Arabidopsis Growth-Promotion and Root Architecture Responses to the Beneficial Rhizobacterium Phyllobacterium brassicacearum Strain STM196 Are Independent of the Nitrate Assimilatory Pathway

Phyllobacterium brassicacearum STM196, a plant growth-promoting rhizobacterium isolated from roots of oilseed rape, stimulates Arabidopsis growth. We have previously shown that the NRT2.5 and NRT2.6 genes are required for this growth promotion response. Since these genes are members of the NRT2 family of nitrate transporters, the nitrogen assimilatory pathway could be involved in growth promotion by STM196. We address this hypothesis using two nitrate reductase mutants, G5 deleted in the major nitrate reductase gene NIA2 and G′4-3 altered in both NIA1 and NIA2 genes. Both mutants had a reduced growth rate and STM196 failed to increase their biomass production on a medium containing NO3− as the sole nitrogen source. However, they both displayed similar growth promotion by STM196 when grown on an NH4+ medium. STM196 was able to stimulate lateral roots development of the mutants under both nutrition conditions. Altogether, our results indicate that the nitrate assimilatory metabolism is not a primary target of STM196 interaction and is not involved in the root developmental response. The NIA1 transcript level was reduced in the shoots of nrt2.5 and nrt2.6 mutants suggesting a role for this nitrate reductase isoform independently from its role in nitrate assimilation.

Identification of mercury‐resistant Streptomyces isolated from Cyperus rotundus L. rhizosphere and molecular cloning of mercury (II) reductase gene

Streptomyces is one of mercury‐resistant bacteria which can convert Hg2+ into nontoxic Hg0 . This study aimed to identify mercury‐resistant Streptomyces present in the Cyperus rotundus rhizosphere from artisanal small‐scale gold mining (ASGM) area and clone merA gene to the cloning and expression vectors. Molecular identification was conducted using 16s rRNA gene with the maximum likelihood algorithms. Results revealed that the AS1 and AS2 strains were a group of Streptomyces ardesiacus and the BR28 strain was closed to Brevibacillus agri. The AS2 merA gene was cloned to pMD20 cloning vectors, pGEX‐5x‐1 and pET‐28c expression vectors. The transformation was successfully performed in BL21 and DH5α competent cells. The full length of the merA gene was confirmed to be 1,425 bp. This study is the first research on identifying mercury‐resistant Streptomyces and cloning the full‐length merA gene in Indonesia.

Effects of Nitrogen Input on Community Structure of the Denitrifying Bacteria with Nitrous Oxide Reductase Gene (nosZ I): A Long-Term Pond Experiment

Excessive nitrogen (N) input is an important factor influencing aquatic ecosystems and has received increasing public attention in the past decades. It remains unclear, however, how N input affects the denitrifying bacterial communities that play a key role in regulating N cycles in various ecosystems. To test our hypothesis – that the abundance and biodiversity of denitrifying bacterial communities decrease with increasing N – we compared the abundance and composition of denitrifying bacteria having nitrous oxide reductase gene (nosZ I) from sediments (0-20 cm) in five experimental ponds with different nitrogen fertilization treatment (TN10, TN20, TN30, TN40, TN50) using quantitative PCR and pyrosequencing techniques. We found that: 1) N addition significantly decreased nosZ I gene abundance, 2) the Invsimpson and Shannon indices (reflecting biodiversity) first increased significantly along with the increasing N loading in TN10~TN40 followed by a decrease in TN50, 3) the beta diversity of the nosZ I denitrifier was clustered into three groups along the TN concentration levels: Cluster I (TN50), Cluster II (TN40), and Cluster III (TN10-TN30), 4) the proportions of Alphaproteobacteria and Betaproteobacteria in the high-N treatment (TN50) were significantly lower than in the lower N treatments (TN10-TN30). 5). The TN concentration was the most important factor driving the alteration of denitrifying bacteria assemblages. Our findings shed new light on the response of denitrification-related bacteria to long-term N loading at pond scale and on the response of denitrifying microorganisms to N pollution.

Aquatic Hyphomycete Taxonomic Relatedness Translates into Lower Genetic Divergence of the Nitrate Reductase Gene

Aquatic hyphomycetes are key microbial decomposers in freshwater that are capable of producing extracellular enzymes targeting complex molecules of leaf litter, thus, being crucial to nutrient cycling in these ecosystems. These fungi are also able to assimilate nutrients (e.g., nitrogen) from stream water, immobilizing these nutrients in the decomposing leaf litter and increasing its nutritional value for higher trophic levels. Evaluating the aquatic hyphomycete functional genetic diversity is, thus, pivotal to understanding the potential impacts of biodiversity loss on nutrient cycling in freshwater. In this work, the inter- and intraspecific taxonomic (ITS1-5.8S-ITS2 region) and functional (nitrate reductase gene) diversity of 40 aquatic hyphomycete strains, belonging to 23 species, was evaluated. A positive correlation was found between the taxonomic and nitrate reductase gene divergences. Interestingly, some cases challenged this trend: Dactylella cylindrospora (Orbiliomycetes) and Thelonectria rubi (Sordariomycetes), which were phylogenetically identical but highly divergent regarding the nitrate reductase gene; and Collembolispora barbata (incertae sedis) and Tetracladium apiense (Leotiomycetes), which exhibited moderate taxonomic divergence but no divergence in the nitrate reductase gene. Additionally, Tricladium chaetocladium (Leotiomycetes) strains were phylogenetically identical but displayed a degree of nitrate reductase gene divergence above the average for the interspecific level. Overall, both inter- and intraspecific functional diversity were observed among aquatic hyphomycetes.

Comparative transcriptome and metabolome analysis reveal glutathione metabolic network and functional genes underlying blue and red-light mediation in maize seedling leaf

Background Light quality severely affects biosynthesis and metabolism-associated process of glutathione. However, the role of specific light is still unclear on the glutathione metabolism. In this article, comparatively transcriptome and metabolome methods are used to fully understand the blue and red-light conditions working on the glutathione metabolism in maize seedling leaf. Results There are 20 differently expressed genes and 4 differently expressed metabolites in KEGG pathway of glutathione metabolism. Among them, 12 genes belong to the glutathione S-transferase family, 3 genes belong to the ascorbate peroxidase gene family and 2 genes belong to the ribonucleoside-diphosphate reductase gene family. Three genes, G6PD, SPDS1, and GPX1 belong to the gene family of glucose 6-phosphate dehydrogenase, spermidine synthase, and glutathione peroxidase, respectively. Four differently expressed metabolites are identified. Three of them, Glutathione disulfide, Glutathione, and l-γ-Glutamyl-L-amino acid are decreased while L-Glutamate is increased. In addition, Through PPI analysis, two annotated genes gst16 and DAAT, and 3 unidentified genes 100381533, pco105094 and umc2770, identified as RPP13-like3, BCAT-like1and GMPS, were obtained. By the analysis of protein sequence and PPI network, we predict that pco105094 and umc2770 were involved in the GSSG-GSH and AsA-GSH cycle in the network of glutathione metabolism. Conclusions Compared to red light, blue light remarkably changed the transcription signal transduction and metabolism of glutathione metabolism. Differently expressed genes and metabolic mapped to the glutathione metabolism signaling pathways. In total, we obtained three unidentified genes, and two of them were predicted in current glutathione metabolism network. This result will contribute to the research of glutathione metabolism of maize.

Genomic Insights into Denitrifying Methane-Oxidizing Bacteria Gemmobacter fulva sp. Nov., Isolated from an Anabaena Culture

The genus Gemmobacter grows phototrophically, aerobically, or anaerobically, and utilizes methylated amine. Here, we present two high-quality complete genomes of the strains con4 and con5T isolated from a culture of Anabaena. The strains possess sMMO (soluble methane monooxygenase)-oxidizing alkanes to carbon dioxide. Functional genes for methane-oxidation (prmAC, mimBD, adh, gfa, fdh) were identified. The genome of strain con5T contains nirB, nirK, nirQ, norB, norC, and norG genes involved in dissimilatory nitrate reduction. The presence of nitrite reductase gene (nirK) and the nitric-oxide reductase gene (norB) indicates that it could potentially use nitrite as an electron acceptor in anoxic environments. Taxonomic investigations were also performed on two strains through polyphasic methods, proposing two isolates as a novel species of the genus Gemmobacter. The findings obtained through the whole genome analyses provide genome-based evidence of complete oxidation of methane to carbon dioxide. This study provides a genetic blueprint of Gemmobacter fulva con5T and its biochemical characteristics, which help us to understand the evolutionary biology of the genus Gemmobacter.

Ribonucleotide Reductase Regulatory Subunit M2 as a Driver of Glioblastoma TMZ-Resistance through Modulation of dNTP Production

Glioblastoma (GBM) remains one of the most resistant and fatal forms of cancer. Previous studies have examined primary and recurrent GBM tumors, but it is difficult to study tumor evolution during therapy where resistance develops. To investigate this, we performed an in vivo single-cell RNA sequencing screen in a patient-derived xenograft (PDX) model. Primary GBM was modeled by mice treated with DMSO control, recurrent GBM was modeled by mice treated with temozolomide (TMZ), and during therapy GBM was modeled by mice euthanized after two of five TMZ treatments. Our analysis revealed the cellular population present during therapy to be distinct from primary and recurrent GBM. We found the Ribonucleotide Reductase gene family to exhibit a unique signature in our data due to an observed subunit switch to favor RRM2 during therapy. GBM cells were shown to rely on RRM2 during therapy causing RRM2-knockdown (KD) cells to be TMZ-sensitive. Using targeted metabolomics, we found RRM2-KDs to produce less dGTP and dCTP than control cells in response to TMZ (p<0.0001). Supplementing RRM2-KDs with deoxycytidine and deoxyguanosine rescued TMZ-sensitivity, suggesting an RRM2-driven mechanism of chemoresistance, established by regulating the production of these nucleotides. In vivo, tumor-bearing mice treated with the RRM2-inhibitor, Triapine, in combination with TMZ, survived longer than mice treated with TMZ alone (p<0.01), indicating promising clinical opportunities in targeting RRM2. Our data present a novel understanding of RRM2 activity, and its alteration during therapeutic stress as response to TMZ-induced DNA damage.