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

Acknowledgements We thank Heather Richmond and Mechthild Bömeke for providing excellent technical assistance. In addition, we thank Jessica Heublein for support with respect to basic soil analyses and Laura Lehtovirta-Morley for useful discussion on cultivation of AO. We also thank Ruth Hartwig-Kruse, Michael Kliesch and the team of the ‘Schutzstation Wattenmeer Langeness’ for support during sampling. FUNDING This work was financially supported by the Deutsche Forschungsgemeinschaft (DFG) (NA 848/1-1).Peer reviewedPostprin

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

Pyrosequencing-Based Assessment of Bacterial Community Structure Along Different Management Types in German Forest and Grassland Soils

BACKGROUND: Soil bacteria are important drivers for nearly all biogeochemical cycles in terrestrial ecosystems and participate in most nutrient transformations in soil. In contrast to the importance of soil bacteria for ecosystem functioning, we understand little how different management types affect the soil bacterial community composition. METHODOLOGY/PRINCIPAL FINDINGS: We used pyrosequencing-based analysis of the V2-V3 16S rRNA gene region to identify changes in bacterial diversity and community structure in nine forest and nine grassland soils from the Schwäbische Alb that covered six different management types. The dataset comprised 598,962 sequences that were affiliated to the domain Bacteria. The number of classified sequences per sample ranged from 23,515 to 39,259. Bacterial diversity was more phylum rich in grassland soils than in forest soils. The dominant taxonomic groups across all samples (>1% of all sequences) were Acidobacteria, Alphaproteobacteria, Actinobacteria, Betaproteobacteria, Deltaproteobacteria, Gammaproteobacteria, and Firmicutes. Significant variations in relative abundances of bacterial phyla and proteobacterial classes, including Actinobacteria, Firmicutes, Verrucomicrobia, Cyanobacteria, Gemmatimonadetes and Alphaproteobacteria, between the land use types forest and grassland were observed. At the genus level, significant differences were also recorded for the dominant genera Phenylobacter, Bacillus, Kribbella, Streptomyces, Agromyces, and Defluviicoccus. In addition, soil bacterial community structure showed significant differences between beech and spruce forest soils. The relative abundances of bacterial groups at different taxonomic levels correlated with soil pH, but little or no relationships to management type and other soil properties were found. CONCLUSIONS/SIGNIFICANCE: Soil bacterial community composition and diversity of the six analyzed management types showed significant differences between the land use types grassland and forest. Furthermore, bacterial community structure was largely driven by tree species and soil pH

Identifizierung und Charakterisierung von mikrobiellen Schlüsselfunktionen in Böden unterschiedlichen Landnutzungs- und Managementtyps der deutschen Biodiversitäts-Exploratorien

Boden beherbergt wahrscheinlich die höchste mikrobielle Artenvielfalt auf der Erde und stellt ein Hauptreservoir für taxonomische, genomische und metabolische Vielfalt von Mikroorganismen dar. Der vielversprechendste Ansatz um Einblicke in die Diversität und Struktur von mikrobiellen Gemeinschaften des Bodens zu gewinnen, ist die Anwendung von kultivierungsunabhängigen Methoden. Außerdem sind diese Methoden wertvoll für die Gewinnung von neuartigen natürlichen Produkten aus Boden. In dieser Studie wurden Wald- und Grünlandbodenproben aus den deutschen Biodiversitäts-Exploratorien Schorfheide-Chorin, Hainich-Dün und Schwäbische Alb mit Hilfe von metagenomischen Methoden analysiert. Aus Ober- und Unterbodenproben, die verschiedene Managementtypen abdecken, wurde Umwelt-DNA isoliert. Durch 16S rRNA Gen-basierte Amplikon Pyrosequenzierung wurde eine phylogenetische Analyse von Grünland- und Waldproben der Schwäbischen Alb (Oberboden) und Grünlandproben des Hainichs (Ober- und Unterboden) ermöglicht. Die bakterielle Diversität war auf der Phylumebene in Grünlandböden größer als in Waldböden. Außerdem konnte ein Effekt der Bodentiefe aufgedeckt werden, diesbezüglich wiesen Oberbodenproben eine höhere Diversität gegenüber Unterbodenproben auf. Innerhalb der Waldböden, die einen breiten pH-Bereich abdeckten, konnte ein signifikanter Einfluss des pH-Wertes sowie der Baumart auf die bakterielle Diversität nachgewiesen werden. Zahlreiche bakterielle Gruppen zeigten auf unterschiedlicher taxonomischer Ebene starke Korrelationen gegenüber dem pH-Wert des Bodens. Dieser pH-Effekt konnte jedoch nicht in den Hainich-Grünlandbodenproben nachgewiesen werden, da ein geringer pH-Bereich durch diese Proben abgedeckt wurde. Stattdessen rief der organische Kohlenstoffgehalt der nahezu neutralen Hainich-Bodenproben statistisch signifikante Effekte gegenüber der Struktur der bakteriellen Gemeinschaften hervor. Unter Verwendung von Umwelt-DNA aus den drei deutschen Biodiversitäts-Exploratorien wurden 14 Plasmid- und 9 Fosmidbanken konstruiert. Die partielle Durchmusterung dieser metagenomischen Genbibliotheken nach lipolytischen und (hemi)cellulolytischen Genen resultierte in der Identifizierung von 37 unterschiedlichen lipolytischen Klonen und 3 verschiedenen (hemi)cellulolytischen Klonen. Die Insert-DNA dieser Klone wurde sequenziert und anschließend analysiert. Es zeigte sich dass 35 Genprodukte der 37 identifizierten lipolytischen Gene bisher unbekannte Vertreter der Lipase-/Esterasefamilien I (echte Lipasen), IV, V, VI und VIII darstellen. Die übrigen beiden Genprodukte repräsentieren Vertreter potentiell neuer Lipase-/Esterasefamilien. Die Insert-DNA von zwei der drei (hemi)cellulolytischen Klone beherbergte Xylanase-kodierende Gene, wogegen die Insert-DNA des übrigen Klones ein Cellulase-kodierendes Gen beinhaltete. Eine Aminosäuresequenzanalyse der Genprodukte zeigte, dass die putative Cellulase eine Kohlenhydratbindedomäne der Familie 9 beherbergt, die zuvor lediglich in Xylanasen detektiert wurde. Durch initiale Charakterisierung der beiden Xylanasen konnte ermittelt werden, dass beide Enzyme eine hohe Aktivität innerhalb eines breiten Temperatur- und pH-Bereichs aufweisen. Außerdem wurde eine biochemische Charakterisierung der Cellulase vorgenommen. Das Enzym war sehr salztolerant und wies eine hohe Aktivität innerhalb eines breiten pH-Bereichs auf

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

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

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.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

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

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

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