First two mitochondrial genomes for the order Filobasidiales reveal novel gene rearrangements and intron dynamics of Tremellomycetes.

In the present study, two mitogenomes from the Filobasidium genus were assembled and compared with other Tremellomycetes mitogenomes. The mitogenomes of F. wieringae and F. globisporum both comprised circular DNA molecules, with sizes of 27,861 bp and 71,783 bp, respectively. Comparative mitogenomic analysis revealed that the genetic contents, tRNAs, and codon usages of the two Filobasidium species differed greatly. The sizes of the two Filobasidium mitogenomes varied greatly with the introns being the main factor contributing to mitogenome expansion in F. globisporum. Positive selection was observed in several protein-coding genes (PCGs) in the Agaricomycotina, Pucciniomycotina, and Ustilaginomycotina species, including cob, cox2, nad2, and rps3 genes. Frequent intron loss/gain events were detected to have occurred during the evolution of the Tremellomycetes mitogenomes, and the mitogenomes of 17 species from Agaricomycotina, Pucciniomycotina, and Ustilaginomycotina have undergone large-scale gene rearrangements. Phylogenetic analyses based on Bayesian inference and the maximum likelihood methods using a combined mitochondrial gene set generated identical and well-supported phylogenetic trees, wherein Filobasidium species had close relationships with Trichosporonales species. This study, which is the first report on mitogenomes from the order Filobasidiales, provides a basis for understanding the genomics, evolution, and taxonomy of this important fungal group.

Global investigation of an engineered nitrogen-fixing Escherichia coli strain reveals regulatory coupling between host and heterologous nitrogen-fixation genes.

Transfer of nitrogen fixation (nif) genes from diazotrophs to amenable heterologous hosts is of increasing interest to genetically engineer nitrogen fixation. However, how the non-diazotrophic host maximizes opportunities to fine-tune the acquired capacity for nitrogen fixation has not been fully explored. In this study, a global investigation of an engineered nitrogen-fixing Escherichia coli strain EN-01 harboring a heterologous nif island from Pseudomonas stutzeri was performed via transcriptomics and proteomics analyses. A total of 1156 genes and 206 discriminative proteins were found to be significantly altered when cells were incubated under nitrogen-fixation conditions. Pathways for regulation, metabolic flux and oxygen protection to nitrogenase were particularly discussed. An NtrC-dependent regulatory coupling between E. coli nitrogen regulation system and nif genes was established. Additionally, pentose phosphate pathway was proposed to serve as the primary route for glucose catabolism and energy supply to nitrogenase. Meanwhile, HPLC analysis indicated that organic acids produced by EN-01 might have negative effects on nitrogenase activity. This study provides a global view of the complex network underlying the acquired nif genes in the recombinant E. coli and also provides clues for the optimization and redesign of robust nitrogen-fixing organisms to improve nitrogenase efficiency by overcoming regulatory or metabolic obstacles.

Interspecies Transfer and Regulation of Pseudomonas stutzeri A1501 Nitrogen Fixation Island in Escherichia coli.

Until now, considerable effort has been made to engineer novel nitrogen-fixing organisms through the transfer of nif genes from various diazotrophs to non-nitrogen fixers; however, regulatory coupling of the heterologous nif genes with the regulatory system of the new host is still not well understood. In this work, a 49 kb nitrogen fixation island from P. stutzeri A1501 was transferred into E. coli using a novel and efficient transformation strategy, and a series of recombinant nitrogen-fixing E. coli strains were obtained. We found that the nitrogenase activity of the recombinant E. coli strain EN-01, similar to the parent strain P. stutzeri A1501, was dependent on external ammonia concentration, oxygen tension, and temperature. We further found that there existed a regulatory coupling between the E. coli general nitrogen regulatory system and the heterologous P. stutzeri nif island in the recombinant E. coli strain. We also provided evidence that the E. coli general nitrogen regulator GlnG protein was involved in the activation of the nif-specific regulator NifA via a direct interaction with the NifA promoter. To the best of our knowledge, this work plays a groundbreaking role in increasing understanding of the regulatory coupling of the heterologous nitrogen fixation system with the regulatory system of the recipient host. Furthermore, it will shed light on the structure and functional integrity of the nif island and will be useful for the construction of novel and more robust nitrogen-fixing organisms through biosynthetic engineering.

1-Aminocyclopropane-1-Carboxylate Deaminase from Pseudomonas stutzeri A1501 Facilitates the Growth of Rice in the Presence of Salt or Heavy Metals.

1-Aminocyclopropane-1-carboxylate (ACC) deaminase, which is encoded by some bacteria, can reduce the amount of ethylene, a root elongation inhibitor, and stimulate the growth of plants under various environmental stresses. The presence of ACC deaminase activity and the regulation of ACC in several rhizospheric bacteria have been reported. The nitrogen-fixing Pseudomonas stutzeri A1501 is capable of endophytic association with rice plants and promotes the growth of rice. However, the functional identification of ACC deaminase has not been performed. In this study, the proposed effect of ACC deaminase in P. stutzeri A1501 was investigated. Genome mining showed that P. stutzeri A1501 carries a single gene encoding ACC deaminase, designated acdS. The acdS mutant was devoid of ACC deaminase activity and was less resistant to NaCl and NiCl2 compared with the wild-type. Furthermore, inactivation of acdS greatly impaired its nitrogenase activity under salt stress conditions. It was also observed that mutation of the acdS gene led to loss of the ability to promote the growth of rice under salt or heavy metal stress. Taken together, this study illustrates the essential role of ACC deaminase, not only in enhancing the salt or heavy metal tolerance of bacteria but also in improving the growth of plants, and provides a theoretical basis for studying the interaction between plant growth-promoting rhizobacteria and plants.

[Concentrations of mercury in ambient air in wastewater irrigated area of Tianjin City and its accumulation in leafy vegetables].

Gaseous Hg can evaporate and enter the plants through the stomata of plat leaves, which will cause a serious threat to local food safety and human health. For the risk assessment, this study aimed to characterize atmospheric mercury (Hg) as well as its accumulation in 5 leafy vegetables (spinach, edible amaranth, rape, lettuce, allium tuberosum) from sewage-irrigated area of Tianjin City. Bio-monitoring sites were located in paddy (wastewater irrigation for 30 a), vegetables (wastewater irrigation for 15 a) and grass (control) fields. Results showed that after long-term wastewater irrigation, the mean values of mercury content in paddy and vegetation fields were significantly higher than the local background value and the national soil environment quality standard value for mercury in grade I, but were still lower than grade II. Soil mercury contents in the studied control grass field were between the local background value and the national soil environment quality standard grade I . Besides, the atmospheric environment of paddy and vegetation fields was subjected to serious mercury pollution. The mean values of mercury content in the atmosphere of paddy and vegetation fields were 71.3 ng x m(-3) and 39.2 ng x m(-3), respectively, which were markedly higher than the reference gaseous mercury value on the north sphere of the earth (1.5-2.0 ng x m(-3)). The mean value of ambient mercury in the control grass fields was 9.4 ng x m(-3). In addition, it was found that the mercury content in leafy vegetables had a good linear correlation with the ambient total gaseous mercury (the data was transformed into logarithms as the dataset did not show a normal distribution). The comparison among 5 vegetables showed that the accumulations of mercury in vegetables followed this order: spinach > edible amaranth > allium tuberosum > rape > lettuce. Median and mean values of mercury contents in spinach and edible amaranth were greater than the hygienic standard for the allowable limit of mercury in food. Spinach appeared to accumulate more mercury than the other four vegetables, in which the median and mean mercury content were both higher than 20 µg x kg(-1). The mercury concentrations in rape, lettuce and allium tuberosum were lower than the standard. Moreover, test results indicated that the Hg content in leafy vegetables was mainly the gaseous mercury through leaf adsorption but not the Hg particulates. This study clearly manifested that there should be a great concern on the pollution risk of both air-and soil borne mercury when cultivating leafy vegetables in long-term wastewater-irrigated area.

Involvement of the ammonium transporter AmtB in nitrogenase regulation and ammonium excretion in Pseudomonas stutzeri A1501.

The nitrogen-fixing Pseudomonas stutzeri strain A1501 contains two ammonium transporter genes, amtB1 and amtB2, linked to glnK. Growth of an amtB1-amtB2 double deletion mutant strain was not impaired compared to that of the wild type under any conditions tested, and it was still capable of taking up ammonium ions at nearly wild-type rates. Nitrogenase activity was repressed in wild-type strain A1501 in response to the addition of ammonium, but nitrogenase activity was only partially impaired in the amtB1 and amtB2 double mutant, suggesting that the two AmtB proteins are involved in regulating expression of nitrogenase or its activity in response to ammonium. An interaction between GlnK and AmtB1 or AmtB2 was observed in a yeast two-hybrid assay. Ammonium was excreted by the amtB double mutant strain under nitrogen fixation conditions, particularly when nifA was expressed constitutively. This suggests that AmtB proteins play a role in controlling the internal pool of ammonia within the cell.

Complete genome sequence of the nitrogen-fixing and rhizosphere-associated bacterium Pseudomonas stutzeri strain DSM4166.

We present here the analysis of the whole-genome sequence of Pseudomonas stutzeri strain DSM4166, a diazotrophic isolate from the rhizosphere of a Sorghum nutans cultivar. To our knowledge, this is the second genome to be sequenced for P. stutzeri. The availability and analysis of the genome provide insight into the evolution of the nitrogen fixation property and identification of rhizosphere competence traits required in interactions with host plants.

Global transcriptional analysis of nitrogen fixation and ammonium repression in root-associated Pseudomonas stutzeri A1501.

BACKGROUND: Biological nitrogen fixation is highly controlled at the transcriptional level by regulatory networks that respond to the availability of fixed nitrogen. In many diazotrophs, addition of excess ammonium in the growth medium results in immediate repression of nif gene transcription. Although the regulatory cascades that control the transcription of the nif genes in proteobacteria have been well investigated, there are limited data on the kinetics of ammonium-dependent repression of nitrogen fixation. RESULTS: Here we report a global transcriptional profiling analysis of nitrogen fixation and ammonium repression in Pseudomonas stutzeri A1501, a root-associated and nitrogen-fixing bacterium. A total of 166 genes, including those coding for the global nitrogen regulation (Ntr) and Nif-specific regulatory proteins, were upregulated under nitrogen fixation conditions but rapidly downregulated as early as 10 min after ammonium shock. Among these nitrogen fixation-inducible genes, 95 have orthologs in each of Azoarcus sp. BH72 and Azotobacter vinelandii AvoP. In particular, a 49-kb expression island containing nif and other associated genes was markedly downregulated by ammonium shock. Further functional characterization of pnfA, a new NifA-sigma54-dependent gene chromosomally linked to nifHDK, is reported. This gene encodes a protein product with an amino acid sequence similar to that of five hypothetical proteins found only in diazotrophic strains. No noticeable differences in the transcription of nifHDK were detected between the wild type strain and pnfA mutant. However, the mutant strain exhibited a significant decrease in nitrogenase activity under microaerobic conditions and lost its ability to use nitrate as a terminal electron acceptor for the support of nitrogen fixation under anaerobic conditions. CONCLUSIONS: Based on our results, we conclude that transcriptional regulation of nif gene expression in A1501 is mediated by the nif-specific and ntr gene regulatory systems. Furthermore, microarray and mutational analyses revealed that many genes of unknown function may play some essential roles in controlling the expression or activity of nitrogenase. The findings presented here establish the foundation for further studies on the physiological function of nitrogen fixation-inducible genes.

A novel RPMXR motif among class II 5-enolpyruvylshikimate-3-phosphate synthases is required for enzymatic activity and glyphosate resistance.

The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase is an attractive target for drugs and herbicides. Here we identified a novel RPMXR motif that is strictly conserved among class II EPSP synthases. Site-directed mutational analysis of this motif showed that substitutions of the four strictly conserved amino acid residues, Arg127, Pro128, Met129, and Arg131, resulted in complete loss of enzymatic activity, whereas changes in the non-conserved Asn130 residue strongly influenced glyphosate resistance (all numbering according to Pseudomonas stutzeri A1501 EPSP synthase). These experimental results, combined with 3D structure modeling of the location and interaction of the RPMXR motif with phosphoenolpyruvate (PEP) and shikimate-3-phosphate (S3P), demonstrate that the novel motif is required for enzymatic activity and glyphosate resistance of class II EPSP synthases.