New Arsenite Oxidase Gene (aioA) PCR Primers for Assessing Arsenite-Oxidizer Diversity in the Environment Using High-Throughput Sequencing

Gene encoding the large subunit of As(III) oxidase (AioA), an important component of the microbial As(III) oxidation system, is a widely used biomarker to characterize As(III)-oxidizing communities in the environment. However, many studies were restricted to a few sequences generated by clone libraries and Sanger sequencing, which may have underestimated the diversity of As(III)-oxidizers in natural environments. In this study, we designed a primer pair, 1109F (5′-ATC TGG GGB AAY RAC AAY TA−3′) and 1548R (5′-TTC ATB GAS GTS AGR TTC AT−3′), targeting gene sequence encoding for the conserved molybdopterin center of the AioA protein, yielding amplicons approximately 450 bp in size that are feasible for highly parallel amplicon sequencing. By utilizing in silico analyses and the experimental construction of clone libraries using Sanger sequencing, the specificity and resolution of 1109F/1548R are approximated with two other previously published and commonly used primers, i.e., M1-2F/M3-2R and deg1F/deg1R. With the use of the 1109F/1548R primer pair, the taxonomic composition of the aioA genes was similar both according to the Sanger and next-generation sequencing (NGS) platforms. Furthermore, high-throughput amplicon sequencing using the primer pair, 1109F/1548R, successfully identified the well-known As(III)-oxidizers in paddy soils and sediments, and they also revealed the differences in the community structure and composition of As(III)-oxidizers in above two biotopes. The random forest analysis showed that the dissolved As(III) had the highest relative influence on the Chao1 index of the aioA genes. These observations demonstrate that the newly designed PCR primers enhanced the ability to detect the diversity of aioA-encoding microorganisms in environments using highly parallel short amplicon sequencing.

A New High-Throughput Screening Method to Detect Antimicrobial Volatiles from Metagenomic Clone Libraries

The ever-growing spread of resistance in medicine and agriculture highlights the need to identify new antimicrobials. Microbial volatile organic compounds (VOCs) are one of the most promising groups of chemicals to meet this need. These rarely exploited molecules exhibit antimicrobial activity and their high vapour pressure makes them ideal for application in surface sterilisation, and in particular, in biofumigation. Therefore, we adapted the previously developed Two Clamp VOCs Assay (TCVA) to a new high-throughput screening for the detection of novel antifungal VOCs from metagenomic clone libraries. As a proof of concept, we tested the new high-throughput TCVA (htTCVA) by sourcing a moss metagenomic library against Fusarium culmorum. This led to the identification of five clones that inhibited the growth of mycelium and spores in vitro by up to 8% and 30% and subsequently, to the identification of VOCs that are potentially, and in part responsible for the clones’ antifungal activity. For these VOCs, the in vitro effect of the pure compounds was as high as 100%. These results demonstrate the robustness and feasibility of the htTCVA, which provides access to completely new and unexplored biosynthetic pathways and their secondary metabolites.

The Role of Agriculture in the Dissemination of Class 1 Integrons, Antimicrobial Resistance, and Diversity of Their Gene Cassettes in Southern China

Integrons are hot spots for acquiring gene cassettes from the environment and play a major role in the bacterial evolution and dissemination of antimicrobial resistance (AMR), thus posing a serious threat. There are currently studies on integrons and antibiotic resistance genes; however, the presence and association of integrons in different agricultural crops and their subsequent dissemination and role in AMR have not been reported previously. This study examines the abundance of integrons, their gene cassette diversity in various crop soils, and their role in the dissemination of AMR in the southern region of China. Samples from different agri-crop soil, such as rice (R.S), sugarcane (S.S), citrus (C.S), banana (B.S), agricultural runoff (the point where the runoff of all sites meet (R.O)), and wild (non-agricultural) soil (W.S), were collected. Quantitative PCR was used to determine the abundance of integrons, and clone libraries were constructed to examine the gene cassette arrays. All the tested samples were found positive for Class-I (CL1) integrons and revealed a higher concentration and higher relative abundance of R.S than the others, with the least found at the W.S site. The W.S CL1 cassette arrays were found empty, and no putative conserved domains were found. The R.O was found to contain a high number of gene cassettes with various functions, while the smallest number of gene cassettes was found in the S.S among the crop soils. Most of the gene cassettes presented by the R.O were primarily shared with other sites, and the antibiotic-resistant genes were consistently observed to be dominant. The constructed clone libraries represented a diverse gene cassette array with 16% novel gene cassettes that play a vital role in pathogenesis, transportation, biosynthesis, and AMR. Most resistance-related gene cassettes were associated with the genes encoding resistance to quaternary ammonium compound (QAC) and aminoglycosides. This study highlights the significant differences in the abundance of integrons among various agricultural soils and offers deep insight into the pools of gene cassettes that play a key role in the dissemination of integrons and AMR.

Fungal diversity of deep-sea sediments in Mid-Oceanic Ridge area of the East Pacific and the South Indian Oceans

In this study, we investigated fungal diversity of six Mid-Oceanic Ridge sediment samples collected in the East Pacific and the South Indian Oceans by culture-dependent as well as culture-independent approaches. A total of 97 fungal isolates were cultured, belonging to 7 genera and 10 species, including Penicillium (2 species), Rhodotorula, Meyerozyma, Ophiocordyceps, Vishniacozyma, Aspergillus (3 species) and Phoma. Forty-six operational taxonomic units (OTUs) were identified from 933 clones selected from 12 clone libraries based on the internal transcribed spacers including 5.8S (ITS) and 18S rDNA. Two cultured fungi and 9 OTUs from the clone libraries showed similarities less than 97% with the existing sequences in GenBank, suggesting possible new fungal taxa. Most of the fungi belonged to the Ascomycota, to a lesser extent the Basidiomycota. Five fungal genera including Aspergillus, Rhodotorula, Ophiocordyceps, Phoma and Penicillium were recovered by both culture-dependent and culture-independent approaches. In addition to some common genera such as Aspergillus, Penicillium, Ophiocordyceps, Purpureocillium, Tolypocladium, Rhodotorula and Fusarium, some unique species were each found in the East Pacific and the South Indian Oceans. It is worth mentioning that Ophiocordyceps heteropoda was found at both oceans by culture-dependent and culture-independent approaches, although it was only previously reported from terrestrial habitats.

Aquatic Thermal Reservoirs of Microbial Life in a Remote and Extreme High Andean Hydrothermal System

Hydrothermal systems are ideal to understand how microbial communities cope with challenging conditions. Lirima, our study site, is a polyextreme, high-altitude, hydrothermal ecosystem located in the Chilean Andean highlands. Herein, we analyze the benthic communities of three nearby springs in a gradient of temperature (42–72 °C represented by stations P42, P53, and P72) and pH, and we characterize their microbial diversity by using bacteria 16S rRNA (V4) gene metabarcoding and 16S rRNA gene clone libraries (bacteria and archaea). Bacterial clone libraries of P42 and P53 springs showed that the community composition was mainly represented by phototrophic bacteria (Chlorobia, 3%, Cyanobacteria 3%, at P42; Chlorobia 5%, and Chloroflexi 5% at P53), Firmicutes (32% at P42 and 43% at P53) and Gammaproteobacteria (13% at P42 and 29% at P53). Furthermore, bacterial communities that were analyzed by 16S rRNA gene metabarcoding were characterized by an overall predominance of Chloroflexi in springs with lower temperatures (33% at P42), followed by Firmicutes in hotter springs (50% at P72). The archaeal diversity of P42 and P53 were represented by taxa belonging to Crenarchaeota, Diapherotrites, Nanoarchaeota, Hadesarchaeota, Thaumarchaeota, and Euryarchaeota. The microbial diversity of the Lirima hydrothermal system is represented by groups from deep branches of the tree of life, suggesting this ecosystem as a reservoir of primitive life and a key system to study the processes that shaped the evolution of the biosphere.

Nitrate Consumers in Arctic Marine Eukaryotic Communities: Comparative Diversities of 18S rRNA, 18S rRNA Genes, and Nitrate Reductase Genes

ABSTRACT For photosynthetic microbial eukaryotes, the rate-limiting step in NO3− assimilation is its reduction to nitrite (NO2−), which is catalyzed by assimilatory nitrate reductase (NR). Oceanic productivity is primarily limited by available nitrogen and, although nitrate is the most abundant form of available nitrogen in oceanic waters, little is known about the identity of microbial eukaryotes that take up nitrate. This lack of knowledge is especially severe for ice-covered seas that are being profoundly affected by climate change. To address this, we examined the distribution and diversity of NR genes in the Arctic region by way of clone libraries and data mining of available metagenomes (total of 4.24 billion reads). We directly compared NR clone phylogenies with the V4 region of the 18S rRNA gene (DNA pool) and 18S rRNA (RNA pool) at two ice-influenced stations in the Canada Basin (Beaufort Sea). The communities from the two nucleic acid templates were similar at the level of major groups, and species identified by way of NR gene phylogeny and microscopy were a subset of the 18S results. Most NR genes from arctic clone libraries matched diatoms and chromist nanoflagellates, including novel clades, while the NR genes in arctic eukaryote metagenomes were dominated by chlorophyte NR, in keeping with the ubiquitous occurrence of Mamiellophyceae in the Arctic Ocean. Overall, these data suggest that a dynamic and mixed eukaryotic community utilizes nitrate across the Arctic region, and they show the potential utility of NR as a tool to identify ongoing changes in arctic photosynthetic communities. IMPORTANCE To better understand the diversity of primary producers in the Arctic Ocean, we targeted a nitrogen cycle gene, NR, which is required for phytoplankton to assimilate nitrate into organic forms of nitrogen macromolecules. We compared this to the more detailed taxonomy from ice-influenced stations using a general taxonomic gene (18S rRNA). NR genes were ubiquitous and could be classified as belonging to diatoms, dinoflagellates, other flagellates, chlorophytes, and unknown microbial eukaryotes, suggesting novel diversity of both species and metabolism in arctic phytoplankton.

Host specificity determines the assemblage of root endophytic bacteria of plants growing in metal contaminated soil

The diversity of endophytic bacteria colonizing the roots of an invasive plant, a pioneer plant, and an endemic plant from varied plant communities developing in a lead-zinc mine tailing pond, southwestern China, was analyzed by the culture-independent method. A total of 1650 16S rDNA sequences were screened for the establishment of four clone libraries, from the pure stands of Arthraxon hispidus (LS-Ah) and Ageratina adenophora (LS-Aa), and a mixed stand of A. adenophora (LC-Aa) and Alnus nepalensis (LC-An) (co-dominant community), respectively. Phylogenetic analysis revealed that the sequences were clustered into at least 17 phylogroups, which consisted of alpha, beta, gamma, delta subclasses of the Proteobacteria, Tenericutes, Bacteroidetes, Chloroflexi, Actinobacteria, Spirochaetes, Chlamydiae, Firmicutes, Deinococcus-Thermus, Planctomycetes, Nitrospirae, Gemmatimonadetes and unclassified bacteria Candidatus Saccharibacteria. The dominant phylum was Proteobacteria (50.49% of the total clones), and the dominant genus was Candidatus Phytoplasma (19.94% of the total clones). The invasive plant (A. adenophora) accumulated more parasitic endophytic bacteria (Phytoplasma) than the other two native plants. Phylogenetic structures of the four 16S rDNA clone libraries were distinct with their similarity indices being less than 0.5. The results also revealed that the dominant phyla and dominant genera in the four clone libraries varied a lot, and the endemic grass harbored a higher diversity of endophytic bacteria than the pioneer and invasive plants, the host-specificity took a more important role in shaping the endophytic bacteria community than the habitats in the metal stressed environment.

Host specificity determines the assemblage of root endophytic bacteria of plants growing in metal contaminated soil

The diversity of endophytic bacteria colonizing the roots of an invasive plant, a pioneer plant, and an endemic plant from varied plant communities developing in a lead-zinc mine tailing pond, southwestern China, was analyzed by the culture-independent method. A total of 1650 16S rDNA sequences were screened for the establishment of four clone libraries, from the pure stands of Arthraxon hispidus (LS-Ah) and Ageratina adenophora (LS-Aa), and a mixed stand of A. adenophora (LC-Aa) and Alnus nepalensis (LC-An) (co-dominant community), respectively. Phylogenetic analysis revealed that the sequences were clustered into at least 17 phylogroups, which consisted of alpha, beta, gamma, delta subclasses of the Proteobacteria, Tenericutes, Bacteroidetes, Chloroflexi, Actinobacteria, Spirochaetes, Chlamydiae, Firmicutes, Deinococcus-Thermus, Planctomycetes, Nitrospirae, Gemmatimonadetes and unclassified bacteria Candidatus Saccharibacteria. The dominant phylum was Proteobacteria (50.49% of the total clones), and the dominant genus was Candidatus Phytoplasma (19.94% of the total clones). The invasive plant (A. adenophora) accumulated more parasitic endophytic bacteria (Phytoplasma) than the other two native plants. Phylogenetic structures of the four 16S rDNA clone libraries were distinct with their similarity indices being less than 0.5. The results also revealed that the dominant phyla and dominant genera in the four clone libraries varied a lot, and the endemic grass harbored a higher diversity of endophytic bacteria than the pioneer and invasive plants, the host-specificity took a more important role in shaping the endophytic bacteria community than the habitats in the metal stressed environment.