Bovine mastitis caused by rapid-growth environmental mycobacteria

Rapid-growth mycobacteria were isolated from two cases of cow mastitis with similar clinical appearance and within a narrow time frame. Mycobacteria were isolated on blood esculine agar. The isolated mycobacteria were Gram stained, Ziehl-Nielsen stained and tested for growth at 25°C, 37°C and 42°C, iron uptake, growth on Löwenstein-Jensen (LJ) agar with and without 5% NaCl, arylsulphatase (3 days), tween 80 hydrolysis, tellurite reduction, nitrate reductase and niacin synthesis. Molecular identification was performed using the Mycobacteria GenoType CM and AS tests (Hain Diagnostika, Nehren, Germany). One isolate was additionally sequenced for the hsp65, rpoB, 16S rRNA gene sequence and transcribed spacer sequence (ITS) DNA. Susceptibility testing of isolates was performed on the Sensititre Rapmycol plate (TREK Diagnostic Systems Ltd.) for trimethoprim/sulfamethoxasole, linezolid, ciprofloxacin, imipenem, moxifloxacin, cefepime, cefoxitin, amoxicillin / clavulanic acid, amikacin, ceftriaxone, doxycycline, minocycline, tigecycline, tobramycine and clarythromycine. Gram-positive acid-resistant rods were observed in stained smears. Both strains grew at 25°C, 37°C and 42°C on LJ medium, and on LJ medium containing 5 % NaCl. The conventional biochemical tests for iron uptake, arylsulphatase (3 days), Tween 80 hydrolysis, tellurite reduction and nitrate reductase were positive, while the niacin test was negative. Both isolates were identified by the GenoType Mycobacterium CM as Mycobacterium fortuitum II/ Mycobacterium mageritense, while application of the GenoType Mycobacterium AS kit identified both isolates as belonging to the species Mycobacterium smegmatis. Analysis of the isolate sequences (strain DS) for 16S ribosomal RNA confirmed a 100% identical result with Mycobacterium smegmatis strain INHR2. According to the CLSI criteria, both strains were sensitive to sulfametoxazole/trimethoprim, linezolid, doxicycline, amikacin and tobramycin. The strains differed in their sensitivity to cefoxitim, and both strains were resistant to clarithromycin. There was a strong difference between the isolates in sensitivity toward cefoxitime and tigecycline.

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.

Dephosphorylation of Nitrate Reductase Protein Regulates Growth of Rice and Adaptability to Low Temperature

Nitrate reductase (NR) is an important enzyme for nitrate assimilation in plants, and its activity is regulated by post-translational phosphorylation. To investigate the effect of NIA1 protein dephosphorylation on the growth of rice and its adaptability to low temperature, we analyzed phenotype, chlorophyll content, nitrogen utilization, and antioxidant capacity at low temperature in lines with a mutated NIA1 phosphorylation site (S532D and S532A), an OsNia1 over-expression line (OE), and wild-type Kitaake rice (WT). Plant height, dry matter weight, and chlorophyll content of S532D and S532A were lower than those of WT and OE under normal growth conditions but were higher than those of WT and OE at low temperature. Compared with WT and OE, the nitrite, H2O2, and MDA contents of S532D and S532A leaves were higher under normal growth conditions. The difference in leaf nitrite content between transgenic lines and WT was narrower at low temperature, especially in S532D and S532A, while H2O2 and MDA contents of S532D and S532A leaves were lower than those in WT and OE leaves. The NH4+-N and amino acid contents of S532D and S532A leaves were higher than those of WT and OE leaves under normal or low temperature. qRT-PCR results revealed that transcription levels of OsNrt2.4, OsNia2, and OsNADH-GOGAT were positively correlated with those of OsNia1, and the transcription levels of OsNrt2.4, OsNia2, and OsNADH-GOGAT were significantly higher in transgenic lines than in WT under both normal and low temperature. Phosphorylation of NR is a steady-state regulatory mechanism of nitrogen metabolism, and dephosphorylation of NIA1 protein improved NR activity and nitrogen utilization efficiency in rice. Excessive accumulation of nitrite under normal growth conditions inhibits the growth of rice; however, accumulation of nitrite is reduced at low temperature, enhancing the cold tolerance of rice.

vRhyme enables binning of viral genomes from metagenomes

Genome binning has been essential for characterization of bacteria, archaea, and even eukaryotes from metagenomes. Yet, no approach exists for viruses. We developed vRhyme, a fast and precise software for construction of viral metagenome-assembled genomes (vMAGs). vRhyme utilizes single- or multi-sample coverage effect size comparisons between scaffolds and employs supervised machine learning to identity nucleotide feature similarities, which are compiled into iterations of weighted networks and refined bins. Using simulated viromes, we displayed superior performance of vRhyme compared to available binning tools in constructing more complete and uncontaminated vMAGs. When applied to 10,601 viral scaffolds from human skin, vRhyme advanced our understanding of resident viruses, highlighted by identification of a Herelleviridae vMAG comprised of 22 scaffolds, and another vMAG encoding a nitrate reductase metabolic gene, representing near-complete genomes post-binning. vRhyme will enable a convention of binning uncultivated viral genomes and has the potential to transform metagenome-based viral ecology.

Inhibition Molecular Mechanism of the Novel Fungicidal N-(naphthalen-1-yl) phenazine-1-carboxamide against Rhizoctonia solani

To explore the molecular mechanism through which the novel fungicide N-(naphthalen-1-yl) phenazine-1-carboxamide (NNPCN) inhibits Rhizoctonia solani, we clarified the target and mode of action, explored lead compounds, and developed novel fungicides. Methods: Growth observation, scanning electron microscopy, transmission electron microscopy, transcriptome sequencing technology, quantitative real-time PCR (qRT-PCR), physiological and biochemical determination, and reverse molecular docking technology were used to study the effects of this compound on the microscopic morphology of R. solani. The differentially expressed genes (DEGs), functions, and metabolic pathways were analyzed. The genes displaying significant differences were randomly selected for qRT-PCR verification and confirmed by physiological and biochemical determination to construct their binding mode with key targets. The results showed that the mycelium treated with NNPCN produced a red secretion and exhibited progressive creeping growth. Under a scanning electron microscope, hyphal swelling, uneven thickness, fractures, deformities, and hyphal surface warts increased. Under a transmission electron microscope, the cell wall was separated, the subcellular organelles were disintegrated, and the septum disappeared. Furthermore, there were 6838 DEGs under NNPCN treatment, including 291 significant DEGs, of which 143 were upregulated and 148 downregulated. Ten DEGs were randomly selected for qRT-PCR verification, and the gene expression trend was consistent with the transcriptome sequencing results. Gene Ontology enrichment analysis showed that the DEGs were significantly enriched in cell wall glucan decomposition and metabolism, cell membrane synthesis, metabolism, composition, organic hydroxyl compounds, oxidoreductase activity, and transition metal ion binding. Metabolic pathway enrichment analysis showed that there were 16 significant metabolic pathways, such as steroid biosynthesis and ABC transporters. Further study found that genes, such as the glycosyl hydrolase family 10 domain-containing protein, which is related to glucan catabolic process function as tied to the cell wall, were downregulated. Lipid oxidation, modification, and other genes related to the cell membrane were also downregulated. Secondly, genes related to lipid modification, lipid metabolism processes, integral components of the membrane, and other ABC transporters were downregulated. Fatty-acid oxidation and carbohydrate metabolic processes, which are related to antioxidant and metabolic functions, displayed significant differences in their target genes. Nitrite reductase [NADH] activity and mitochondrial organization gene expression were downregulated. These results revealed that target genes may involved in the cell wall, cell membrane, antioxidant and metabolism, nitrogen metabolism, and mitochondria. The results of the physiological and biochemical tests showed that NNPCN decreased the β-1,3-glucanase, malondialdehyde, and ATPase activities and nucleic acid leakage but increased the activity of nitrate reductase. The results of the reverse molecular docking showed that NNPCN could freely bind to target proteins such as β-1,3-glucanase, ABC transporter, and NADPH nitrate reductase, whereby NNPCN could bind to glucanase via van der Waals and electrostatic forces and to ABC transporter and NADPH nitrate reductase via hydrogen bonding. Conclusion: The mechanism via which NNPCN inhibits R. solani may be related to the cell wall structure, cell membrane damage, antioxidant activity, and metabolism.

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.