Functional Metagenomics Approach for the Discovery of Novel Genes Encoding Phosphatase Activity.

Phosphate release from inorganic and organic phosphorus compounds can be enzymatically mediated. Phosphate-releasing enzymes, comprising acid and alkaline phosphatases, are recognized as useful biocatalysts in applications such as plant and animal nutrition, bioremediation, and diagnostic analysis. Here, we describe a functional metagenomics approach enabling rapid identification of genes encoding these enzymes. The target genes are detected based on small- and large-insert metagenomic libraries derived from diverse environments. This approach has the potential to unveil entirely new phosphatase families or subfamilies and members of known enzyme classes that hydrolyze phosphomonoester bonds such as phytases. Additionally, we provide a strategy for efficient heterologous expression of phosphatase genes.

The ubiquitous soil verrucomicrobial clade 'Candidatus Udaeobacter' shows preferences for acidic pH.

Members of the verrucomicrobial clade 'Candidatus Udaeobacter' rank among the most dominant bacterial phylotypes in soil. Nevertheless, despite this global prevalence, in-depth analyses with respect to pH preferences of 'Ca. Udaeobacter' representatives are still lacking. Here, we utilized a recently designed primer pair, specifically targeting 'Ca. Udaeobacter', to investigate links between soil pH and the abundance as well as phylotype composition of this largely unexplored verrucomicrobial clade. Based on 150 forest and 150 grassland soils, comprising a broad pH range, we determined the highest total abundance of 'Ca. Udaeobacter' in strongly acidic soil (pH, ~5.1) and, noteworthy, in ultra-acidic soil (pH < 3.5) and at a pH ≥ 7, its abundance drastically declined. When we analysed the six most dominant amplicon sequence variants affiliated with 'Ca. Udaeobacter' separately, their abundances peaked within a pH range of approximately 4.7-5.2, and only in one case at slightly acidic soil pH (pH, 6.1). Our study benefits from a combination of quantitative real-time PCR and high-throughput amplicon sequencing, enabling for the first time a highly specific abundance analysis of representatives affiliated with 'Ca. Udaeobacter', which revealed that this globally abundant verrucomicrobial clade shows preferences for acidic soil.

Sasso Pisano Geothermal Field Environment Harbours Diverse Ktedonobacteria Representatives and Illustrates Habitat-Specific Adaptations.

The hydrothermal steam environment of Sasso Pisano (Italy) was selected to investigate the associated microbial community and its metabolic potential. In this context, 16S and 18S rRNA gene partial sequences of thermophilic prokaryotes and eukaryotes inhabiting hot springs and fumaroles as well as mesophilic microbes colonising soil and water were analysed by high-throughput amplicon sequencing. The eukaryotic and prokaryotic communities from hot environments clearly differ from reference microbial communities of colder soil sites, though Ktedonobacteria showed high abundances in various hot spring samples and a few soil samples. This indicates that the hydrothermal steam environments of Sasso Pisano represent not only a vast reservoir of thermophilic but also mesophilic members of this Chloroflexi class. Metabolic functional profiling revealed that the hot spring microbiome exhibits a higher capability to utilise methane and aromatic compounds and is more diverse in its sulphur and nitrogen metabolism than the mesophilic soil microbial consortium. In addition, heavy metal resistance-conferring genes were significantly more abundant in the hot spring microbiome. The eukaryotic diversity at a fumarole indicated high abundances of primary producers (unicellular red algae: Cyanidiales), consumers (Arthropoda: Collembola sp.), and endoparasite Apicomplexa (Gregarina sp.), which helps to hypothesise a simplified food web at this hot and extremely nutrient-deprived acidic environment.

Novel Soil-Derived Beta-Lactam, Chloramphenicol, Fosfomycin and Trimethoprim Resistance Genes Revealed by Functional Metagenomics.

Antibiotic resistance genes (ARGs) in soil are considered to represent one of the largest environmental resistomes on our planet. As these genes can potentially be disseminated among microorganisms via horizontal gene transfer (HGT) and in some cases are acquired by clinical pathogens, knowledge about their diversity, mobility and encoded resistance spectra gained increasing public attention. This knowledge offers opportunities with respect to improved risk prediction and development of strategies to tackle antibiotic resistance, and might help to direct the design of novel antibiotics, before further resistances reach hospital settings or the animal sector. Here, metagenomic libraries, which comprise genes of cultivated microorganisms, but, importantly, also those carried by the uncultured microbial majority, were screened for novel ARGs from forest and grassland soils. We detected three new beta-lactam, a so far unknown chloramphenicol, a novel fosfomycin, as well as three previously undiscovered trimethoprim resistance genes. These ARGs were derived from phylogenetically diverse soil bacteria and predicted to encode antibiotic inactivation, antibiotic efflux, or alternative variants of target enzymes. Moreover, deduced gene products show a minimum identity of ~21% to reference database entries and confer high-level resistance. This highlights the vast potential of functional metagenomics for the discovery of novel ARGs from soil ecosystems.

Metagenome-Assembled Genome Sequences of a Biofilm Derived from Marsberg Copper Mine.

We sequenced the metagenome of a biofilm collected near a leachate stream of the Marsberg copper mine (Germany) and reconstructed eight metagenome-assembled genomes. These genomes yield copper resistance through Cu(I) oxidation via multiple copper oxidases and extrusion through copper-exporting P-type ATPases.

Metagenome-Assembled Genome Sequences from an Anoxygenic Photosynthetic Consortium Involved in Sulfur Cycling.

We sequenced the metagenome of an anoxygenic photosynthetic consortium originating from pond water and reconstructed four metagenome-assembled genomes. These genomes include Desulfocapsa, Paludibacter, Lamprocystis, and Rhodocyclaceae representatives and indicate the presence of genes for dissimilatory sulfate reduction and oxidation of reduced sulfur compounds.

Metagenome Sequences of a Wastewater Treatment Plant Digester Sludge-Derived Enrichment Culture.

We sequenced the metagenome of a microbial community enriched under strictly anaerobic conditions from wastewater treatment plant-derived digester sludge. The metagenomic analysis of the enrichment revealed that Acetobacterium and methanogenic archaea belonged to the dominant prokaryotes, and genes encoding components of the Wood-Ljungdahl pathway were identified.

Globally Abundant "Candidatus Udaeobacter" Benefits from Release of Antibiotics in Soil and Potentially Performs Trace Gas Scavenging.

Verrucomicrobia affiliated with "Candidatus Udaeobacter" belong to the most abundant soil bacteria worldwide. Although the synthesis of antibiotics presumably evolved in soil, and environmental pollution with antimicrobials increases, the impact of these complex molecules on "Ca Udaeobacter" remains to be elucidated. In this study, we demonstrate that "Ca. Udaeobacter" representatives residing in grassland as well as forest soil ecosystems show multidrug resistance and even take advantage of antibiotics release. Soils treated with up to six different antibiotics exhibited a higher "Ca. Udaeobacter" abundance than corresponding controls after 3, 8, and 20 days of incubation. In this context, we provide evidence that "Ca. Udaeobacter" representatives may utilize nutrients which are released due to antibiotic-driven lysis of other soil microbes and thereby reduce energetically expensive synthesis of required biomolecules. Moreover, genomic analysis revealed the presence of genes conferring resistance to multiple classes of antibiotics and indicated that "Ca. Udaeobacter" representatives most likely oxidize the trace gas H2 to generate energy. This energy might be required for long-term persistence in terrestrial habitats, as already suggested for other dominant soil bacteria. Our study illustrates, for the first time, that globally abundant "Ca. Udaeobacter" benefits from release of antibiotics, which confers advantages over other soil bacteria and represents a so-far overlooked fundamental lifestyle feature of this poorly characterized verrucomicrobial genus. Furthermore, our study suggests that "Ca. Udaeobacter" representatives can utilize H2 as an alternative electron donor.IMPORTANCE Soil bacteria have been investigated for more than a century, but one of the most dominant terrestrial groups on Earth, "Candidatus Udaeobacter," remains elusive and largely unexplored. Its natural habitat is considered a major reservoir of antibiotics, which directly or indirectly impact phylogenetically diverse microorganisms. Here, we found that "Ca. Udaeobacter" representatives exhibit multidrug resistance and not only evade harmful effects of antimicrobials but even benefit from antibiotic pressure in soil. Therefore, "Ca. Udaeobacter" evidently affects the composition of soil resistomes worldwide and might represent a winner of rising environmental pollution with antimicrobials. In addition, our study indicates that "Ca. Udaeobacter" representatives utilize H2 and thereby contribute to global hydrogen cycling. The here-reported findings provide insights into elementary lifestyle features of "Ca. Udaeobacter," potentially contributing to its successful global dissemination.

Distribution of Medically Relevant Antibiotic Resistance Genes and Mobile Genetic Elements in Soils of Temperate Forests and Grasslands Varying in Land Use.

Antibiotic-resistant pathogens claim the lives of thousands of people each year and are currently considered as one of the most serious threats to public health. Apart from clinical environments, soil ecosystems also represent a major source of antibiotic resistance determinants, which can potentially disseminate across distinct microbial habitats and be acquired by human pathogens via horizontal gene transfer. Therefore, it is of global importance to retrieve comprehensive information on environmental factors, contributing to an accumulation of antibiotic resistance genes and mobile genetic elements in these ecosystems. Here, medically relevant antibiotic resistance genes, class 1 integrons and IncP-1 plasmids were quantified via real time quantitative PCR in soils derived from temperate grasslands and forests, varying in land use over a large spatial scale. The generated dataset allowed an analysis, decoupled from regional influences, and enabled the identification of land use practices and soil characteristics elevating the abundance of antibiotic resistance genes and mobile genetic elements. In grassland soils, the abundance of the macrolide resistance gene mefA as well as the sulfonamide resistance gene sul2 was positively correlated with organic fertilization and the abundance of aac(6')-lb, conferring resistance to different aminoglycosides, increased with mowing frequency. With respect to forest soils, the beta-lactam resistance gene blaIMP-12 was significantly correlated with fungal diversity which might be due to the fact that different fungal species can produce beta-lactams. Furthermore, except blaIMP-5 and blaIMP-12, the analyzed antibiotic resistance genes as well as IncP-1 plasmids and class-1 integrons were detected less frequently in forest soils than in soils derived from grassland that are commonly in closer proximity to human activities.

Functional Metagenomics Reveals a New Catalytic Domain, the Metallo-ß-Lactamase Superfamily Domain, Associated with Phytase Activity.

Inositol-6-phosphate, also known as phytic acid, is a phosphorus source that plays several important roles in the phosphorus cycle and in cell metabolism. The known characterized enzymes responsible for its degradation, the phytases, are mostly derived from cultured individual microorganisms. The catalytic signatures of phytases are restricted to the molecular domains of four protein superfamilies: histidine phosphatases, protein tyrosine phosphatases, the purple acid phosphatases and the ß-propeller phosphatases. During function-based screening of previously generated forest soil metagenomic libraries for Escherichia coli clones conferring phytase activity, two positive clones harboring the plasmids pLP05 and pLP12 were detected. Analysis of the insert sequences revealed the absence of classic phosphatase/phytase signatures of the proteins deduced from the putative genes, but the genes mblp01 (pLP05) and mblp02 (pLP12) encoded putative metallo-ß-lactamases (MBLs). Several MBL representatives are promiscuous proteins with phosphoesterase activity, but phytase activity was previously not reported. Both mblp01 and mblp02 were subcloned, expressed, and analyzed. Mblp01 and Mblp02 are members of the lactamase B2 family. Protein modeling showed that the closest structural homologue of both proteins was ZipD of E. coli Mblp01 and Mblp02 showed activity toward the majority of the tested phosphorylated substrates, including phytate. The maximal enzyme activities were recorded for Mblp01 at 50°C under acidic conditions and for Mblp02 at 35°C and a neutral pH. In the presence of Cu2+ or SDS, the activities of Mblp01 and Mblp02 were strongly inhibited. Analyses of the minimal inhibitory concentrations of several ß-lactam antibiotics revealed that recombinant E. coli cells carrying mblp01 or mblp02 showed reduced sensitivity toward ß-lactam antibiotics.IMPORTANCE Phytic acid is a phosphorus storage molecule in many plant tissues, a source of phosphorus alternative to phosphate rocks, but it can also be a problematic antinutrient. In comparison to other phosphorus sources, phytic acid exhibits reduced bioavailability. Additionally, it influences functions of secondary messengers and acts as antioxidant in tumor growth prevention. The enzymatic capability to process phytate has been reported for a limited number of protein families. This might be due to the almost exclusive use of proteins derived from individual microorganisms to analyze phytase activity. With such a restriction, the study of the complexity and diversity of the phytases remains incomplete. By using metagenome-derived samples, this study demonstrates the existence of phytase activity in one of the most promiscuous superfamilies, the metallo-ß-lactamases. Our results increase the general knowledge on phytase diversity in environmental samples and could provide new avenues for the study and engineering of new biocatalysts.

Discovery of Novel Antibiotic Resistance Determinants in Forest and Grassland Soil Metagenomes.

Soil represents a significant reservoir of antibiotic resistance genes (ARGs), which can potentially spread across distinct ecosystems and be acquired by pathogens threatening human as well as animal health. Currently, information on the identity and diversity of these genes, enabling anticipation of possible future resistance development in clinical environments and the livestock sector, is lacking. In this study, we applied functional metagenomics to discover novel sulfonamide as well as tetracycline resistance genes in soils derived from forest and grassland. Screening of soil metagenomic libraries revealed a total of eight so far unknown ARGs. The recovered genes originate from phylogenetically diverse soil bacteria (e.g., Actinobacteria, Chloroflexi, or Proteobacteria) and encode proteins with a minimum identity of 46% to other antibiotic resistance determinants. In particular forest soil ecosystems have so far been neglected in studies focusing on antibiotic resistance. Here, we detected for the first time non-mobile dihydropteroate synthase (DHPS) genes conferring resistance to sulfonamides in forest soil with no history of exposure to these synthetic drugs. In total, three sulfonamide resistant DHPSs, differing in taxonomic origin, were discovered in beech or pine forest soil. This indicates that sulfonamide resistance naturally occurs in forest-resident soil bacterial communities. Besides forest soil-derived sulfonamide resistance proteins, we also identified a DHPS affiliated to Chloroflexi in grassland soil. This enzyme and the other recovered DHPSs confer reduced susceptibility toward sulfamethazine, which is widely used in food animal production. With respect to tetracycline resistance, four efflux proteins affiliated to the major facilitator superfamily (MFS) were identified. Noteworthy, one of these proteins also conferred reduced susceptibility toward lincomycin.

Characteristics of the First Protein Tyrosine Phosphatase with Phytase Activity from a Soil Metagenome.

Protein tyrosine phosphatases (PTPs) fulfil multiple key regulatory functions. Within the group of PTPs, the atypical lipid phosphatases (ALPs) are known for their role as virulence factors associated with human pathogens. Another group of PTPs, which is capable of using inositol-hexakisphosphate (InsP6) as substrate, are known as phytases. Phytases play major roles in the environmental phosphorus cycle, biotechnology, and pathogenesis. So far, all functionally characterized PTPs, including ALPs and PTP-phytases, have been derived exclusively from isolated microorganisms. In this study, screening of a soil-derived metagenomic library resulted in identification of a gene (pho16B), encoding a PTP, which shares structural characteristics with the ALPs. In addition, the characterization of the gene product (Pho16B) revealed the capability of the protein to use InsP6 as substrate, and the potential of soil as a source of phytases with so far unknown characteristics. Thus, Pho16B represents the first functional environmentally derived PTP-phytase. The enzyme has a molecular mass of 38 kDa. The enzyme is promiscuous, showing highest activity and affinity toward naphthyl phosphate (Km 0.966 mM). Pho16B contains the HCXXGKDR[TA]G submotif of PTP-ALPs, and it is structurally related to PtpB of Mycobacterium tuberculosis. This study demonstrates the presence and functionality of an environmental gene codifying a PTP-phytase homologous to enzymes closely associated to bacterial pathogenicity.

Functional Metagenomics Reveals an Overlooked Diversity and Novel Features of Soil-Derived Bacterial Phosphatases and Phytases.

Phosphatases, including phytases, play a major role in cell metabolism, phosphorus cycle, biotechnology, and pathogenic processes. Nevertheless, their discovery by functional metagenomics is challenging. Here, soil metagenomic libraries were successfully screened for genes encoding phosphatase activity. In this context, we report the largest number and diversity of phosphatase genes derived from functional metagenome analysis. Two of the detected gene products carry domains which have never been associated with phosphatase activity before. One of these domains, the SNARE-associated domain DedA, harbors a so-far-overlooked motif present in numerous bacterial SNARE-associated proteins. Our analysis revealed a previously unreported phytase activity of the alkaline phosphatase and sulfatase superfamily (cl23718) and of purple acid phosphatases from nonvegetal origin. This suggests that the classical concept comprising four classes of phytases should be modified and indicates high performance of our screening method for retrieving novel types of phosphatases/phytases hidden in metagenomes of complex environments.IMPORTANCE Phosphorus (P) is a key element involved in numerous cellular processes and essential to meet global food demand. Phosphatases play a major role in cell metabolism and contribute to control the release of P from phosphorylated organic compounds, including phytate. Apart from the relationship with pathogenesis and the enormous economic relevance, phosphatases/phytases are also important for reduction of phosphorus pollution. Almost all known functional phosphatases/phytases are derived from cultured individual microorganisms. We demonstrate here for the first time the potential of functional metagenomics to exploit the phosphatase/phytase pools hidden in environmental soil samples. The recovered diversity of phosphatases/phytases comprises new types and proteins exhibiting largely unknown characteristics, demonstrating the potential of the screening method for retrieving novel target enzymes. The insights gained into the unknown diversity of genes involved in the P cycle highlight the power of function-based metagenomic screening strategies to study Earth's phosphatase pools.

Comparative Genomics and Description of Putative Virulence Factors of Melissococcus plutonius, the Causative Agent of European Foulbrood Disease in Honey Bees.

In Europe, approximately 84% of cultivated crop species depend on insect pollinators, mainly bees. Apis mellifera (the Western honey bee) is the most important commercial pollinator worldwide. The Gram-positive bacterium Melissococcus plutonius is the causative agent of European foulbrood (EFB), a global honey bee brood disease. In order to detect putative virulence factors, we sequenced and analyzed the genomes of 14 M. plutonius strains, including two reference isolates. The isolates do not show a high diversity in genome size or number of predicted protein-encoding genes, ranging from 2.021 to 2.101 Mbp and 1589 to 1686, respectively. Comparative genomics detected genes that might play a role in EFB pathogenesis and ultimately in the death of the honey bee larvae. These include bacteriocins, bacteria cell surface- and host cell adhesion-associated proteins, an enterococcal polysaccharide antigen, an epsilon toxin, proteolytic enzymes, and capsule-associated proteins. In vivo expression of three putative virulence factors (endo-alpha-N-acetylgalactosaminidase, enhancin and epsilon toxin) was verified using naturally infected larvae. With our strain collection, we show for the first time that genomic differences exist between non-virulent and virulent typical strains, as well as a highly virulent atypical strain, that may contribute to the virulence of M. plutonius. Finally, we also detected a high number of conserved pseudogenes (75 to 156) per genome, which indicates genomic reduction during evolutionary host adaptation.

Links between seawater flooding, soil ammonia oxidiser communities and their response to changes in salinity.

Coastal areas worldwide are challenged by climate change-associated increases in sea level and storm surge quantities that potentially lead to more frequent flooding of soil ecosystems. Currently, little is known of the effects of inundation events on microorganisms controlling nitrification in these ecosystems. The goal of this study was to investigate the impact of seawater flooding on the abundance, community composition and salinity tolerance of soil ammonia oxidisers. Topsoil was sampled from three islands flooded at different frequencies by the Wadden Sea. Archaeal ammonia oxidiser amoA genes were more abundant than their betaproteobacterial counterparts, and the distribution of archaeal and bacterial ammonia oxidiser amoA and 16S rRNA gene sequences significantly differed between the islands. The findings indicate selection of ammonia oxidiser phylotypes with greater tolerance to high salinity and slightly alkaline pH (e.g. Nitrosopumilus representatives) in frequently flooded soils. A cluster phylogenetically related to gammaproteobacterial ammonia oxidisers was detected in all samples analysed in this survey. Nevertheless, no gammaprotebacterial amoA genes could be amplified via PCR and only betaproteobacterial ammonia oxidisers were detected in enrichment cultures. A slurry-based experiment demonstrated the tolerance of both bacterial and archaeal ammonia oxidisers to a wide range of salinities (e.g. Wadden Sea water salinity) in soil naturally exposed to seawater at a high frequency.

Genome Sequence of Creatinine-Fermenting Tissierella creatinophila Strain KRE 4T (DSM 6911).

Tissierella creatinophila strain KRE 4T (DSM 6911) is a strictly anaerobic, creatinine-fermenting, and creatine-fermenting organism, which has been isolated from sewage sludge. The draft genome consists of one circular chromosome (2.5 Mb) and harbors 2,533 predicted protein-encoding genes.

Function-Based Metagenomic Library Screening and Heterologous Expression Strategy for Genes Encoding Phosphatase Activity.

The release of phosphate from inorganic and organic phosphorus compounds can be mediated enzymatically. Phosphate-releasing enzymes, comprising acid and alkaline phosphatases, are recognized as useful biocatalysts in applications such as plant and animal nutrition, bioremediation and diagnostic analysis. Metagenomic approaches provide access to novel phosphatase-encoding genes. Here, we describe a function-based screening approach for rapid identification of genes conferring phosphatase activity from small-insert and large-insert metagenomic libraries derived from various environments. This approach bears the potential for discovery of entirely novel phosphatase families or subfamilies and members of known enzyme classes hydrolyzing phosphomonoester bonds such as phytases. In addition, we provide a strategy for efficient heterologous phosphatase gene expression.

Driving forces of soil bacterial community structure, diversity, and function in temperate grasslands and forests.

Soil bacteria provide a large range of ecosystem services such as nutrient cycling. Despite their important role in soil systems, compositional and functional responses of bacterial communities to different land use and management regimes are not fully understood. Here, we assessed soil bacterial communities in 150 forest and 150 grassland soils derived from three German regions by pyrotag sequencing of 16S rRNA genes. Land use type (forest and grassland) and soil edaphic properties strongly affected bacterial community structure and function, whereas management regime had a minor effect. In addition, a separation of soil bacterial communities by sampling region was encountered. Soil pH was the best predictor for bacterial community structure, diversity and function. The application of multinomial log-linear models revealed distinct responses of abundant bacterial groups towards pH. Predicted functional profiles revealed that differences in land use not only select for distinct bacterial populations but also for specific functional traits. The combination of 16S rRNA data and corresponding functional profiles provided comprehensive insights into compositional and functional adaptations to changing environmental conditions associated with differences in land use and management.

Estimates of Soil Bacterial Ribosome Content and Diversity Are Significantly Affected by the Nucleic Acid Extraction Method Employed.

Modern sequencing technologies allow high-resolution analyses of total and potentially active soil microbial communities based on their DNA and RNA, respectively. In the present study, quantitative PCR and 454 pyrosequencing were used to evaluate the effects of different extraction methods on the abundance and diversity of 16S rRNA genes and transcripts recovered from three different types of soils (leptosol, stagnosol, and gleysol). The quality and yield of nucleic acids varied considerably with respect to both the applied extraction method and the analyzed type of soil. The bacterial ribosome content (calculated as the ratio of 16S rRNA transcripts to 16S rRNA genes) can serve as an indicator of the potential activity of bacterial cells and differed by 2 orders of magnitude between nucleic acid extracts obtained by the various extraction methods. Depending on the extraction method, the relative abundances of dominant soil taxa, in particular Actino bacteria and Proteobacteria, varied by a factor of up to 10. Through this systematic approach, the present study allows guidelines to be deduced for the selection of the appropriate extraction protocol according to the specific soil properties, the nucleic acid of interest, and the target organisms.

Fine Spatial Scale Variation of Soil Microbial Communities under European Beech and Norway Spruce.

The complex interactions between trees and soil microbes in forests as well as their inherent seasonal and spatial variations are poorly understood. In this study, we analyzed the effects of major European tree species (Fagus sylvatica L. and Picea abies (L.) Karst) on soil bacterial and fungal communities. Mineral soil samples were collected from different depths (0-10, 10-20 cm) and at different horizontal distances from beech or spruce trunks (0.5, 1.5, 2.5, 3.5 m) in early summer and autumn. We assessed the composition of soil bacterial and fungal communities based on 16S rRNA gene and ITS DNA sequences. Community composition of bacteria and fungi was most strongly affected by soil pH and tree species. Different ectomycorrhizal fungi (e.g., Tylospora) known to establish mutualistic associations with plant roots showed a tree species preference. Moreover, bacterial and fungal community composition showed spatial and seasonal shifts in soil surrounding beech and spruce. The relative abundance of saprotrophic fungi was higher at a depth of 0-10 vs. 10-20 cm depth. This was presumably a result of changes in nutrient availability, as litter input and organic carbon content decreased with soil depth. Overall bacterial community composition showed strong variations under spruce with increasing distance from the tree trunks, which might be attributed in part to higher fine root biomass near spruce trunks. Furthermore, overall bacterial community composition was strongly affected by season under deciduous trees.