Diazotrophic Community Variation Underlies Differences in Nitrogen Fixation Potential in Paddy Soils Across a Climatic Gradient in China.

Biological nitrogen (N2) fixation as a source of new N input into the soil by free-living diazotrophs is important for achieving sustainable rice agriculture. However, the dominant environmental drivers or factors influencing N2 fixation and the functional significance of the diazotroph community structure in paddy soil across a climatic gradient are not yet well understood. Thus, we characterized the diazotroph community and identified the ecological predictors of N2 fixation potential in four different climate zones (mid-temperate, warm-temperate, subtropical, and tropical paddy soils) in eastern China. Comprehensive nifH gene sequencing, functional activity detection, and correlation analysis with environmental factors were estimated. The potential nitrogenase activity (PNA) was highest in warm-temperate regions, where it was 6.2-, 2.9-, and 2.2-fold greater than in the tropical, subtropical, and mid-temperate regions, respectively; nifH gene abundance was significantly higher in warm-temperate and subtropical zones than in the tropical or mid-temperate zones. Diazotroph diversity was significantly higher in the tropical climate zone and significantly lower in the mid-temperate zone. Non-metric multidimensional scaling and canonical correlation analysis indicated that paddy soil diazotroph populations differed significantly among the four climate zones, mainly owing to differences in climate and soil pH. Structural equation models and automatic linear models revealed that climate and nutrients indirectly affected PNA by affecting soil pH and diazotroph community, respectively, while diazotroph community, C/P, and nifH gene abundance directly affected PNA. And C/P ratio, pH, and the diazotroph community structure were the main predictors of PNA in paddy soils. Collectively, the differences in diazotroph community structure have ecological significance, with important implications for the prediction of soil N2-fixing functions under climate change scenarios.

Publisher Correction: Nitric oxide-dependent anaerobic ammonium oxidation.

The original HTML version of this Article had an incorrect Published online date of 20 March 2019; it should have been 18 March 2019. This has been corrected in the HTML version of the Article. The PDF version was correct from the time of publication.

Nitric oxide-dependent anaerobic ammonium oxidation.

Nitric oxide (NO) has important functions in biology and atmospheric chemistry as a toxin, signaling molecule, ozone depleting agent and the precursor of the greenhouse gas nitrous oxide (N2O). Although NO is a potent oxidant, and was available on Earth earlier than oxygen, it is unclear whether NO can be used by microorganisms for growth. Anaerobic ammonium-oxidizing (anammox) bacteria couple nitrite reduction to ammonium oxidation with NO and hydrazine as intermediates, and produce N2 and nitrate. Here, we show that the anammox bacterium Kuenenia stuttgartiensis is able to grow in the absence of nitrite by coupling ammonium oxidation to NO reduction, and produce only N2. Under these growth conditions, the transcription of proteins necessary for NO generation is downregulated. Our work has potential implications in the control of N2O and NO emissions from natural and manmade ecosystems, where anammox bacteria contribute significantly to N2 release to the atmosphere. We hypothesize that microbial NO-dependent ammonium oxidation may have existed on early Earth.

Prognostic value of tumor-infiltrating Foxp3+ regulatory T cells in patients with breast cancer: a meta-analysis.

PURPOSE: The prognostic value of tumor-infiltrating Foxp3+ regulatory T cells (Tregs) in breast cancer remains controversial. Therefore, we performed this meta-analysis to determine the impact of Foxp3+ Tregs infiltration on survival outcomes. METHODS: Relevant literature was retrieved from Pubmed, Web of science and Cocohrane until May 30, 2016. Meta-analysis was performed using hazard ratios (HRs), odds ratio (OR) and 95 % confidence intervals (CI) as effect measures. RESULTS: Fourteen studies (10,259 patients) were included. Meta-analysis showed that high Foxp3+ Tregs infiltration was correlated with high histological grade (OR= 2.96, 95%CI [2.03-4.31]), estrogen receptor (ER) negativity (OR= 0.38, 95%CI [0.23-0.60]), human epidermal growth factor receptor type 2 (HER2) positivity (OR=2.43, 95%CI [1.69-3.51]). The detection of FOXP3+ Tregs was significantly associated with recurrence-free survival (RFS) of patients (HR = 1.58, 95 % CI [1.03-2.44]). CONCLUSION: Our meta-analysis suggests that high Foxp3+ Tregs infiltration is associated with poor RFS in breast cancer patients and predicts histological grade, estrogen receptor and HER-2 status.

Interleukin-8 upregulates integrin ß3 expression and promotes estrogen receptor-negative breast cancer cell invasion by activating the PI3K/Akt/NF-κB pathway.

Interleukin-8 (IL-8) possesses tumorigenic and proangiogenic properties and is overexpressed in many human cancers. The integrin family regulates a diverse array of cellular functions crucial to the initiation, progression and metastasis of solid tumors. However, the mechanisms of action of IL-8 and integrin in estrogen receptor-negative breast cancer are largely unknown. In this study, IL-8 and integrin ß3 expression in human breast cancer cells and tissues was examined by real-time PCR, Western blot and immunochemistry analysis. Integrin ß3 expression, invasive ability and the activation of PI3K/Akt and NF-κB pathways in IL-8 knockdown breast cancer cells were evaluated. In addition, reporter assay and ChIP were performed to assess integrin ß3 promoter activity in IL-8 knockdown cells. We observed a positive correlation between integrin ß3 and IL-8 expression, which was inversely correlated with ER status in breast cancer cell lines and tissues. IL-8 siRNA decreased the invasion and integrin ß3 expression in human breast cancer cells. Moreover, IL-8 siRNA attenuated the phosphorylation of PI3K and Akt and inhibited NF-κB activity and binding on integrin ß3 promoter. IL-8 siRNA diminished NF-κB nuclear translocation via blocking IκB phosphorylation in the cytoplasm. In conclusion, IL-8 activates the PI3K/Akt pathway, which in turn activates NF-κB, resulting in the upregulation of integrin ß3 expression and increased invasion of estrogen receptor-negative breast cancer cells. IL-8/PI3K/Akt/NF-κB/integrin ß3 axis may be exploited for therapeutic intervention to breast cancer metastasis.

Community size and composition of ammonia oxidizers and denitrifiers in an alluvial intertidal wetland ecosystem.

Global nitrogen cycling is mainly mediated by the activity of microorganisms. Nitrogen cycle processes are mediated by functional groups of microorganisms that are affected by constantly changing environmental conditions and substrate availability. In this study, we investigated the temporal and spatial patterns of nitrifier and denitrifier communities in an intertidal wetland. Soil samples were collected over four distinct seasons from three locations with different vegetative cover. Multiple environmental factors and process rates were measured and analyzed together with the community size and composition profiles. We observed that the community size and composition of the nitrifiers and denitrifiers are affected significantly by seasonal factors, while vegetative cover affected the community composition. The seasonal impacts on the community size of ammonia oxidizing archaea (AOA) are much higher than that of ammonia oxidizing bacteria (AOB). The seasonal change was a more important indicator for AOA community composition patterns, while vegetation was more important for the AOB community patterns. The microbial process rates were correlated with both the community size and composition.

The first complete genome sequence of the class Fimbriimonadia in the phylum Armatimonadetes.

In this study, we present the complete genome of Fimbriimonas ginsengisoli Gsoil 348T belonging to the class Fimbriimonadia of the phylum Armatimonadetes, formerly called as candidate phylum OP10. The complete genome contains a single circular chromosome of 5.23 Mb including a 45.5 kb prophage. Of the 4820 open reading frames (ORFs), 3,000 (62.2%) genes could be classified into Clusters of Orthologous Groups (COG) families. With the split of rRNA genes, strain Gsoil 348T had no typical 16S-23S-5S ribosomal RNA operon. In this genome, the GC skew inversion which was usually observed in archaea was found. The predicted gene functions suggest that the organism lacks the ability to synthesize histidine, and the TCA cycle is incomplete. Phylogenetic analyses based on ribosomal proteins indicated that strain Gsoil 348T represents a deeply branching lineage of sufficient divergence with other phyla, but also strongly involved in superphylum Terrabacteria.

High specific activity for anammox bacteria enriched from activated sludge at 10°C.

Anammox in the water line of a waste water treatment plant (WWTP) saves energy for aeration and allows for recovering biogas from organic material. Main challenges for applying the anammox process in the water line are related to the low temperature of <20°C, causing a significant drop in the specific anammox activity. The aim of this research was to enrich a cold-adapted anammox species, with a high specific activity. This was achieved in a 4.2L reactor operated at 10°C, fed with 61 mg (NH4+NO2)-N/L and inoculated with activated sludge from two selected municipal WWTPs. Candidatus Brocadia fulgida was the dominant species in the enriched biomass, with a specific activity was 30-44 mg N/(g VS d). This is two times higher than previously reported at 10°C, which is beneficial for full scale application. Biomass yield was 0.046 g biomass/g N converted, similar to that at higher temperatures.

Lysozyme and penicillin inhibit the growth of anaerobic ammonium-oxidizing planctomycetes.

Anaerobic ammonium-oxidizing (anammox) planctomycetes oxidize ammonium in the absence of molecular oxygen with nitrite as the electron acceptor. Although planctomycetes are generally assumed to lack peptidoglycan in their cell walls, recent genome data imply that the anammox bacteria have the genes necessary to synthesize peptidoglycan-like cell wall structures. In this study, we investigated the effects of two antibacterial agents that target the integrity and synthesis of peptidoglycan (lysozyme and penicillin G) on the anammox bacterium Kuenenia stuttgartiensis. The effects of these compounds were determined in both short-term batch incubations and long-term (continuous-cultivation) growth experiments in membrane bioreactors. Lysozyme at 1 g/liter (20 mM EDTA) lysed anammox cells in less than 60 min, whereas penicillin G did not have any observable short-term effects on anammox activity. Penicillin G (0.5, 1, and 5 g/liter) reversibly inhibited the growth of anammox bacteria in continuous-culture experiments. Furthermore, transcriptome analyses of the penicillin G-treated reactor and the control reactor revealed that penicillin G treatment resulted in a 10-fold decrease in the ribosome levels of the cells. One of the cell division proteins (Kustd1438) was downregulated 25-fold. Our results suggested that anammox bacteria contain peptidoglycan-like components in their cell wall that can be targeted by lysozyme and penicillin G-sensitive proteins were involved in their synthesis. Finally, we showed that a continuous membrane reactor system with free-living planktonic cells was a very powerful tool to study the physiology of slow-growing microorganisms under physiological conditions.

Nitrogen removal with the anaerobic ammonium oxidation process.

Anaerobic ammonium-oxidizing (anammox) bacteria convert ammonium to N2 with nitrite as the terminal electron acceptor in the absence of O2. Nitritation-anammox bioreactors provide a cost-effective and environment-friendly alternative to conventional nitrification/denitrification nitrogen removal systems. Currently, this process is only applied for ammonium removal from wastewater with high ammonium load and temperature. Nevertheless, recent results obtained with laboratory-scale bioreactors suggest new possible routes of application of the Nitritation-anammox technology including (1) municipal wastewater treatment, removal of (2) methane in combination with nitrite-reducing methane-oxidizing bacteria, (3) nitrate coupled to organic acid oxidation and (4) nitrogen oxides. The current review summarizes the state-of-the-art of the application of Nitritation-anammox systems and discusses the possibilities of utilizing these recent results for wastewater treatment.

Nitrogen removal by a nitritation-anammox bioreactor at low temperature.

Currently, nitritation-anammox (anaerobic ammonium oxidation) bioreactors are designed to treat wastewaters with high ammonium concentrations at mesophilic temperatures (25 to 40°C). The implementation of this technology at ambient temperatures for nitrogen removal from municipal wastewater following carbon removal may lead to more-sustainable technology with energy and cost savings. However, the application of nitritation-anammox bioreactors at low temperatures (characteristic of municipal wastewaters except in tropical and subtropical regions) has not yet been explored. To this end, a laboratory-scale (5-liter) nitritation-anammox sequencing batch reactor was adapted to 12°C in 10 days and operated for more than 300 days to investigate the feasibility of nitrogen removal from synthetic pretreated municipal wastewater by the combination of aerobic ammonium-oxidizing bacteria (AOB) and anammox. The activities of both anammox and AOB were high enough to remove more than 90% of the supplied nitrogen. Multiple aspects, including the presence and activity of anammox, AOB, and aerobic nitrite oxidizers (NOB) and nitrous oxide (N2O) emission, were monitored to evaluate the stability of the bioreactor at 12°C. There was no nitrite accumulation throughout the operational period, indicating that anammox bacteria were active at 12°C and that AOB and anammox bacteria outcompeted NOB. Moreover, our results showed that sludge from wastewater treatment plants designed for treating high-ammonium-load wastewaters can be used as seeding sludge for wastewater treatment plants aimed at treating municipal wastewater that has a low temperature and low ammonium concentrations.

Metagenome Analysis of a Complex Community Reveals the Metabolic Blueprint of Anammox Bacterium "Candidatus Jettenia asiatica".

Anaerobic ammonium-oxidizing (anammox) bacteria are key players in the global nitrogen cycle and responsible for significant global nitrogen loss. Moreover, the anammox process is widely implemented for nitrogen removal from wastewaters as a cost-effective and environment-friendly alternative to conventional nitrification-denitrification systems. Currently, five genera of anammox bacteria have been identified, together forming a deep-branching order in the Planctomycetes-Verrucomicrobium-Chlamydiae superphylum. Members of all genera have been detected in wastewater treatment plants and have been enriched in lab-scale bioreactors, but genome information is not yet available for all genera. Here we report the metagenomic analysis of a granular sludge anammox reactor dominated (∼50%) by "Candidatus Jettenia asiatica." The metagenome was sequenced using both Illumina and 454 pyrosequencing. After de novo assembly 37,432 contigs with an average length of 571 nt were obtained. The contigs were then analyzed by BLASTx searches against the protein sequences of "Candidatus Kuenenia stuttgartiensis" and a set of 25 genes essential in anammox metabolism were detected. Additionally all reads were mapped to the genome of an anammox strain KSU-1 and de novo assembly was performed again using the reads that could be mapped on KSU-1. Using this approach, a gene encoding copper-containing nitrite reductase NirK was identified in the genome, instead of cytochrome cd(1)-type nitrite reductase (NirS, present in "Ca. Kuenenia stuttgartiensis" and "Ca. Scalindua profunda"). Finally, the community composition was investigated through MetaCluster analysis, 16S rRNA gene analysis and read mapping, which showed the presence of other important community members such as aerobic ammonia-oxidizing bacteria, methanogens, and the denitrifying methanotroph "Ca. Methylomirabilis oxyfera", indicating a possible active methane and nitrogen cycle in the bioreactor under the prevailing operational conditions.

Description of Fimbriimonas ginsengisoli gen. nov., sp. nov. within the Fimbriimonadia class nov., of the phylum Armatimonadetes.

Strain Gsoil 348(T) was isolated from a ginseng field soil sample by selecting micro-colonies from one-fifth strength modified R2A agar medium after a long incubation period. 16S rRNA gene sequence analysis indicated that the strain is related to members of the phylum Armatimonadetes (formerly called candidate phylum OP10). Strain Gsoil 348(T) is mesophilic, strictly aerobic, non-motile and rod-shaped. It only grows in low nutrient media. The major respiratory quinones are menaquinones MK-11 and MK-10, and the main fatty acids are iso-C(15:0), iso-C(17:0), C(16:0) and C(16:1) ω11c. The G+C content is 61.4 mol%. The 16S rRNA gene sequences in public databases belonging to the phylum Armatimonadetes were clustered here into 6 groups. Five of these groups constituted a coherent cluster distinct from the sequences of other phyla in phylogenetic trees that were constructed using multiple-outgroup sequences from 49 different phyla. On the basis of polyphasic taxonomic analyses, it is proposed that strain Gsoil 348(T) (= KACC 14959(T) = JCM 17079(T)) should be placed in Fimbriimonas ginsengisoli gen. nov., sp. nov., as the cultured representative of the Fimbriimonadia class. nov., corresponding with Group 4 of the phylum Armatimonadetes.

Rapid and simple cryopreservation of anaerobic ammonium-oxidizing bacteria.

A quick and simple protocol for long-term cryopreservation of anaerobic ammonium-oxidizing bacteria (anammox bacteria) was developed. After 29 weeks of preservation at -80°C, activity recovery for all tested cultures under at least one of the applied sets of preservation conditions was observed. Growth recovery was also demonstrated for a single-cell culture of "Candidatus Kuenenia stuttgartiensis."

Bacterial diversity of water and sediment in the Changjiang estuary and coastal area of the East China Sea.

The Changjiang estuary and the coastal area of the East China Sea (ECS) represent important interfaces of terrestrial and marine environments. This study included analyses of water and sediments collected during different seasons in these regions to determine the composition of microbial assemblages by means of 16S rRNA gene clone libraries. We retrieved 1946 sequences and 779 distinct operational taxonomic units from 36 clone libraries. Shannon-Weaver diversity index values and rarefaction analysis indicated that bacterial diversity in the sediment samples was much higher than in the water samples. Proteobacteria (72.9%) was the most abundant phylum, followed by Firmicutes (6.4%), Bacteroidetes (4.6%) and Actinobacteria (4.1%). In the water, clone sequences related to Alphaproteobacteria were the most abundant, whereas in the sediment samples, sequences affiliated with Gammaproteobacteria were predominant. Principal coordinate analysis showed that water samples collected from the Changjiang estuary and the ECS clustered separately. However, this spatial pattern could not be observed in sediment samples, which were mainly distinguished from one another by the season. Bacterial diversity in the Changjiang estuary was higher than that in the ECS, which may be the result of the mixing of bacterial communities from the Changjiang River, the estuary and the coastal ocean.