteria, 1,536 clones were identified using phyloge- 1976). Differences in the distribution of bacterial species netic taxon probes to classify arrays of 16S rRNA genes. in different aggregate size classes have also been revealed Among the predominant taxonomic groups were the α- using biochemical profiling and molecular methods (Kabir Proteobacteria, Planctomycetes, and Acidobacteria. When et al. 1994; Ranjard et al. 2000; Schutter and Dick 2002; compared across all phyla, the taxonomic compositions and Blackwood et al. 2006;Mummeyetal.2006). Low- distributions of bacterial taxa associated with the inner and resolution molecular methods include community profiling outer fractions were nearly identical. Our results suggest by denaturing gradient gel electrophoresis of 16S rRNA that the ephemeral nature of soil aggregates in desert genes (Kandeler et al. 2000) and terminal restriction agricultural soils may reduce differences in the spatial fragment length polymorphism analyses (Sessitsch et al. distribution of bacterial populations as compared to that 2001). The most recent work has used cloning and which occur in soils with more stable aggregates. sequence analysis of 16S rRNA genes to elucidate the relationship between inner- and outer-aggregate fractions Keywords 16S rRNA . Microaggregates . Macroarray . and microbial community structure (Mummey et al. 2006). Soil aggregate . Microbial community While many studies, so far, have revealed differences in the species richness of inner- and outer-aggregate fractions with inner-aggregates generally harboring greater species diver- Introduction sity, other studies using terminal restriction fragment length polymorphism have shown that differences in bacterial The size, stability, and chemical properties of soil aggre- community composition between the exterior and interior gates are important factors that influence the spatial fractions in some soils are marginal or not significant (Blackwood et al. 2006). This suggests that the influence of microsite location is dependent on the chemical and J.-S. Kim : D. Crowley (*) Department of Environmental Sciences, University of California, physical properties of the soils from which the aggregates Riverside, CA 92521, USA are formed. e-mail: [email protected] Here, we were interested in characterizing the difference in bacterial community structure and species composition R. S. Dungan Northwest Irrigation and Soils Research Laboratory, USDA-ARS, for inner- and outer-aggregate fractions from a desert Kimberly, ID 83341, USA agricultural soil in California’s Imperial Valley. Soils of 1004 Biol Fertil Soils (2008) 44:1003–1011 the Southwestern US are particularly difficult to manage On a mass basis, the inner-aggregate fraction accounted for due to the low amounts of organic matter and rapid 96.1% of the total aggregate mass. decomposition of organic materials that lead to low soil aggregation and poor soil structure (Martens et al. 2005). Soil DNA extraction This area receives less than 10 cm of rain per year and relies entirely on irrigation. Many farmers in the Imperial DNA was extracted from the inner- and outer-aggregate and Coachella Valley are using organic amendments and fractions using the Fast DNA Spin Kit for soil (Bio 101, green manures to improve soil structure. Hence, there is QBiogene, Vista, CA, USA). The DNA extraction proce- interest in whether organic amendments are also improving dure was conducted according to the manufacturer’s or affecting soil microbial diversity, presumably by im- protocol. The DNA was then concentrated using a Speed- proving the diversity of microhabitats that are provided by vac (AES 1000, Savant, Holbrook, NY, USA), gel eluted, improved soil aggregation using organic amendments. and purified with a QIAquick gel extraction kit (Qiagen, Nonetheless, the ephemeral nature of aggregates that are Chatsworth, CA, USA). formed in these loamy sand soils with low of amounts of clay may preclude development of distinct microbial 16S rRNA clone library construction communities since the organic matter in these fractions is relatively unprotected and short lived. To address this A clone library of the 16S rRNA genes from the inner- and question, we examined the distribution of different phylo- outer-aggregate fractions was generated by polymerase genetic groups of the inner- and outer-aggregate fractions of chain reaction (PCR) of the purified DNA templates. The the Imperial soil using a DNA approach, combined with PCR reactions were performed using the 27f (5′-GAG CTC sequencing of 16S rRNA gene clones. AGA GTT TGA TCM TGG CTC AG-3′) and 1492r (3′- CAC GYT ACC TTG TTA CGA CTT-5′) primers (Lane 1991). Reaction mixtures contained 20 pmol of each primer, 5 U of Ampli Taq DNA polymerase (Applied Materials and methods Biosystems, Foster, CA, USA), 2.5 μl of the thermophilic DNA polymerase 10× reaction buffer, 200 μM of each Soil sampling, characterization, and fractionation deoxynucleoside triphosphate, 10 μL of soil DNA com- posed of equal volumes of DNA from each soil replicate, Nine 10-cm soil cores were collected from within a 100-m2 and sterile deionized water to a final volume of 25 μl. The quadrant of a field planted with lettuce in the Imperial PCR temperature conditions were as follows: 94°C for Valley, California and were immediately transported to the 2 min, followed by 35 cycles of 94°C for 5 s, 48°C for 20 s, laboratory where they were stored at 4°C for 72 h. The and 72°C for 40 s, and a final primer extension at 72°C for Imperial soil was classified as a loamy sand (fine, smectitic, 2 min. The PCR products were visualized on 1% agarose calcareous, hyperthermic Vertic Torrifluvents). The soil has gels by UV transillumination with ethidium bromide a pH of 7.2, electrical conductivity of 20.9 dS m-1, organic staining. Bands were removed and purified with a QIA- carbon content of 1.7%, total nitrogen content of 0.1%, and quick gel extraction kit (Qiagen). particle size of 79% sand, 7% silt, and 14% clay. Prior to The PCR products were ligated into pGEM-T Easy fractionation, the soil samples were passed through a 2-mm Vector, transformed into competent Escherichia coli JM109 sieve using a Tyler Ro-Tap Sieve Shaker (Mentor, OH, (Promega, Madison, WI, USA), and plated on Luria- USA) and then pooled. Bertani (LB) agar plates for selection of transformants. The soil was treated to obtain the inner and outer fraction Construction of 16S rRNA clone library arrays was of soil aggregates as described by Ranjard et al. (1997). The accomplished by spotting near full length 16S rRNA gene inner fraction of the aggregate contains micropores with a sequences onto nylon membranes as described by Valinsky diameter of 2 to 6 μm, while the outer fraction is et al. (2002). Microdroplets (0.5 μl) of freshly grown represented by the macropores with a diameter >6 μm overnight cultures of the rRNA gene clones were added to (Hattori 1988). The entire fractionation procedure was the PCR reagents using a 384-pin solid pin replicator (V & performed twice on 30 g soil (dry weight) and involved P Scientific, San Diego, CA, USA). The PCR was 15 successive soil washes in sterile 0.8% NaCl solution performed using a PTC-200 thermal cycler (Bio-Rad). with shaking at 100 rpm on a rotary shaker for 1 min. The The PCR conditions were as follows: 94°C for 10 min; supernatants, containing bacteria that are easily washed 35 cycles of 94°C for 1 min, 65°C for 1 min and 72oC for from the outer fraction of the aggregates, were pooled and 2 min, and 72°C for 10 min. The products were printed centrifuged at 9,800×g for 20 min. The remaining soil onto dry Hybond N + membranes (GE Healthcare, Piscat- represented bacteria adhered to the inner-aggregate fraction. away, NJ, USA) with a QPix robot (Genetix, Hampshire, Biol Fertil Soils (2008) 44:1003–1011 1005

UK) using a 384-pin gridding head and a 23 nl slot pin. A signal intensities was performed with control probes. The total of 1,536 clones were spotted onto the 11×14 cm array. results were entered in an Excel file as binary data with the One membrane was made for each oligonucleotide probe criteria of that zero values had values below the maximum tested in this study. of the negative controls and 1 values having a minimum of Hybridization of the oligonucleotide probes was con- the value for the positive control sequences (Valinsky et al. ducted as described by Valinsky et al.( 2002). The DNA 2002). oligonucleotide probes (Table 1) were synthesized by Operon Biotechnologies (Huntsville, Al, USA) and were Phylogenetic analysis by 16S rRNA clone sequencing 33P end-labeled with T4 polynucleotide kinase (T4 PNK; New England Biolabs, Ipswich, MA, USA). The hybrid- To determine the phylogenetic composition of the microbial ization solutions containing 1 nM DNA oligonucleotide communities from the aggregate fractions and confirm the probe were applied to the membranes and incubated specificity of the array approach, clones were randomly overnight at 11°C. The membranes were, then, washed selected from the soil 16S rRNA gene clone libraries, and twice in 0.1× to 4× SSC buffer for 5 to 30 min at 11°C. The their nucleotide sequences were determined at the UCR membranes were then exposed overnight to Storage Center for Genomics using an ABI PRISM BigDye Phosphor Screens (GE Healthcare). Images were acquired terminator v3.0 cycle sequencing kit (Applied Biosystems, by scanning the membranes with a Typhoon model no. Foster City, CA, USA). The sequencing primers were T7 9140 (GE Healthcare). Signal intensities with background and SP6. Sequence identities were determined using the correction were obtained by using ImaGene 5.0 software basic local alignment search tool to identify the nearest (BioDiscovery, El Segundo, CA, USA). Normalization of matches with published sequences at the National Center

Table 1 16S rRNA gene targeted oligonucleotide probes used for microarray

Target group Probe or target site Sequence (antisense5′–3′) Reference

Cytophaga-Flexibacter-Bacteroides 562–580 5'-WCC CTT TAA ACC CAR T-3′ O’Sullivan et al. 2002 31F-Acidobacterium 15–31 5′-GAT TCT GAG CCA GGA TC-3′ Barns et al. 1999 5′-GAT CCT TGG CTC AGA ATC-3′ (target) A-sub-Acidobacterium 293–306 5′-GYG CCC TCT CAG GC-3′ Barns et al. 1999 5′-GCC TGA GAG GGC RC-3′ (target) G-sub-Acidobacterium 379–393 5′-GTC GTC AGG CTT GCG-3′ Barns et al. 1999 5′-CGC AAG CCT GAC GAC-3′ (target) O-sub-Acidobacterium 480–497 5′-ACG CAA GGT ACC GTC G-3′ Barns et al. 1999 5′-CGA CGG TAC CTT GCG T-3′ (target) Y-sub-Acidobacterium 484–503 5′-GAS CTT ACA AAC RGT ACC-3′ Barns et al. 1999 5′-GGT ACY GTT TGT AAG STC-3′ (target) Alpha-Proteobacteria 682–697 5′-ATT TCA CCT CTA CAC T-3′ Ashelford et al. 2002 Beta-Proteobacteria 359–378 5′-CCC ATT GTC CAA AAT TCC CC-3′ Ashelford et al. 2002 Gamma-Proteobacteria 730–747 5′-TCG AGC CAG GAG GCC GCC-3′ Eilers et al. 2001 Delta-Proteobacteria 96–115 5′-CAC CCG TGC GCC RCT YTA CT-3′ Ashelford et al. 2002 Bacillus 1295–1317 5′-GCA GCC TAC AAT CCG AAC TGA GA-3′ Daly and Shirazi-Beechey 2003 Clostridium 1421–1442 5′-CTA CGG ACT TCG GGT GTT CCC G-3′ Daly and Shirazi-Beechey 2003 Actinomycetes 226–243 5′-TAG GCC GCG GGC TCA TCC-3′ Heuer et al. 1997 5′-GGA TGA GCC CGC GGC CTA-3′ (target) Planctomycetes 886–904 5′-GCC TTG CGA CCA TAC TCC C-3′ Neef et al. 1998 Verrucomicrobia 47–63 5′-GAC TTG CAT GTC TTA WC-3′ Buckley and Schmidt 2001 Pseudomonas 289–308 5′-ACT GAT CAT CCT CTC AGA CC-3′ Widmer et al. 1998 5′-GGT CTG AGA GGA TGA TCA GT-3′ (target) Rhizobium 1246–1261 5′-TCG CTG CCC ACT GTC-3′ Cyanobacteria 781–805 5′-AAA GGG ATT AGA TAC CCC TGT AGT C-3′ Nubel et al. 1997 Ammonia-oxidizing bacteria 218–235 5′-CGG CCG CTC CAA AAG CAT-3′ Gieseke et al. 2001 Sulfate-reducing bacteria 385–402 5′-CGG CGT TGC TGC GTC AGG-3′ Rabus et al. 1996 Control probe 8–26 5′-CTGAGCCAGGATCAAAGCT-3′ Amann et al. 1995 Control probe 338–355 5′-GCTGCCTCCCGTAGGAGT-3′ Amann et al. 1995 Control probe 518–533 5′-TTACCGCGGCSGCTGG-3′ Control probe 1392–1406 5′-ACGGGCGGTGTGTAC-3′ Amann et al. 1995 1006 Biol Fertil Soils (2008) 44:1003–1011 for Biotechnology Information database (http://www.ncbi. A summary of the results for the community composi- nlm.nih.gov/). After obtaining raw sequences using Chro- tion of the inner- and outer-aggregate fractions is presented mas 2 (Technelysium Pty Ltd, Tewantin, Queensland, in Fig. 1. The proportion of bacteria in the inner fraction to Australia), which were edited with GCG Seqlab (Accelrys, that of the outer fraction was relatively similar for each San Diego, CA, USA), putative chimeric sequences were phylogenetic group. However, some differences between identified using Bellerophon (Huber et al. 2004), and the the inner and outer fractions were noted; particularly 16S rRNA sequences were aligned using the nearest sequences representing Planctomycetes were present at alignment space termination aligner and the aligned approximately 10% greater numbers in the inner fraction sequences were compared to the Lane mask (Lane 1991) than in the outer fraction, while approximately 25% more using the Greengenes web site (DeSantis et al. 2006). Rhizobia and Cyanobacteria were located in the outer Evolutionary distances were calculated with the Kimura 2- fraction than the inner fraction. Overall, our results suggest parameter, and a phylogenetic tree was constructed by the that Proteobacteria,especiallyα-Proteobacteria,predominate neighbor-joining method (Saitou and Nei 1987) with both the inner- and outer-aggregate fractions of the Imperial MEGA3 for Windows (Kumar et al. 2004). Bootstrap soil. The second most abundant group was the Planctomy- analyses of the neighbor-joining data were conducted based cetes, followed by Actinobacteria and then Acidobacteria. on 1,000 samples to assess the stability of the phylogenetic Mummey et al. (2006) examined three diverse soils and found relationships. that Proteobacteria, Gemmatimonadetes,andActinobacteria All nucleotide sequences have been deposited in GenBank were highly abundant in inner-microaggregates. In contrast, under accession numbers AY493903-48, AY493950-57, clones affiliated with Acidobacteria were enriched in libraries AY493960-83, and AY493985. derived from macroaggregate fractions but poorly represented in inner-microaggregate fractions (Mummey et al. 2006). The results of our study are in general agreement with a Results and discussion range of values that have been detected in prior surveys of soil bacterial communities. In a tabulation of multiple One of the major questions in soil microbial ecology today surveys, Proteobacteria have previously been shown to be is the identification of soil chemical and physical factors dominant in many soils, comprising an average of 50% of that function as the major determinants of soil microbial the total bacteria, with an observed range of 20% to 58% in community structure at different scales and how these are different soil types (DeSantis et al. 2006; Janssen 2006). In influenced by temporal and climatic variability, as well as the Imperial soil analyzed here, Proteobacteria comprised soil management practices. Aggregates serve as the primary 54% of the total clones, among which the α-Proteobacteria organizational structure for spatial arrangement of bacteria were predominant. In a study by Mummey and Stahl at very fine scales. In desert soils where macroaggregate (2004), Proteobacteria, also predominantly α-Proteobace- formation is impeded by low organic matter and high ria, comprised 57% of whole microaggregate clones from carbon turnover rates, there is a predominance of small microaggregates as the main organizational structure. The ephemeral nature of soil aggregates under agricultural conditions is particularly problematic in desert agricultural Planctomycetes Verrucomicrobia Inner soils where warm soil and humid conditions from irrigation, Sulfate-reducing bacteria Outer along with high fertilizer inputs and disturbance from Ammonia-oxidizing bacteria Rhizobia tillage, all favor the rapid mineralization of soil organic Pseudomonas matter (Martens et al. 2005). δ-Proteobacteria γ-Proteobacteria In this study, we focused on the microbial community β -Proteobacteria composition of inner- and outer-aggregate fractions from a α -Proteobacteria Cyanobacteria desert agricultural soil located in the Imperial Valley of Bacteriodetes California. The population distributions of the predominant Actinobacteria Clostridium phylogenetic groups were examined using a macroarray Bacillus analysis of 16S rRNA gene clones that were identified at O-Acidobacteria G-Acidobacteria taxon levels above genus using a set of 20 oligonucleotide A-Acidobacteria probes. The oligonucleotide probes were designed to target the most common taxonomic groups that have been 0 5 10 15 20 25 Abundance (%) previously described in soils (Table 1). Of the 20 probes, Fig. 1 Distribution of phylogenetic groups in the inner- and outer- 18 generated positive signals, with the exception of the aggregate fractions from the Imperial loamy sand soil as determined 31F-Acidobacteria and Y-subgroup-Acidobacteria. by macroarray analysis of the 16S rRNA clones Biol Fertil Soils (2008) 44:1003–1011 1007 undisturbed soil libraries. Other bacterial phyla that were very limited and will require systematic surveys to within the ranges described by Janssen (2006)were determine the population distributions that are normal for Acidobacteria, which comprised 8% of the clones, and soils having different soil textures and physical and Actinobacteria, which comprised 7% of the clones (Fig. 1). chemical properties. Currently, there is very little informa- Planctomycetes, which comprised 13% of the clones, were tion on the microbial community composition of most slightly outside the range limit of 7.8% as described by important agricultural soils. As such surveys are conducted, Janssen (2006). one question will be to decide the appropriate level of As discussed by Janssen (2006), certain phylum-level taxonomic resolution that should be used to describe groups are known to respond to changes in environmental microbial communities. The higher taxon probes used here conditions; for example, Verrumicrobia, which are affected provide information on broad scale structure but do not by soil moisture (Buckley and Schmidt 2001) and Acid- yield information on the individual genera and species. obacteria division 1, which are influenced by pH (Barns et In the analysis of individual clones selected from the al. 1999). However, for the most part our understanding of arrayed sequences, phylogenetic trees were generated for the determinants of microbial community structure is still three groups including Acidobacteria (Fig. 2), Proteobac-

Fig. 2 Acidobacteria diversity 535-in 100 in the inner- and outer-aggregate 537-in fractions from Imperial loamy 21-out sand soil 85 284-in 1283-out 99 529-in

95 soil clone DA008 (Y12597) soil clone C028 (AF013527) 6 100 572-in 90 85 soil clone kb2426 (Z95732) 117-out 8 100 Geothrix fermentans (U41563) Holophaga foetida (X77215)

88 7 soil clone 32-20 (Z95713) 869-out 99 100 soil clone iii1-8 (Z95729) 4 soil clone S111 (AF013560) soil clone 11-25 (Z95709) 100 soil clone RB30 (Z95720) 100 539-in 195-out

100 soil clone S023 (AF013550) 199-out 100 5 88 201-out 100 1460-out soil clone11-14 (Z95707) 1 Ellin345 (AF498727) 88 100 Ellin337 (AF498719) 2 Ellin7137 (AY673303) soil clone DA052 (Y07646)

100 Ellin6076 (AY234728) Ellin371 (AF498753) 3 355-in 78 100 soil cloneWD254 (AJ292582) ground water clone GOUTB8 (AY050595) 1526-in soil clone C002 (AF013515) 351-in

100 445-in

0.02 1008 Biol Fertil Soils (2008) 44:1003–1011 teria (Fig. 3), and Bacteria from all other phylogenetic by four clones and Actinobacteria with five clones. Another groups (Fig. 4). The 18 clones that belonged to the three clones were affiliated with Nitrospira, four with Acidobacteria were widely distributed among subdivisions unclassified Verrucomicrobia, and each two with Plancto- 3, 4, 5, 6, and 7. The 21 clones that belong to the mycetes and Chloroflexi. Proteobacteria were likewise affiliated across all of the Within each of the phylogenetic trees, there was a mostly major divisions of α-, β-, γ-, and δ-Proteobacteria and had even distribution of clones from both the inner- and outer- from 92% to 99% similarities to known species and genera. aggregate fractions within clusters of taxonomically related Forty clones from the phylogenetic tree for “other” bacteria bacteria. One exception was with the Acidobacteria in affiliated with the Firmicutes, of which ten belonged to which the four clones belonging to subdivision 3 were only Bacillus, one to Clostridia, and two unknown clones. Other associated with the inner aggregate fraction, and three major groups included the Gemmatimonadetes, represented clones belonging to subdivision 5 were associated only

Fig. 3 Proteobacteria diversity 119-out 100 of inner- and outer-aggregate 79 Massilia timonae (U54470) fractions from Imperial loamy Nitrosospira multiformis (L35509) sand soil Sterolibacterium denitrificans (AJ306683) Spirillum volutans (M34131) Thiobacter subterraneus (AB180657) 433-in 460-in 100 beta 100 533-in 100 Burkholderia cenocepacia (AF148556) 99-in Ramlibacter henchirensis (AF439400) Rubrivivax gelatinosus (D16213) 99 11-out 96 Schlegelella thermodepolymerans (AY152824) Xanthomonas melonis (Y10756) 1289-out 100 Fulvimonas soli (AJ311653) Thioalkalispira microaerophila (AF481118) 597-out 100 100 330-out Rheinheimera baltica (AJ441080) 105-out Piscirickettsia salmonis (U36941) gamma Cellvibrio japonicus (AF452103) 94 Pseudomonas fluorescens (AJ308308) 89 1451-out 100 455-in Thialkalivibrio denitrificans (AF126545) 99 99 Ectothiorhodospira shaposhnikovii (M59151) 1350-out 1508-in 100 Stella vacuolata (AJ535711) 15-out alpha 100 100 Candidatus Odyssella thessalonicensis (AF069496) 1299-in 100 Haliangium tepidum (AB062751) 604-out

100 Chondromyces apiculatus (AJ233938) 439-in Geobacter grbiciae (AF335183) Desulfuromonas palmitatis (U28172) 99 delta 95 Pelobacter venetianus (U41562) Cystobacter fuscus (M94276) 1213-in Candidatus Entotheonella palauensis (AF130847) 213-out 100 343-in 86

0.02 Biol Fertil Soils (2008) 44:1003–1011 1009

Fig. 4 Phylogenetic tree of 100 1370-out other bacteria in the inner- and 1142-in Bacillus bataviensis (AJ542508) outer-aggregate fractions from 94 Bacillus vireti (AJ542509) 83 Bacillus niacini (AB021194) Imperial loamy sand soil 7-out 92 1469-in 100 939-in 1385-out 99 Bacillus shackletonii (AJ250318) 681-out 88 100 Bacillus subtilis (AF074970) 103-out 98 449-in Firmicutes 99 Paenibacillus macerans (X60624) 94 1047-in 100 Brevibacillus borstelensis (D78456) 397-out 100 Tepidibacter formicigenes (AY245527) Thermacetogenium phaeum (AB020336) Ammonifex degensii (U34975) Thermaerobacter nagasakiensis (AB061441) 100 Thermaerobacter subterraneus (AF343566) 107-out 100 97-out Anaerolinea thermophila (AB046413) 1-out 100 197-out Chloroflexi Rubrobacter radiotolerans (X87134) 1373-out 100 100 359-in 100 1494-in Acidimicrobium ferrooxidans (U75647) Actinobacteria 100 451-in 98 Microlunatus phosphovorus (D26169) 98 341-in 100 Streptomyces albus (AJ621602) 100 1328-in Gemmatimonas aurantiaca (AB072735) 99 353-in Gemmatimonadetes 3-out 100 100 5-out 100 9-out Rhodothermus marinus (X80994) 100 441-in 100 453-in 89 99 1484-out Hongiella mannitolivorans (AY264838) Bacteroidetes 95 435-in 100 1417-in 100 Flexibacter japonensis (AB078055) 100 Cytophaga arvensicola (D12657) 99 605-out 339-in 100 1183-out Nitrospira Nitrospira moscoviensis (X82558) 100 193-out 97 Pirellula staleyi (AJ231183) 1430-in Planctomycetes 101-in OD1 100 Verrucomicrobium spinosum (X90515) Prosthecobacter dejongeii (U60012) 100 409-out 100 139-out Verrucomicrobia 100 337-in 97 357-in

0.02

with the outer aggregate fraction (Fig. 2). However, many predominant phylogenetic groups that are present. In the more clones from different samples would be needed to most thorough study to date surveying the bacterial determine if there is, in fact, a preferential distribution of compositions of inner- and outer-aggregates of three Acidobacteria or other phylogenetic groups by aggregate different soils, a total of 230 clones were analyzed location. (Mummey et al. 2006), as compared to the 1,536 clones At a higher level of resolution aimed at identification of analyzed here with the array. specific clones, our analysis of the 16S rRNA gene Currently, there are contrasting reports in the literature sequences from representative clones from the array on the degree of influence of aggregates on microbial confirmed the phylogenetic assignments provided by the community structure. Studies showing distinct community higher taxon probes. However, the limited number of development for inner and outer aggregate fractions include clones that were sequenced provided only a narrow view those by Hattori and Hattori (1976), Chenu et al. (2001), of the broad-scale taxonomic structure of the bacterial Ranjard and Richaume (2001), Sessitsch et al. (2001), and communities associated with the aggregates as compared to Mummey et al. 2006. In contrast, Blackwood and cow- the much larger data set analyzed by the array. One of the orkers (2006) showed there was a relatively high degree of difficulties in using direct sequencing to discriminate soil similarity in the taxonomic composition of bacterial bacterial communities from different samples is that large communities within soil aggregates and suggest that other numbers of clones must be analyzed to determine the components of soil structure, such as arrangement of 1010 Biol Fertil Soils (2008) 44:1003–1011 aggregates in relation to plant residues, roots, and macro- DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, pores, may be more important than aggregate size or intra- Huber T, Dalevi D, Hu P, Andersen GL (2006) Greengenes, a chimera-checked 16S rRNA gene database and workbench aggregate regions in determining the types of microbial compatible with ARB. Appl Environ Microbiol 72:5069–5072 communities present in aggregates. Nevertheless, there are Eilers H, Pernthaler J, Peplies J, Glöckner FO, Gerdts G, Amann R not yet definitive explanations for how aggregates affect (2001) Isolation of novel pelagic bacteria from the German bight community structure in different soils. Our results support and their seasonal contributions to surface picoplankton. Appl Environ Microbiol 67:5134–5142 the idea that aggregates from soils that form in sandy, desert Gieseke A, Purkhold U, Wagner M, Amann R, Schramm A (2001) agricultural soils with low organic matter content and high Community structure and activity dynamics of nitrifying bacteria turnover do not manifest distinct microbial communities in a phosphate-removing biofilm. Appl Environ Microbiol between the interior and exterior regions of the soil 67:1351–1362 Hattori T (1988) Soil aggregates as microhabitats of microorganisms aggregates. An open question is whether or not the Report of the Institute for Agricultural Research, vol. 37. Tohoku chemical properties of soil organic matter fractions vary University, Sendai, pp 23–36 for interior and exterior aggregate fractions in desert soils as Hattori T, Hattori R (1976) The physical environment in soil has been shown for other soils (Stewart et al. 2008). Soil microbiology an attempt to extend principles of microbiology to soil microorganisms. Crit Rev Microbiol 4:423–461 organic carbon can be fractionated into six different pools Heuer H, Krsek M, Baker P, Smalla K, Wellington EM (1997) that include unprotected organic matter, a biochemically Analysis of actinomycete communities by specific amplification protected pool, microaggregate protected organic matter, of genes encoding 16S rRNA and gel-electrophoretic separation and hydrolyzable and nonhydrolyzable fractions associated in denaturing gradients. Appl Environ Microbiol 63:3233–3241 Huber T, Faulkner G, Hugenholtz P (2004) Bellerophon: a program to with silt and clay (Six et al. 2002; Stewart et al. 2008). In detect chimeric sequences in multiple sequence alignments. the ephemeral aggregates that form in loamy sand desert Bioinform 20:2317–2319 soils, it would be expected that much of the organic matter Janssen P (2006) Identifying the dominant soil bacterial taxa in has relatively little physical protection within microaggre- libraries of 16S rRNA and 16S rRNA genes. Appl Environ Microbiol 72:1719–1728 gates that undergo rapid turnover and that have very little Kabir M, Chotte JL, Rahman M, Bally R, Monrozier LJ (1994) chemical protection by association with clay surfaces. Full Distribution of soil fractions and location of soil bacteria in a understanding of the factors that control community structure vertisol under cultivation and perennial grass. Plant Soil within aggregates will require combined approaches using 163:243–255 Kandeler E, Tscherko D, Bruce KD, Stemmer M, Hobbs PJ, Bardgett molecular analysis of microbial community structures in RD, Amelung W (2000) Structure and function of the soil combination with chemical analyses of soil organic carbon microbial community in microhabitats of a heavy metal polluted fractions in micro- and macroaggregates across a range of soil. Biol Fert Soils 32:390–400 soil types. Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163 Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic Acid Techniques in Bacterial References Systematics. Wiley, New York, pp 115–175 Martens DA, Emmerich W, McLain JET, Johnsen TN (2005) Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification Atmospheric carbon mitigation potential of agricultural management and in situ detection of individual microbial cells without in the southwestern USA. Soil Tillage Res 83:95–119 cultivation. Microbiol Rev 59:143–169 Mummey DL, Stahl PD (2004) Analysis of soil whole- and inner- Ashelford KE, Weightman AJ, Fry JC (2002) PRIMROSE: a computer microaggregate bacterial communities. Microb Ecol 48:41–50 program for generating and estimating the phylogenetic range of Mummey D, Holben W, Six J, Stahl P (2006) Spatial stratification of 16S rRNA oligonucleotide probes and primers in conjunction with soil bacterial populations in aggregates of diverse soils. Microb the RDP-II database. Nucl Acids Res 30:3481–3489 Ecol 51:404–411 Barns SM, Takala SL, Kuske CR (1999) Wide distribution and Neef A, Amann R, Schlesner H, Schleifer KH (1998) Monitoring a diversity of members of the bacterial kingdom Acidobacterium in widespread bacterial group: in situ detection of planctomycetes the environment. Appl Environ Microbiol 65:1731–1737 with 16S rRNA-targeted probes. Microbiol 144:3257–3266 Blackwood CB, Dell CJ, Smucker AJM, Paul EA (2006) Eubacterial Nubel U, Garcia-Pichel F, Muyzer G (1997) PCR primers to amplify communities in different soil macroaggregate environments and 16S rRNA genes from cyanobacteria. Appl Environ Microbiol cropping systems. Soil Biol Biochem 38:720–728 63:3327–3332 Buckley DH, Schmidt TM (2001) Environmental factors influencing O’Sullivan LA, Weightman AJ, Fry JC (2002) New degenerate the distribution of rRNA from Verrucomicrobia in soil. FEMS Cytophaga-Flexibacter-Bacteroides-specific 16S ribosomal DNA Microbiol Ecol 35:105–112 targeted oligonucleotide probes reveal high bacterial diversity in Chenu C, Hassink J, Bloem J (2001) Short-term changes in the spatial river Taff epilithon. Appl Environ Microbiol 68:201–210 distribution of microorganisms in soil aggregates as affected by Rabus R, Fukui M, Wilkes H, Widdle F (1996) Degradative capacities glucose addition. Biol Fertil Soils 34:349–356 and 16S rRNA-targeted whole-cell hybridization of sulfate-reducing Daly K, Shirazi-Beechey SP (2003) Design and evaluation of group- bacteria in an anaerobic enrichment culture utilizing alkylbenzenes specific oligonucleotide probes for quantitative analysis of from crude oil. Appl Environ Microbiol 62:3605–3613 intestinal ecosystems: their application to assessment of equine Ranjard L, Richaume A (2001) Quantitative and qualitative micro- colonic microflora. FEMS Microbiol Ecol 44:243–252 scale distribution of bacteria in soil. Res Microbiol 152:707–716 Biol Fertil Soils (2008) 44:1003–1011 1011

Ranjard L, Richaume A, Jocteur-Monrozier L, Nazaret S (1997) Six J, Elliot ET, Paul EA, Paustian K (2002) Stabilization mechanisms Response of soil bacteria to Hg (II) in relation to soil characteristics of soil organic matter: implications for C-saturation of soils. Plant and cell location. FEMS Microbiol Ecol 24:321–331 Soil 241:155–176 Ranjard L, Poly F, Combrisson J, Richaume A, Gourbiere F, Stewart CE, Plante AF, Paustian K, Conant RT, Six J (2008) Soil Thioulouse J, Nazaret S (2000) Heterogeneous cell density and carbon saturation: linking concept and measurable carbon pools. genetic structure of bacterial pools associated with various soil Soil Sci Am J. 72:379–392 microenvironments as determined by enumeration and DNA Valinsky L, Della Vedova G, Scupham AJ, Alvey S, Figueroa A, Yin B, fingerprinting approach (RISA). Microb Ecol 39:263–272 Hartin RJ, Chrobak M, Crowley DE, Jiang T, Borneman J (2002) Saitou N, Nei M (1987) The neighbor-joining method a new method Analysis of bacterial community composition by oligonucleotide fin- for reconstructing phylogenetic trees. Molec Biol Evol 4:406– gerprinting of rRNA genes. Appl Environ Microbiol 68:3243–3250 425 Van Gestel M, Merckx R, Vlassek K (1996) Spatial distribution of Schutter ME, Dick RP (2002) Microbial community profiles and microbial biomass in microaggregates of a silty-loam soil and the activities among aggregates of winter fallow and cover-cropped relation with the resistance of microorganisms to soil drying. Soil soil. Soil Sci Soc Am J 66:142–153 Biol Biochem 28:503–510 Sessitsch A, Weilharter A, Gerzabek MH, Kirchmann H, Kandeler E Widmer F, Seidler RJ, Gillevet PM, Watrud LS, Di Giovanni GD (2001) Microbial population structures in soil particle size (1998) A highly selective PCR protocol for detecting 16S rRNA fractions of a long-term fertilizer field experiment. Appl Environ genes of the genus Pseudomonas (sensu stricto) in environmental Microbiol 67:4215–4224 samples. Appl Environ Microbiol 64:2545–2553