Identification of small molecule inhibitors of the Chloracidobacterium thermophilum type IV pilus protein PilB by ensemble virtual screening.

Antivirulence strategy has been explored as an alternative to traditional antibiotic development. The bacterial type IV pilus is a virulence factor involved in host invasion and colonization in many antibiotic resistant pathogens. The PilB ATPase hydrolyzes ATP to drive the assembly of the pilus filament from pilin subunits. We evaluated Chloracidobacterium thermophilum PilB (CtPilB) as a model for structure-based virtual screening by molecular docking and molecular dynamics (MD) simulations. A hexameric structure of CtPilB was generated through homology modeling based on an existing crystal structure of a PilB from Geobacter metallireducens. Four representative structures were obtained from molecular dynamics simulations to examine the conformational plasticity of PilB and improve docking analyses by ensemble docking. Structural analyses after 1 µs of simulation revealed conformational changes in individual PilB subunits are dependent on ligand presence. Further, ensemble virtual screening of a library of 4234 compounds retrieved from the ZINC15 database identified five promising PilB inhibitors. Molecular docking and binding analyses using the four representative structures from MD simulations revealed that top-ranked compounds interact with multiple Walker A residues, one Asp-box residue, and one arginine finger, indicating these are key residues in inhibitor binding within the ATP binding pocket. The use of multiple conformations in molecular screening can provide greater insight into compound flexibility within receptor sites and better inform future drug development for therapeutics targeting the type IV pilus assembly ATPase.

Acidification induce chemical and microbial variation in tea plantation soils and bacterial degradation of the key acidifying phenolic acids.

Camellia sinensis is an important economic plant grown in southern subtropical hilly areas, especially in China, mainly for the production of tea. Soil acidification is a significant cause of the reduction of yield and quality and continuous cropping obstacles in tea plants. Therefore, chemical and microbial properties of tea growing soils were investigated and phenolic acid-degrading bacteria were isolated from a tea plantation. Chemical and ICP-AES investigations showed that the soils tested were acidic, with pH values of 4.05-5.08, and the pH negatively correlated with K (p < 0.01), Al (p < 0.05), Fe and P. Aluminum was the highest (47-584 mg/kg) nonessential element. Based on high-throughput sequencing, a total of 34 phyla and 583 genera were identified in tea plantation soils. Proteobacteria and Acidobacteria were the main dominant phyla and the highest abundance of Acidobacteria was found in three soils, with nearly 22% for the genus Gp2. Based on the functional abundance values, general function predicts the highest abundance, while the abundance of amino acids and carbon transport and metabolism were higher in soils with pH less than 5. According to Biolog Eco Plate™ assay, the soil microorganisms utilized amino acids well, followed by polymers and phenolic acids. Three strains with good phenolic acid degradation rates were obtained, and they were identified as Bacillus thuringiensis B1, Bacillus amyloliquefaciens B2 and Bacillus subtilis B3, respectively. The three strains significantly relieved the inhibition of peanut germination and growth by ferulic acid, p-coumaric acid, p-hydroxybenzoic acid, cinnamic acid, and mixed acids. Combination of the three isolates showed reduced relief of the four phenolic acids due to the antagonist of B2 against B1 and B3. The three phenolic acid degradation strains isolated from acidic soils display potential in improving the acidification and imbalance in soils of C. sinensis.

Legume-microbiome interactions unlock mineral nutrients in regrowing tropical forests.

Legume trees form an abundant and functionally important component of tropical forests worldwide with N2-fixing symbioses linked to enhanced growth and recruitment in early secondary succession. However, it remains unclear how N2-fixers meet the high demands for inorganic nutrients imposed by rapid biomass accumulation on nutrient-poor tropical soils. Here, we show that N2-fixing trees in secondary Neotropical forests triggered twofold higher in situ weathering of fresh primary silicates compared to non-N2-fixing trees and induced locally enhanced nutrient cycling by the soil microbiome community. Shotgun metagenomic data from weathered minerals support the role of enhanced nitrogen and carbon cycling in increasing acidity and weathering. Metagenomic and marker gene analyses further revealed increased microbial potential beneath N2-fixers for anaerobic iron reduction, a process regulating the pool of phosphorus bound to iron-bearing soil minerals. We find that the Fe(III)-reducing gene pool in soil is dominated by acidophilic Acidobacteria, including a highly abundant genus of previously undescribed bacteria, Candidatus Acidoferrum, genus novus. The resulting dependence of the Fe-cycling gene pool to pH determines the high iron-reducing potential encoded in the metagenome of the more acidic soils of N2-fixers and their nonfixing neighbors. We infer that by promoting the activities of a specialized local microbiome through changes in soil pH and C:N ratios, N2-fixing trees can influence the wider biogeochemical functioning of tropical forest ecosystems in a manner that enhances their ability to assimilate and store atmospheric carbon.

Uptake of Phytoplankton-Derived Carbon and Cobalamins by Novel Acidobacteria Genera in Microcystis Blooms Inferred from Metagenomic and Metatranscriptomic Evidence.

Interactions between bacteria and phytoplankton can influence primary production, community composition, and algal bloom development. However, these interactions are poorly described for many consortia, particularly for freshwater bloom-forming cyanobacteria. Here, we assessed the gene content and expression of two uncultivated Acidobacteria from Lake Erie Microcystis blooms. These organisms were targeted because they were previously identified as important catalase producers in Microcystis blooms, suggesting that they protect Microcystis from H2O2. Metatranscriptomics revealed that both Acidobacteria transcribed genes for uptake of organic compounds that are known cyanobacterial products and exudates, including lactate, glycolate, amino acids, peptides, and cobalamins. Expressed genes for amino acid metabolism and peptide transport and degradation suggest that use of amino acids and peptides by Acidobacteria may regenerate nitrogen for cyanobacteria and other organisms. The Acidobacteria genomes lacked genes for biosynthesis of cobalamins but expressed genes for its transport and remodeling. This indicates that the Acidobacteria obtained cobalamins externally, potentially from Microcystis, which has a complete gene repertoire for pseudocobalamin biosynthesis; expressed them in field samples; and produced pseudocobalamin in axenic culture. Both Acidobacteria were detected in Microcystis blooms worldwide. Together, the data support the hypotheses that uncultured and previously unidentified Acidobacteria taxa exchange metabolites with phytoplankton during harmful cyanobacterial blooms and influence nitrogen available to phytoplankton. Thus, novel Acidobacteria may play a role in cyanobacterial physiology and bloom development. IMPORTANCE Interactions between heterotrophic bacteria and phytoplankton influence competition and successions between phytoplankton taxa, thereby influencing ecosystem-wide processes such as carbon cycling and algal bloom development. The cyanobacterium Microcystis forms harmful blooms in freshwaters worldwide and grows in buoyant colonies that harbor other bacteria in their phycospheres. Bacteria in the phycosphere and in the surrounding community likely influence Microcystis physiology and ecology and thus the development of freshwater harmful cyanobacterial blooms. However, the impacts and mechanisms of interaction between bacteria and Microcystis are not fully understood. This study explores the mechanisms of interaction between Microcystis and uncultured members of its phycosphere in situ with population genome resolution to investigate the cooccurrence of Microcystis and freshwater Acidobacteria in blooms worldwide.

Genomic Analysis of Family UBA6911 (Group 18 Acidobacteria) Expands the Metabolic Capacities of the Phylum and Highlights Adaptations to Terrestrial Habitats.

Approaches for recovering and analyzing genomes belonging to novel, hitherto-unexplored bacterial lineages have provided invaluable insights into the metabolic capabilities and ecological roles of yet-uncultured taxa. The phylum Acidobacteria is one of the most prevalent and ecologically successful lineages on Earth, yet currently, multiple lineages within this phylum remain unexplored. Here, we utilize genomes recovered from Zodletone Spring, an anaerobic sulfide and sulfur-rich spring in southwestern Oklahoma, as well as from multiple disparate soil and nonsoil habitats, to examine the metabolic capabilities and ecological role of members of family UBA6911 (group 18) Acidobacteria. The analyzed genomes clustered into five distinct genera, with genera Gp18_AA60 and QHZH01 recovered from soils, genus Ga0209509 from anaerobic digestors, and genera Ga0212092 and UBA6911 from freshwater habitats. All genomes analyzed suggested that members of Acidobacteria group 18 are metabolically versatile heterotrophs capable of utilizing a wide range of proteins, amino acids, and sugars as carbon sources, possess respiratory and fermentative capacities, and display few auxotrophies. Soil-dwelling genera were characterized by larger genome sizes, higher numbers of CRISPR loci, an expanded carbohydrate active enzyme (CAZyme) machinery enabling debranching of specific sugars from polymers, possession of a C1 (methanol and methylamine) degradation machinery, and a sole dependence on aerobic respiration. In contrast, nonsoil genomes encoded a more versatile respiratory capacity for oxygen, nitrite, sulfate, and trimethylamine N-oxide (TMAO) respiration, as well as the potential for utilizing the Wood-Ljungdahl (WL) pathway as an electron sink during heterotrophic growth. Our results not only expand our knowledge of the metabolism of a yet-uncultured bacterial lineage but also provide interesting clues on how terrestrialization and niche adaptation drive metabolic specialization within the Acidobacteria. IMPORTANCE Members of the Acidobacteria are important players in global biogeochemical cycles, especially in soils. A wide range of acidobacterial lineages remain currently unexplored. We present a detailed genomic characterization of genomes belonging to family UBA6911 (also known as group 18) within the phylum Acidobacteria. The genomes belong to different genera and were obtained from soil (genera Gp18_AA60 and QHZH01), freshwater habitats (genera Ga0212092 and UBA6911), and an anaerobic digestor (genus Ga0209509). While all members of the family shared common metabolic features, e.g., heterotrophic respiratory abilities, broad substrate utilization capacities, and few auxotrophies, distinct differences between soil and nonsoil genera were observed. Soil genera were characterized by expanded genomes, higher numbers of CRISPR loci, a larger carbohydrate active enzyme (CAZyme) repertoire enabling monomer extractions from polymer side chains, and methylotrophic (methanol and methylamine) degradation capacities. In contrast, nonsoil genera encoded more versatile respiratory capacities for utilizing nitrite, sulfate, TMAO, and the WL pathway, in addition to oxygen as electron acceptors. Our results not only broaden our understanding of the metabolic capacities within the Acidobacteria but also provide interesting clues on how terrestrialization shaped Acidobacteria evolution and niche adaptation.

Soil Acidobacteria Strain AB23 Resistance to Oxidative Stress Through Production of Carotenoids.

Metagenomic studies revealed the prevalence of Acidobacteria in soils, but the physiological and ecological reasons for their success are not well understood. Many Acidobacteria exhibit carotenoid-related pigments, which may be involved in their tolerance of environmental stress. The aim of this work was to investigate the role of the orange pigments produced by Acidobacteria strain AB23 isolated from a savannah-like soil and to identify putative carotenoid genes in Acidobacteria genomes. Phylogenetic analysis revealed that strain AB23 belongs to the Occallatibacter genus from the class Acidobacteriia (subdivision 1). Strain AB23 produced carotenoids in the presence of light and vitamins; however, the growth rate and biomass decreased when cells were exposed to light. The presence of carotenoids resulted in tolerance to hydrogen peroxide. Comparative genomics revealed that all members of Acidobacteriia with available genomes possess the complete gene cluster for phytoene production. Some Acidobacteriia members have an additional gene cluster that may be involved in the production of colored carotenoids. Both colored and colorless carotenoids are involved in tolerance to oxidative stress. These results show that the presence of carotenoid genes is widespread among Acidobacteriia. Light and atmospheric oxygen stimulate carotenoid synthesis, but there are other natural sources of oxidative stress in soils. Tolerance to environmental oxidative stress provided by carotenoids may offer a competitive advantage for Acidobacteria in soils.

Efficiency and mechanisms of cadmium removal via core-shell zeolite/Zn-layer double hydroxides.

To investigate the removal mechanisms of cadmium (Cd) by Zn-layer double hydroxides-modified zeolites substrates in constructed rapid infiltration systems (CRIS), the ZnAl-LDHs and ZnFe-LDHs were synthesized and in-situ coated on the original zeolites through co-precipitation method. The prepared Zn-LDHs-modified and original zeolites were characterized by scanning electron microscope (SEM) and energy dispersive spectroscopy (EDS) methods, whose results provided the evidences that the Zn-LDHs were successfully coated on the original zeolites. From the results of purification experiments, the average Cd removal rates of ZnAl-LDHs-modified, ZnFe-LDHs-modified and original zeolites were 88.40, 86.00 and 32.52%, respectively; demonstrating that the removal rates of zeolites could significantly improve. Additionally, the modification of Zn-LDHS could enhance the theoretical adsorption ability. According to the results of isothermal adsorption and desorption tests, the desorption rates of Zn-LDHs-modified zeolites were higher than that of original zeolites. Cd adsorption capacity of ZnFe-LDHs-modified zeolites was 1428.57 mg kg-1 and original zeolites was 434.783 mg kg-1. In the adsorption kinetic studies, the pseudo-second-order models were used to well describe the experimental results of Zn-LDHs-modified zeolites, indicating that their adsorption types were attributed to be more stable chemisorption. Besides, the relevant microbial tests also confirmed that microbial enzymatic activity and extracellular polymeric substances (EPS) were significantly promoted on surface of Zn-LDHs-modified zeolites. The contents of EPS on the surface of zeolites were as following: ZnAl-LDHs-modified zeolites (78.58128 µg/g) > ZnFe-LDHs-modified zeolites (71.85445 µg/g) > original zeolites (68.69904 µg/g). Meanwhile, the results of high-throughput sequencing showed that modification by Zn-LDHs improved microbial diversity and relative abundance. The Proteobacteria was the dominant phylum and the Acidobacteria was conducive to Cd removal. Overall, it could be concluded that ZnAl-LDHs-modified zeolites might be applied as an efficient substrate for Cd removal in CRIS.

Species interactions and distinct microbial communities in high Arctic permafrost affected cryosols are associated with the CH4 and CO2 gas fluxes.

Microbial metabolism of the thawing organic carbon stores in permafrost results in a positive feedback loop of greenhouse gas emissions. CO2 and CH4 fluxes and the associated microbial communities in Arctic cryosols are important in predicting future warming potential of the Arctic. We demonstrate that topography had an impact on CH4 and CO2 flux at a high Arctic ice-wedge polygon terrain site, with higher CO2 emissions and lower CH4 uptake at troughs compared to polygon interior soils. The pmoA sequencing suggested that USCα cluster of uncultured methanotrophs is likely responsible for observed methane sink. Community profiling revealed distinct assemblages across the terrain at different depths. Deeper soils contained higher abundances of Verrucomicrobia and Gemmatimonadetes, whereas the polygon interior had higher Acidobacteria and lower Betaproteobacteria and Deltaproteobacteria abundances. Genome sequencing of isolates from the terrain revealed presence of carbon cycling genes including ones involved in serine and ribulose monophosphate pathways. A novel hybrid network analysis identified key members that had positive and negative impacts on other species. Operational Taxonomic Units (OTUs) with numerous positive interactions corresponded to Proteobacteria, Candidatus Rokubacteria and Actinobacteria phyla, while Verrucomicrobia and Acidobacteria members had negative impacts on other species. Results indicate that topography and microbial interactions impact community composition.

Reaction centers of the thermophilic microaerophile, Chloracidobacterium thermophilum (Acidobacteria) I: biochemical and biophysical characterization.

Chloracidobacterium thermophilum is a microaerophilic, anoxygenic member of the green chlorophototrophic bacteria. This bacterium is the first characterized oxygen-requiring chlorophototroph with chlorosomes, the FMO protein, and homodimeric type-1 reaction centers (RCs). The RCs of C. thermophilum are also unique because they contain three types of chlorophylls, bacteriochlorophyll aP esterified with phytol, Chl aPD esterified with Δ2,6-phytadienol, and Zn-BChl aP' esterified with phytol, in the approximate molar ratio 32:24:4. The light-induced difference spectrum of these RCs had a bleaching maximum at 839 nm and also revealed an electrochromic bandshift that is probably derived from a BChl a molecule near P840+. The FX [4Fe-4S] cluster had a midpoint potential of ca. - 581 mV, and the spectroscopic properties of the P+ F X - spin-polarized radical pair were very similar to those of reaction centers of heliobacteria and green sulfur bacteria. The data further indicate that electron transfer occurs directly from A0- to FX, as occurs in other homodimeric type-1 RCs. Washing experiments with isolated membranes suggested that the PscB subunit of these reaction centers is more tightly bound than PshB in heliobacteria. Thus, the reaction centers of C. thermophilum have some properties that resemble other homodimeric reaction centers but also have specific properties that are more similar to those of Photosystem I. These differences probably contribute to protection of the electron transfer chain from oxygen, contributing to the oxygen tolerance of this microaerophile.

The type IV pilus assembly motor PilB is a robust hexameric ATPase with complex kinetics.

The bacterial type IV pilus (T4P) is a versatile nanomachine that functions in pathogenesis, biofilm formation, motility, and horizontal gene transfer. T4P assembly is powered by the motor ATPase PilB which is proposed to hydrolyze ATP by a symmetrical rotary mechanism. This mechanism, which is deduced from the structure of PilB, is untested. Here, we report the first kinetic studies of the PilB ATPase, supporting co-ordination among the protomers of this hexameric enzyme. Analysis of the genome sequence of Chloracidobacterium thermophilum identified a pilB gene whose protein we then heterologously expressed. This PilB formed a hexamer in solution and exhibited highly robust ATPase activity. It displays complex steady-state kinetics with an incline followed by a decline over an ATP concentration range of physiological relevance. The incline is multiphasic and the decline signifies substrate inhibition. These observations suggest that variations in intracellular ATP concentrations may regulate T4P assembly and T4P-mediated functions in vivo in accordance with the physiological state of bacteria with unanticipated complexity. We also identified a mutant pilB gene in the genomic DNA of C. thermophilum from an enrichment culture. The mutant PilB variant, which is significantly less active, exhibited similar inhibition of its ATPase activity by high concentrations of ATP. Our findings here with the PilB ATPase from C. thermophilum provide the first line of biochemical evidence for the co-ordination among PilB protomers consistent with the symmetrical rotary model of catalysis based on structural studies.

Influence of application of manganese ore in constructed wetlands on the mechanisms and improvement of nitrogen and phosphorus removal.

Vertical up-flow constructed wetlands (CWs) with manganese ore (Mn ore) as media (M-CWs) were developed to treat simulated polluted river water. The results showed that the average removal efficiencies for NH4-N, NO3-N, TN and TP were 91.74%, 83.29%, 87.47% and 65.12% in M-CWs, respectively, which were only 79.12%, 72.90%, 75.85% and 43.23% in the CWs without Mn ore (C-CWs). Nutrient mass balance showed that nitrogen (N) removal was improved by enhanced microbial processes, media storage and plant uptake in M-CWs. Moreover, almost 50% of phosphorus (P) was retained by media storage because of the adsorption processes on Mn ore. It was found that addition of Mn ore enhanced denitrification as the relative abundance of denitrifying bacteria increased. The produced Mn(II) and more abundant Gammaproteobacteria confirmed alternative N removal pathways including anoxic nitrification coupled to Mn ore reduction and denitrification using Mn(II) as electron donor. Mn(II) concentration in the effluent of M-CWs was below the drinking water limit of 0.1 mg/L, which makes them environmentally-friendly.

Genomic insights into the Acidobacteria reveal strategies for their success in terrestrial environments.

Members of the phylum Acidobacteria are abundant and ubiquitous across soils. We performed a large-scale comparative genome analysis spanning subdivisions 1, 3, 4, 6, 8 and 23 (n = 24) with the goal to identify features to help explain their prevalence in soils and understand their ecophysiology. Our analysis revealed that bacteriophage integration events along with transposable and mobile elements influenced the structure and plasticity of these genomes. Low- and high-affinity respiratory oxygen reductases were detected in multiple genomes, suggesting the capacity for growing across different oxygen gradients. Among many genomes, the capacity to use a diverse collection of carbohydrates, as well as inorganic and organic nitrogen sources (such as via extracellular peptidases), was detected - both advantageous traits in environments with fluctuating nutrient environments. We also identified multiple soil acidobacteria with the potential to scavenge atmospheric concentrations of H2 , now encompassing mesophilic soil strains within the subdivision 1 and 3, in addition to a previously identified thermophilic strain in subdivision 4. This large-scale acidobacteria genome analysis reveal traits that provide genomic, physiological and metabolic versatility, presumably allowing flexibility and versatility in the challenging and fluctuating soil environment.

15N photo-CIDNP MAS NMR analysis of reaction centers of Chloracidobacterium thermophilum.

Photochemically induced dynamic nuclear polarization (photo-CIDNP) has been observed in the homodimeric, type-1 photochemical reaction centers (RCs) of the acidobacterium, Chloracidobacterium (Cab.) thermophilum, by 15N magic-angle spinning (MAS) solid-state NMR under continuous white-light illumination. Three light-induced emissive (negative) signals are detected. In the RCs of Cab. thermophilum, three types of (bacterio)chlorophylls have previously been identified: bacteriochlorophyll a (BChl a), chlorophyll a (Chl a), and Zn-bacteriochlorophyll a' (Zn-BChl a') (Tsukatani et al. in J Biol Chem 287:5720-5732, 2012). Based upon experimental and quantum chemical 15N NMR data, we assign the observed signals to a Chl a cofactor. We exclude Zn-BChl because of its measured spectroscopic properties. We conclude that Chl a is the primary electron acceptor, which implies that the primary donor is most likely Zn-BChl a'. Chl a and 81-OH Chl a have been shown to be the primary electron acceptors in green sulfur bacteria and heliobacteria, respectively, and thus a Chl a molecule serves this role in all known homodimeric type-1 RCs.

Microbial community changes during a toxic cyanobacterial bloom in an alkaline Hungarian lake.

The Carpathian Basin is a lowland plain located mainly in Hungary. Due to the nature of the bedrock, alluvial deposits, and a bowl shape, many lakes and ponds of the area are characterized by high alkalinity. In this study, we characterized temporal changes in eukaryal and bacterial community dynamics with high throughput sequencing and relate the changes to environmental conditions in Lake Velence located in Fejér county, Hungary. The sampled Lake Velence microbial populations (algal and bacterial) were analyzed to identify potential correlations with other community members and environmental parameters at six timepoints over 6 weeks in the Spring of 2012. Correlations between community members suggest a positive relationship between certain algal and bacterial populations (e.g. Chlamydomondaceae with Actinobacteria and Acidobacteria), while other correlations allude to changes in these relationships over time. During the study, high nitrogen availability may have favored non-nitrogen fixing cyanobacteria, such as the toxin-producing Microcystis aeruginosa, and the eutrophic effect may have been exacerbated by high phosphorus availability as well as the high calcium and magnesium content of the Carpathian Basin bedrock, potentially fostering exopolymer production and cell aggregation. Cyanobacterial bloom formation could have a negative environmental impact on other community members and potentially affect overall water quality as well as recreational activities. To our knowledge, this is the first prediction for relationships between photoautotrophic eukaryotes and bacteria from an alkaline, Hungarian lake.

Persistence of the dominant soil phylum Acidobacteria by trace gas scavenging.

The majority of microbial cells in global soils exist in a spectrum of dormant states. However, the metabolic processes that enable them to survive environmental challenges, such as nutrient-limitation, remain to be elucidated. In this work, we demonstrate that energy-starved cultures of Pyrinomonas methylaliphatogenes, an aerobic heterotrophic acidobacterium isolated from New Zealand volcanic soils, persist by scavenging the picomolar concentrations of H2 distributed throughout the atmosphere. Following the transition from exponential to stationary phase due to glucose limitation, the bacterium up-regulates by fourfold the expression of an eight-gene operon encoding an actinobacteria-type H2-uptake [NiFe]-hydrogenase. Whole-cells of the organism consume atmospheric H2 in a first-order kinetic process. Hydrogen oxidation occurred most rapidly under oxic conditions and was weakly associated with the cell membrane. We propose that atmospheric H2 scavenging serves as a mechanism to sustain the respiratory chain of P. methylaliphatogenes when organic electron donors are scarce. As the first observation of H2 oxidation to our knowledge in the Acidobacteria, the second most dominant soil phylum, this work identifies new sinks in the biogeochemical H2 cycle and suggests that trace gas oxidation may be a general mechanism for microbial persistence.

Distinct Anaerobic Bacterial Consumers of Cellobiose-Derived Carbon in Boreal Fens with Different CO2/CH4 Production Ratios.

Northern peatlands in general have high methane (CH4) emissions, but individual peatlands show considerable variation as CH4 sources. Particularly in nutrient-poor peatlands, CH4 production can be low and exceeded by carbon dioxide (CO2) production from unresolved anaerobic processes. To clarify the role anaerobic bacterial degraders play in this variation, we compared consumers of cellobiose-derived carbon in two fens differing in nutrient status and the ratio of CO2 to CH4 produced. After [13C]cellobiose amendment, the mesotrophic fen produced equal amounts of CH4 and CO2 The oligotrophic fen had lower CH4 production but produced 3 to 59 times more CO2 than CH4 RNA stable-isotope probing revealed that in the mesotrophic fen with higher CH4 production, cellobiose-derived carbon was mainly assimilated by various recognized fermenters of Firmicutes and by Proteobacteria The oligotrophic peat with excess CO2 production revealed a wider variety of cellobiose-C consumers, including Firmicutes and Proteobacteria, but also more unconventional degraders, such as Telmatobacter-related Acidobacteria and subphylum 3 of Verrucomicrobia Prominent and potentially fermentative Planctomycetes and Chloroflexi did not appear to process cellobiose-C. Our results show that anaerobic degradation resulting in different levels of CH4 production can involve distinct sets of bacterial degraders. By distinguishing cellobiose degraders from the total community, this study contributes to defining anaerobic bacteria that process cellulose-derived carbon in peat. Several of the identified degraders, particularly fermenters and potential Fe(III) or humic substance reducers in the oligotrophic peat, represent promising candidates for resolving the origin of excess CO2 production in peatlands. IMPORTANCE: Peatlands are major sources of the greenhouse gas methane (CH4), yet in many peatlands, CO2 production from unresolved anaerobic processes exceeds CH4 production. Anaerobic degradation produces the precursors of CH4 production but also represents competing processes. We show that anaerobic degradation leading to high or low CH4 production involved distinct sets of bacteria. Well-known fermenters dominated in a peatland with high CH4 production, while novel and unconventional degraders could be identified in a site where CO2 production greatly exceeds CH4 production. Our results help identify and assign functions to uncharacterized bacteria that promote or inhibit CH4 production and reveal bacteria potentially producing the excess CO2 in acidic peat. This study contributes to understanding the microbiological basis for different levels of CH4 emission from peatlands.

Acidicapsa ferrireducens sp. nov., Acidicapsa acidiphila sp. nov., and Granulicella acidiphila sp. nov.: novel acidobacteria isolated from metal-rich acidic waters.

Four novel strains of Acidobacteria were isolated from water samples taken from pit lakes at two abandoned metal mines in the Iberian Pyrite Belt mining district, south-west Spain. Three of the isolates belong to the genus Acidicapsa (MCF9T, MCF10T, and MCF14) and one of them to the genus Granulicella (MCF40T). All isolates are moderately acidophilic (pH growth optimum 3.8-4.1) and mesophilic (temperature growth optima 30-32 °C). Isolates MCF10T and MCF40T grew at pH lower (<3.0) than previously reported for all other acidobacteria. All four strains are obligate heterotrophs and metabolised a wide range of sugars. While all four isolates are obligate aerobes, MCF9T, MCF10T, and MCF14 catalysed the reductive dissolution of the ferric iron mineral schwertmannite when incubated under micro-aerobic conditions. Isolates MCF9T and MCF14 shared 99.5% similarity of their 16 S rRNA genes, and were considered to be strains of the same species. The major quinone of strains MCF10T, MCF9T, and MCF40T is MK-8, and their DNA G + C contents are 60.0, 59.7, and 62.1 mol%, respectively. Based on phylogenetic and phenotypic data, three novel species, Acidicapsa ferrireducens strain MCF9T (=DSM 28997T = NCCB 100575T), Acidicapsa acidiphila strain MCF10T (=DSM 29819T = NCCB 100576T), and Granulicella acidiphila strain MCF40T (DSM 28996T = NCCB 100577T), are proposed.

The Relative Abundance and Transcriptional Activity of Marine Sponge-Associated Microorganisms Emphasizing Groups Involved in Sulfur Cycle.

During the last decades, our knowledge about the activity of sponge-associated microorganisms and their contribution to biogeochemical cycling has gradually increased. Functional groups involved in carbon and nitrogen metabolism are well documented, whereas knowledge about microorganisms involved in the sulfur cycle is still limited. Both sulfate reduction and sulfide oxidation has been detected in the cold water sponge Geodia barretti from Korsfjord in Norway, and with specimens from this site, the present study aims to identify extant versus active sponge-associated microbiota with focus on sulfur metabolism. Comparative analysis of small subunit ribosomal RNA (16S rRNA) gene (DNA) and transcript (complementary DNA (cDNA)) libraries revealed profound differences. The transcript library was predominated by Chloroflexi despite their low abundance in the gene library. An opposite result was found for Acidobacteria. Proteobacteria were detected in both libraries with representatives of the Alpha- and Gammaproteobacteria related to clades with presumably thiotrophic bacteria from sponges and other marine invertebrates. Sequences that clustered with sponge-associated Deltaproteobacteria were remotely related to cultivated sulfate-reducing bacteria. The microbes involved in sulfur cycling were identified by the functional gene aprA (adenosine-5'-phosphosulfate reductase) and its transcript. Of the aprA sequences (DNA and cDNA), 87 % affiliated with sulfur-oxidizing bacteria. They clustered with Alphaproteobacteria and with clades of deep-branching Gammaproteobacteria. The remaining sequences clustered with sulfate-reducing Archaea of the phylum Euryarchaeota. These results indicate an active role of yet uncharacterized Bacteria and Archaea in the sponge's sulfur cycle.

Maize growth responses to soil microbes and soil properties after fertilization with different green manures.

The use of green manures in agriculture can provide nutrients, affect soil microbial communities, and be a more sustainable management practice. The activities of soil microbes can effect crop growth, but the extent of this effect on yield remains unclear. We investigated soil bacterial communities and soil properties under four different green manure fertilization regimes (Vicia villosa, common vetch, milk vetch, and radish) and determined the effects of these regimes on maize growth. Milk vetch showed the greatest potential for improving crop productivity and increased maize yield by 31.3 %. This change might be related to changes in soil microbes and soil properties. The entire soil bacterial community and physicochemical properties differed significantly among treatments, and there were significant correlations between soil bacteria, soil properties, and maize yield. In particular, abundance of the phyla Acidobacteria and Verrucomicrobia was positively correlated with maize yield, while Proteobacteria and Chloroflexi were negatively correlated with yield. These data suggest that the variation of maize yield was related to differences in soil bacteria. The results also indicate that soil pH, alkali solution nitrogen, and available potassium were the key environmental factors shaping soil bacterial communities and determining maize yields. Both soil properties and soil microbes might be useful as indicators of soil quality and potential crop yield.

The first acidobacterial laccase-like multicopper oxidase revealed by metagenomics shows high salt and thermo-tolerance.

Metagenomics is a powerful tool that allows identifying enzymes with novel properties from the unculturable component of microbiomes. However, thus far only a limited number of laccase or laccase -like enzymes identified through metagenomics has been subsequently biochemically characterized. This work describes the successful bio-mining of bacterial laccase-like enzymes in an acidic bog soil metagenome and the characterization of the first acidobacterial laccase-like multicopper oxidase (LMCO). LMCOs have hitherto been mostly studied in fungi and some have already found applications in diverse industries. However, improved LMCOs are in high demand. Using molecular screening of a small metagenomic library (13,500 clones), a gene encoding a three-domain LMCO (LacM) was detected, showing the highest similarity to putative copper oxidases of Candidatus Solibacter (Acidobacteria). The encoded protein was expressed in Escherichia coli, purified by affinity chromatography and biochemically characterized. LacM oxidized a variety of phenolic substrates, including two standard laccase substrates (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), k cat/k M  = 8.45 s-1 mM-1; 2,6-dimethoxyphenol (2,6-DMP), k cat/k M  = 6.42 s-1 mM-1), next to L-3,4-dihydroxyphenylalanine (L-DOPA), vanillic acid, syringaldazine, pyrogallol, and pyrocatechol. With respect to the latter two lignin building blocks, LacM showed the highest catalytic activity (k cat/k M  = 173.6 s-1 mM-1) for pyrogallol, with ca. 20% activity preserved even at pH 8.0. The enzyme was thermostable and heat-activated in the interval 40-60 °C, with an optimal activity on ABTS at 50 °C. It was rather stable at high salt concentration (e.g., 34% activity preserved at 500 mM NaCl) and in the presence of organic solvents. Remarkably, LacM decolored azo and triphenylmethane dyes, also in the absence of redox mediators.