Identification of a novel glycoside hydrolase family 8 xylanase from Deinococcus geothermalis and its application at low temperatures.

Xylanase is the most important hydrolase in the xylan hydrolase system, the main function of which is ß-1,4-endo-xylanase, which randomly cleaves xylans to xylo-oligosaccharides and xylose. Xylanase has wide ranging of applications, but there remains little research on the cold-adapted enzymes required in some low-temperature industries. Glycoside hydrolase family 8 (GH8) xylanases have been reported to have cold-adapted enzyme activity. In this study, the xylanase gene dgeoxyn was excavated from Deinococcus geothermalis through sequence alignment. The recombinant xylanase DgeoXyn encodes 403 amino acids with a theoretical molecular weight of 45.39 kDa. Structural analysis showed that DgeoXyn has a (α/α)6-barrel fold structure typical of GH8 xylanase. At the same time, it has strict substrate specificity, is only active against xylan, and its hydrolysis products include xylobiose, xylotrinose, xytetranose, xylenanose, and a small amount of xylose. DgeoXyn is most active at 70 â„ƒ and pH 6.0. It is very stable at 10, 20, and 30 â„ƒ, retaining more than 80% of its maximum enzyme activity. The enzyme activity of DgeoXyn increased by 10% after the addition of Mn2+ and decreased by 80% after the addition of Cu2+. The Km and Vmax of dgeox were 42 mg/ml and 20,000 U/mg, respectively, at a temperature of 70 â„ƒ and pH of 6.0 using 10 mg/ml beechwood xylan as the substrate. This research on DgeoXyn will provide a theoretical basis for the development and application of low-temperature xylanase.

A Novel Glycoside Hydrolase DogH Utilizing Soluble Starch to Maltose Improve Osmotic Tolerance in Deinococcus radiodurans.

Deinococcus radiodurans is a microorganism that can adjust, survive or thrive in hostile conditions and has been described as "the strongest microorganism in the world". The underlying mechanism behind the exceptional resistance of this robust bacterium still remains unclear. Osmotic stress, caused by abiotic stresses such as desiccation, salt stress, high temperatures and freezing, is one of the main stresses suffered by microorganisms, and it is also the basic response pathway by which organisms cope with environmental stress. In this study, a unique trehalose synthesis-related gene, dogH (Deinococcus radiodurans orphan glycosyl hydrolase-like family 10), which encodes a novel glycoside hydrolase, was excavated using a multi-omics combination method. The content accumulation of trehalose and its precursors under hypertonic conditions was quantified by HPLC-MS. Ours results showed that the dogH gene was strongly induced by sorbitol and desiccation stress in D. radiodurans. DogH glycoside hydrolase hydrolyzes α-1,4-glycosidic bonds by releasing maltose from starch in the regulation of soluble sugars, thereby increasing the concentration of TreS (trehalose synthase) pathway precursors and trehalose biomass. The maltose and alginate content in D. radiodurans amounted to 48 µg mg protein-1 and 45 µg mg protein-1, respectively, which were 9 and 28 times higher than those in E. coli, respectively. The accumulation of greater intracellular concentrations of osmoprotectants may be the true reason for the higher osmotic stress tolerance of D. radiodurans.

Development and Regulation of the Extreme Biofilm Formation of Deinococcus radiodurans R1 under Extreme Environmental Conditions.

To grow in various harsh environments, extremophiles have developed extraordinary strategies such as biofilm formation, which is an extremely complex and progressive process. However, the genetic elements and exact mechanisms underlying extreme biofilm formation remain enigmatic. Here, we characterized the biofilm-forming ability of Deinococcus radiodurans in vitro under extreme environmental conditions and found that extremely high concentrations of NaCl or sorbitol could induce biofilm formation. Meantime, the survival ability of biofilm cells was superior to that of planktonic cells in different extreme conditions, such as hydrogen peroxide stress, sorbitol stress, and high UV radiation. Transcriptome profiles of D. radiodurans in four different biofilm development stages further revealed that only 13 matched genes, which are involved in environmental information processing, carbohydrate metabolism, or stress responses, share sequence homology with genes related to the biofilm formation of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Overall, 64% of the differentially expressed genes are functionally unknown, indicating the specificity of the regulatory network of D. radiodurans. The mutation of the drRRA gene encoding a response regulator strongly impaired biofilm formation ability, implying that DrRRA is an essential component of the biofilm formation of D. radiodurans. Furthermore, transcripts from both the wild type and the drRRA mutant were compared, showing that the expression of drBON1 (Deinococcus radioduransBON domain-containing protein 1) significantly decreased in the drRRA mutant during biofilm development. Further analysis revealed that the drBON1 mutant lacked the ability to form biofilm and DrRRA, and as a facilitator of biofilm formation, could directly stimulate the transcription of the biofilm-related gene drBON1. Overall, our work highlights a molecular mechanism mediated by the response regulator DrRRA for controlling extreme biofilm formation and thus provides guidance for future studies to investigate novel mechanisms that are used by D. radiodurans to adapt to extreme environments.

Insight rifampicin-resistant (rpoB) mutation in Pseudomonas stutzeri leads to enhance the biosynthesis of secondary metabolites to survive against harsh environments.

In this study, a wild-type and five distinct rifampicin-resistant (Rifr) rpoB mutants of Pseudomonas stutzeri (i.e., Q518R, D521Y, D521V, H531R and I614T) ability were investigated against harsh environments (particularly nutritional complexity). Among these, the robust Rifr phenotype of P. Stutzeri was associated only with base replacements of the amino deposits. The use of carboxylic and amino acids significantly increased in various Rifr mutants than that of wild type of P. stutzeri. The assimilation of carbon and nitrogen (N) sources of Rifr mutants' confirmed that the organism maintains the adaptation in nutritionally complex environments. Acetylene reduction assay at different times also found the variability for N-fixation in all strains. Among them, the highest nitrogenase activity was determined in mutant 'D521V'. The assimilation of carbon and nitrogen sources of P. stutzeri and its Rifr mutants ensures that the organism maintains the adaptability in nutritionally complex environments through fixing more nitrogen.

Devosia salina sp. nov., isolated from South China Sea sediment.

An aerobic, Gram-stain-negative, rod-shaped and motile strain, designated SCS-3T, was isolated from deep-sea sediment of the South China Sea. Phylogenetic analysis based on the 16S rRNA gene sequence similarities revealed that strain SCS-3T represented a novel species of the genus Devosia, with closely related strains 'Devosia sediminis' MSA67T (98.61 %), Devosia riboflavina IFO13584T (98.22 %) and Devosia indica IO390501T (97.72 %). The G+C content of the genomic DNA is 63.44 mol%. The digital DNA-DNA hybridization values with 'D. sediminis' MSA67T, D. riboflavina IFO13584T and D. indica IO390501T were 24.50, 21.8 and 24.80 %, respectively. The major polar lipids of strain SCS-3T were diphosphatidylglycerol, phosphatidylglycerol and three unidentified glycolipids. Ubiquinone-10 was the sole isoprenoid quinone, and C16 : 0, C18 : 1 ω7c 11-methyl and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) were the major fatty acids. Based on polyphasic taxonomic data, strain SCS-3T represents a novel species of the genus Devosia, for which the name Devosia salina sp. nov. is proposed. The type strain is SCS-3T (=JCM 34403T=GDMCC 1.2221T).

Post-transcriptional control of bacterial nitrogen metabolism by regulatory noncoding RNAs.

Nitrogen metabolism is the most basic process of material and energy metabolism in living organisms, and processes involving the uptake and use of different nitrogen sources are usually tightly regulated at the transcriptional and post-transcriptional levels. Bacterial regulatory noncoding RNAs are novel post-transcriptional regulators that repress or activate the expression of target genes through complementarily pairing with target mRNAs; therefore, these noncoding RNAs play an important regulatory role in many physiological processes, such as bacterial substance metabolism and stress response. In recent years, a study found that noncoding RNAs play a vital role in the post-transcriptional regulation of nitrogen metabolism, which is currently a hot topic in the study of bacterial nitrogen metabolism regulation. In this review, we present an overview of recent advances that increase our understanding on the regulatory roles of bacterial noncoding RNAs and describe in detail how noncoding RNAs regulate biological nitrogen fixation and nitrogen metabolic engineering. Furthermore, our goal is to lay a theoretical foundation for better understanding the molecular mechanisms in bacteria that are involved in environmental adaptations and metabolically-engineered genetic modifications.

Mapping and validation of a major QTL for primary root length of soybean seedlings grown in hydroponic conditions.

BACKGROUND: The root system provides nutrient absorption and is closely related to abiotic stress tolerance, but it is difficult to study the roots under field conditions. This study was conducted to identify quantitative trait loci (QTL) associated with primary root length (PRL) during soybean seedling growth in hydroponic conditions. A total of 103 F7 recombinant inbred lines (RILs) derived from a cross between K099 (short primary root) and Fendou 16 (long primary root) were used to identify QTL for PRL in soybean. The RIL population was genotyped with 223 simple sequence repeats markers covering 20 chromosomes. Phenotyping for primary root length was performed for 3-weeks plants grown in hydoponic conditions. The identified QTL was validated in near isogenic lines and in a separate RIL population. RESULTS: QTL analysis using inclusive composite interval mapping method identified a major QTL on Gm16 between SSR markers Sat_165 and Satt621, explaining 30.25 % of the total phenotypic variation. The identified QTL, qRL16.1, was further confirmed in a segregating population derived from a residual heterozygous line (RHLs-98). To validate qRL16.1 in a different genetic background, QTL analysis was performed in another F6 RIL population derived from a cross between Union (medium primary root) and Fendou 16, in which a major QTL was detected again in the same genomic region as qRL16.1, explaining 14 % of the total phenotypic variation for PRL. In addition, the effect of qRL16.1 was confirmed using two pair of near-isogenic lines (NILs). PRL was significantly higher in NILs possessing the qRL16.1 allele from Fendou 16 compared to allele from K099. CONCLUSIONS: The qRL16.1 is a novel QTL for primary root length in soybean which provides important information on the genetic control of root development. Identification of this major QTL will facilitate positional cloning and DNA marker-assisted selection for root traits in soybean.

Bioelectrochemical Fixation of Nitrogen to Extracellular Ammonium by Pseudomonas stutzeri.

Diazotrophs can produce bioavailable nitrogen from inert N2 gas by bioelectrochemical nitrogen fixation (e-BNF), which is emerging as an energy-saving and highly selective strategy for agriculture and industry. However, current e-BNF technology is impeded by requirements for NH4+ assimilation inhibitors to facilitate intracellular ammonia secretion and precious metal catalysts to generate H2 as the energy-carrying intermediate. Here, we initially demonstrate inhibitor- and catalystless extracellular NH4+ production by the diazotroph Pseudomonas stutzeri A1501 using an electrode as the sole electron donor. Multiple lines of evidence revealed that P. stutzeri produced 2.32 ± 0.25 mg/liter extracellular NH4+ at a poised potential of -0.3 V (versus standard hydrogen electrode [SHE]) without the addition of inhibitors or expensive catalysts. The electron uptake mechanism was attributed to the endogenous electron shuttle phenazine-1-carboxylic acid, which was excreted by P. stutzeri and mediated electron transfer from electrodes into cells to directly drive N2 fixation. The faradaic efficiency was 20% ± 3%, which was 2 to 4 times that of previous e-BNF attempts using the H2-mediated pathway. This study reports a diazotroph capable of producing secretable NH4+ via extracellular electron uptake, which has important implications for optimizing the performance of e-BNF systems and exploring the novel nitrogen-fixing mode of syntrophic microbial communities in the natural environment. IMPORTANCE Ammonia greatly affects global ecology, agriculture, and the food industry. Diazotrophs with an enhanced capacity of extracellular NH4+ excretion have been proven to be more beneficial to the growth of microalgae and plants, whereas most previously reported diazotrophs produce intracellular organic nitrogen in the absence of chemical suppression and genetic manipulation. Here, we demonstrate that Pseudomonas stutzeri A1501 is capable of extracellular NH4+ production without chemical suppression or genetic manipulation when the extracellular electrode is used as the sole electron donor. We also reveal the electron uptake pathway from the extracellular electron-donating partner to P. stutzeri A1501 via redox electron shuttle phenazines. Since both P. stutzeri A1501 and potential electron-donating partners (such as electroactive microbes and natural semiconductor minerals) are abundant in diverse soils and sediments, P. stutzeri A1501 has broader implications on the improvement of nitrogen fertilization in the natural environment.

Pseudomonas nanhaiensis sp. nov., a lipase-producing bacterium isolated from deep-sea sediment of the South China Sea.

A bacterial lipase producing bacterium, designated SCS 2-3, was isolated from deep-sea sediment of the South China Sea. Phylogenetic analysis based on the 16S rRNA sequence revealed that strain SCS2-3 belonged to the genus Pseudomonas and had 98.56% similarity to P. xinjiangensis NRRL B-51270T as the closest relative strain. MLSA using four protein-coding genes (dnaK, gyrA, recA, and rpoB) showed strain SCS 2-3 to form a separate branch. ANI and in silico DDH values between strain SCS 2-3 and related type strains of Pseudomonas were less than 81.51% and 23.80%, respectively. Genome comparison showed that strain SCS 2-3 shared 1875 core gene families with other eight closely related type strains in Pseudomonas, and the number of strain-unique genes was 263. Through gene annotations, genes related to lipase were found in the genome. Furthermore, a combination of phenotypic, chemotaxonomic, phylogenetic and genotypic data clearly indicated that strain SCS 2-3 represents a novel species of the genus Pseudomonas, for which the name Pseudomonas nanhaiensis sp. nov. is proposed. The type strain is SCS 2-3T (= GDMCC 1.2219T = JCM 34440T).

Master regulator NtrC controls the utilization of alternative nitrogen sources in Pseudomonas stutzeri A1501.

Pseudomonas stutzeri A1501 is a model strain used to study associative nitrogen fixation, and it possesses the nitrogen regulatory NtrC protein in the core genome. Nitrogen sources represent one of the important factors affecting the efficiency of biological nitrogen fixation in the natural environment. However, the regulation of NtrC during nitrogen metabolism in P. stutzeri A1501 has not been clarified. In this work, a phenotypic analysis of the ntrC mutant characterized the roles of NtrC in nitrogen metabolism and the oxidative stress response of P. stutzeri A1501. To systematically identify NtrC-controlled gene expression, RNA-seq was performed to further analyse the gene expression differences between the wild-type strain and the ∆ntrC mutant under nitrogen fixation conditions. A total of 1431 genes were found to be significantly altered by ntrC deletion, among which 147 associative genes had NtrC-binding sites, and the pathways for nitrogen fixation regulation, nitrogenous compound acquisition and catabolism and nitrate assimilation were discussed. Furthermore, the oxidative stress-related gene (katB), which was upregulated by ntrC deletion, was suggested to be a potential target gene of NtrC, thus highlighting the importance of NtrC in nitrogenase protection against oxygen damage. Based on these findings, we propose that NtrC is a high-ranking element in the regulatory network of P. stutzeri A1501 that controls a variety of nitrogen metabolic and oxidative stress responsive traits required for adaptation to complex rhizosphere environments.

An electrochemical DNA biosensor based on nitrogen-doped graphene nanosheets decorated with gold nanoparticles for genetically modified maize detection.

A reliable electrochemical biosensor is reported based on nitrogen-doped graphene nanosheets and gold nanoparticle (Au/N-G) nanocomposites for the event-specific detection of GM maize MIR162. The differential pulse voltammetry response of methylene blue (MB) was chosen to monitor the target DNA hybridization event. Under the optimum conditions, the peak current increased linearly with the logarithm of the concentration of DNA in the range 1.0 × 10-14 to 1.0 × 10-8 M, and the detection limit was 2.52 × 10-15 M (S/N = 3). It is also demonstrated that the DNA biosensor has high selectivity, good stability, and fabrication reproducibility. The biosensor has been effectively applied to detect MIR162 in real samples, showing its potential as an effective tool for GM crop analysis. These results will contribute to the development of new portable transgenic detection systems. Graphical abstract .

The Novel ncRNA OsiR Positively Regulates Expression of katE2 and Is Required for Oxidative Stress Tolerance in Deinococcus radiodurans.

Deinococcus radiodurans is a polyextremophilic bacterium well known for its extreme resistance to irradiation, oxidative stress, and other damaging conditions. Many small noncoding RNAs (ncRNAs) in D. radiodurans have been identified by deep sequencing analysis and computational predictions. However, the precise roles of ncRNAs and their target genes in the oxidative stress response have not been investigated. Here, we report the identification and characterization of a novel ncRNA named OsiR (for oxidative stress-induced ncRNA). Oxidative stress tolerance analysis showed that deleting osiR significantly decreased viability, total antioxidant capacity, and catalase activity in D. radiodurans under oxidative stress conditions. Comparative phenotypic and qRT-PCR analyses of an osiR mutant identify a role of OsiR in regulating the expression of the catalase gene katE2. Microscale thermophoresis and genetic complementation showed that a 21-nt sequence in the stem-loop structure of OsiR (204-244 nt) directly base pairs with its counterpart in the coding region of katE2 mRNA (843-866 nt) via a 19 nt region. In addition, deletion of katE2 caused a significant reduction of catalase activity and oxidative stress tolerance similar to that observed in an osiR mutant. Our results show that OsiR positively regulates oxidative stress tolerance in D. radiodurans by increasing the mRNA stability and translation efficiency of katE2. This work provides a new regulatory pathway mediated by ncRNA for the oxidative stress response that most likely contributes to the extreme tolerances of D. radiodurans.

Characterization of high- and low-molecular-weight glutenin subunits from Chinese Xinjiang wheat landraces and historical varieties.

Landraces and historical varieties are necessary germplasms for genetic improvement of modern cereals. Allelic variations at the Glu-1 and Glu-3 loci in 300 common wheat landraces and 43 historical varieties from Xinjiang, China, were evaluated by Sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and allele-specific molecular markers. Among the materials investigated, three, nine, and seven alleles were identified from the Glu-A1, Glu-B1, and Glu-D1 loci, respectively, and a total of 26 high-molecular-weight glutenin subunit (HMW-GS) combinations were found, of which 18 combinations were identified in landraces and historical varieties. Allelic frequency of HMW-GS combinations null, 7 + 8, 2 + 12 was found to be the highest in both the landraces (63.3%) and historical varieties (39.5%). Besides, some distinctive HMW-GS alleles, such as the novel Glu-B1 allele 6.1* + 8.1* and Glu-D1 alleles 2.6 + 12, 2.1 + 10.1, and 5** + 10 were observed in Xinjiang wheat landraces. Among the Glu-A3 and Glu-B3 loci of landraces and historical varieties, a total of eight and nine alleles were found, respectively. At each locus, two novel alleles were identified. A total of 33 low-molecular-weight glutenin subunit (LMW-GS) combinations of Glu-A3 and Glu-B3 were identified, with 31 and 14 combinations occurring in landraces and historical varieties, respectively, but only 10 combinations shared by both of them. As Glu-D1, Glu-A3, and Glu-B3 have highest contribution to the end-use quality and processing properties as compared to Glu-A1, Glu-B1, and Glu-D3 locus, the novel or distinctive HMW-GS and LMW-GS alleles in these loci could potentially be utilized for the improvement in the quality of modern wheat.

The Pseudomonas stutzeri-Specific Regulatory Noncoding RNA NfiS Targets katB mRNA Encoding a Catalase Essential for Optimal Oxidative Resistance and Nitrogenase Activity.

Pseudomonas stutzeri A1501 is a versatile nitrogen-fixing bacterium capable of living in diverse environments and coping with various oxidative stresses. NfiS, a regulatory noncoding RNA (ncRNA) involved in the control of nitrogen fixation in A1501, was previously shown to be required for optimal resistance to H2O2; however, the precise role of NfiS and the target genes involved in the oxidative stress response is entirely unknown. In this work, we systematically investigated the NfiS-based mechanisms underlying the response of this bacterium to H2O2 at the cellular and molecular levels. A mutant strain carrying a deletion of nfiS showed significant downregulation of oxidative stress response genes, especially katB, a catalase gene, and oxyR, an essential regulator for transcription of catalase genes. Secondary structure prediction revealed two binding sites in NfiS for katB mRNA. Complementation experiments using truncated nfiS genes showed that each of two sites is functional, but not sufficient, for NfiS-mediated regulation of oxidative stress resistance and nitrogenase activities. Microscale thermophoresis assays further indicated direct base pairing between katB mRNA and NfiS at both sites 1 and 2, thus enhancing the half-life of the transcript. We also demonstrated that katB expression is dependent on OxyR and that both OxyR and KatB are essential for optimal oxidative stress resistance and nitrogenase activities. H2O2 at low concentrations was detoxified by KatB, leaving O2 as a by-product to support nitrogen fixation under O2-insufficient conditions. Moreover, our data suggest that the direct interaction between NfiS and katB mRNA is a conserved and widespread mechanism among P. stutzeri strains.IMPORTANCE Protection against oxygen damage is crucial for survival of nitrogen-fixing bacteria due to the extreme oxygen sensitivity of nitrogenase. This work exemplifies how the small ncRNA NfiS coordinates oxidative stress response and nitrogen fixation via base pairing with katB mRNA and nifK mRNA. Hence, NfiS acts as a molecular link to coordinate the expression of genes involved in oxidative stress response and nitrogen fixation. Our study provides the first insight into the biological functions of NfiS in oxidative stress regulation and adds a new regulation level to the mechanisms that contribute to the oxygen protection of the MoFe nitrogenase.

NfiR, a New Regulatory Noncoding RNA (ncRNA), Is Required in Concert with the NfiS ncRNA for Optimal Expression of Nitrogenase Genes in Pseudomonas stutzeri A1501.

Expression of nitrogenase genes (nifHDK) is strictly regulated at both transcriptional and posttranscriptional levels. Efficient nitrogenase activity requires maintaining sufficient levels of nif mRNAs, yet the underlying mechanism is not fully understood due to its complexity. We have previously shown that a novel regulatory noncoding RNA (ncRNA), NfiS, optimizes nitrogen fixation through targeting nifK mRNA in Pseudomonas stutzeri A1501. Here, we report the identification and characterization of a second ncRNA inducible under nitrogen fixation conditions (nitrogen-free and microaerobic conditions), termed NfiR (for nitrogen fixation condition-inducible ncRNA), the expression of which is dependent on two global regulators, NtrC and Hfq. Comparative phenotypic and proteomic analyses of an nfiR mutant identify a role of NfiR in regulating the expression of nitrogenase genes. Further microscale thermophoresis and genetic complementation showed that an 11-nucleotide (nt) sequence in the stem-loop structure of NfiR (nucleotides 12 to 22) pairs with its counterpart in the coding region of nifD mRNA (nucleotides 1194 to 1207) by eight nucleotides. Significantly, deletion of nfiR caused a 60% reduction of nitrogenase activity, and the half-life of nifD mRNA was reduced from 20 min for the wild type to 15 min for the ΔnfiR mutant. With regard to nitrogenase activity and stability of the nifD and nifK transcripts, phenotypes were more severe for the double deletion mutant lacking nfiR and nfiS, suggesting that NfiR, in concert with NfiS, optimizes nitrogenase production at the posttranscriptional level.IMPORTANCE Biological nitrogen fixation is an energy-expensive process requiring the hydrolysis of 16 ATPs. Consequently, the expression of nif genes is highly regulated at both transcriptional and posttranscriptional levels through complex regulatory networks. Global regulation involves a number of regulatory proteins, such as the nif-specific activator NifA and the global nitrogen regulator NtrC, as well as various regulatory ncRNAs. We show that the two P. stutzeri ncRNAs, namely NfiS and NfiR (for nitrogen fixation condition-inducible ncRNA), optimize nitrogen fixation and environmental stress responses. NfiS and NfiR respond differently to various environmental signals and differ in their secondary structures. In addition, the two ncRNAs target the mRNAs of nifK and nifD, respectively. Such ncRNA-based posttranscriptional regulation of nitrogenase expression might be an evolved survival strategy, particularly in nitrogen-limiting environments. This study not only highlights the significant roles of regulatory ncRNAs in the coordination and fine tuning of various physiological processes but also provides a new paradigm for posttranscriptional regulation in nitrogen-fixing bacteria.

The novel regulatory ncRNA, NfiS, optimizes nitrogen fixation via base pairing with the nitrogenase gene nifK mRNA in Pseudomonas stutzeri A1501.

Unlike most Pseudomonas, the root-associated bacterium Pseudomonas stutzeri A1501 fixes nitrogen after the horizontal acquisition of a nitrogen-fixing (nif) island. A genome-wide search for small noncoding RNAs (ncRNAs) in P. stutzeri A1501 identified the novel P. stutzeri-specific ncRNA NfiS in the core genome, whose synthesis was significantly induced under nitrogen fixation or sorbitol stress conditions. The expression of NfiS was RNA chaperone Hfq-dependent and activated by the sigma factor RpoN/global nitrogen activator NtrC/nif-specific activator NifA regulatory cascade. The nfiS-deficient mutant displayed reduced nitrogenase activity, as well as increased sensitivity to multiple stresses, such as osmotic and oxidative stresses. Secondary structure prediction and complementation studies confirmed that a stem-loop structure was essential for NfiS to regulate the nitrogenase gene nifK mRNA synthesis and thus nitrogenase activity. Microscale thermophoresis and physiological analysis showed that NfiS directly pairs with nifK mRNA and ultimately enhances nitrogenase activity by increasing the translation efficiency and the half-life of nifK mRNA. Our data also suggest structural and functional divergence of NfiS evolution in diazotrophic and nondiazotrophic backgrounds. It is proposed that NfiS was recruited by nifK mRNA as a novel regulator to integrate the horizontally acquired nif island into host global networks.

Identification and validation of QTLs for 100-seed weight using chromosome segment substitution lines in soybean.

The 100-seed weight (100SW) is one of the most important traits that control soybean yield. To identify the quantitative trait loci (QTL) of 100SW, 120 BC3F5 chromosome segment substitution lines (CSSLs) were cultivated over three years. The CSSLs were developed from a cross between the cultivated soybean variety 'Jackson' and the wild soybean accession 'JWS156-1', followed by continuous backcrossing using 'Jackson' variety as a recurrent parent. A total of nine QTLs (qSW8.1, qSW9.1, qSW12.1, qSW13.1, qSW14.1, qSW16.1, qSW17.1, qSW17.2, and qSW20.1) were detected on eight chromosomes. Of these, qSW12.1 (LOD = 6.78-12.31) was detected over the three successive years on chromosome 12 as a novel, stable, and major QTL. To validate the effect of qSW12.1, a residual heterozygous line (RHL), RHL564, which showed heterozygous at the qSW12.1 region, was selected from the BC3F5 population. Of the two homologous genotypes in the progenies produced by self-pollination of RHL564, a higher seed weight was observed in the 'Jackson' genotype plants than that in the 'JWS156-1' genotype plants. qSW12.1 was delimited in an interval of approximately 1,348 kb between the BARCSOYSSR_12_1282 and BARCSOYSSR_12_1347 markers on chromosome 12.

The genome of Paenibacillus sabinae T27 provides insight into evolution, organization and functional elucidation of nif and nif-like genes.

BACKGROUND: Most biological nitrogen fixation is catalyzed by the molybdenum nitrogenase. This enzyme is a complex which contains the MoFe protein encoded by nifDK and the Fe protein encoded by nifH. In addition to nifHDK, nifHDK-like genes were found in some Archaea and Firmicutes, but their function is unclear. RESULTS: We sequenced the genome of Paenibacillus sabinae T27. A total of 4,793 open reading frames were predicted from its 5.27 Mb genome. The genome of P. sabinae T27 contains fifteen nitrogen fixation (nif) genes, including three nifH, one nifD, one nifK, four nifB, two nifE, two nifN, one nifX and one nifV. Of the 15 nif genes, eight nif genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX and nifV) and two non-nif genes (orf1 and hesA) form a complete nif gene cluster. In addition to the nif genes, there are nitrogenase-like genes, including two nifH-like genes and five pairs of nifDK-like genes. IS elements on the flanking regions of nif and nif-like genes imply that these genes might have been obtained by horizontal gene transfer. Phylogenies of the concatenated 8 nif gene (nifB, nifH, nifD, nifK, nifE, nifN, nifX and nifV) products suggest that P. sabinae T27 is closely related to Frankia. RT-PCR analysis showed that the complete nif gene cluster is organized as an operon. We demonstrated that the complete nif gene cluster under the control of σ70-dependent promoter enabled Escherichia coli JM109 to fix nitrogen. Also, here for the first time we demonstrated that unlike nif genes, the transcriptions of nifHDK-like genes were not regulated by ammonium and oxygen, and nifH-like or nifD-like gene could not restore the nitrogenase activity of Klebsiella pneumonia nifH- and nifD- mutant strains, respectively, suggesting that nifHDK-like genes were not involved in nitrogen fixation. CONCLUSIONS: Our data and analysis reveal the contents and distribution of nif and nif-like genes and contribute to the study of evolutionary history of nitrogen fixation in Paenibacillus. For the first time we demonstrated that the transcriptions of nifHDK-like genes were not regulated by ammonium and oxygen and nifHDK-like genes were not involved in nitrogen fixation.

Integrated Hfq-interacting RNAome and transcriptomic analysis reveals complex regulatory networks of nitrogen fixation in root-associated Pseudomonas stutzeri A1501.

The RNA chaperone Hfq acts as a global regulator of numerous biological processes, such as carbon/nitrogen metabolism and environmental adaptation in plant-associated diazotrophs; however, its target RNAs and the mechanisms underlying nitrogen fixation remain largely unknown. Here, we used enhanced UV cross-linking immunoprecipitation coupled with high-throughput sequencing to identify hundreds of Hfq-binding RNAs probably involved in nitrogen fixation, carbon substrate utilization, biofilm formation, and other functions. Collectively, these processes endow strain A1501 with the requisite capabilities to thrive in the highly competitive rhizosphere. Our findings revealed a previously uncharted landscape of Hfq target genes. Notable among these is nifM, encoding an isomerase necessary for nitrogenase reductase solubility; amtB, encoding an ammonium transporter; oprB, encoding a carbohydrate porin; and cheZ, encoding a chemotaxis protein. Furthermore, we identified more than 100 genes of unknown function, which expands the potential direct regulatory targets of Hfq in diazotrophs. Our data showed that Hfq directly interacts with the mRNA of regulatory proteins (RsmA, AlgU, and NifA), regulatory ncRNA RsmY, and other potential targets, thus revealing the mechanistic links in nitrogen fixation and other metabolic pathways. IMPORTANCE: Numerous experimental approaches often face challenges in distinguishing between direct and indirect effects of Hfq-mediated regulation. New technologies based on high-throughput sequencing are increasingly providing insight into the global regulation of Hfq in gene expression. Here, enhanced UV cross-linking immunoprecipitation coupled with high-throughput sequencing was employed to identify the Hfq-binding sites and potential targets in the root-associated Pseudomonas stutzeri A1501 and identify hundreds of novel Hfq-binding RNAs that are predicted to be involved in metabolism, environmental adaptation, and nitrogen fixation. In particular, we have shown Hfq interactions with various regulatory proteins' mRNA and their potential targets at the posttranscriptional level. This study not only enhances our understanding of Hfq regulation but, importantly, also provides a framework for addressing integrated regulatory network underlying root-associated nitrogen fixation.

Comparative Genomic Analysis of a Thermophilic Protease-Producing Strain Geobacillus stearothermophilus H6.

The genus Geobacillus comprises thermophilic gram-positive bacteria which are widely distributed, and their ability to withstand high temperatures makes them suitable for various applications in biotechnology and industrial production. Geobacillus stearothermophilus H6 is an extremely thermophilic Geobacillus strain isolated from hyperthermophilic compost at 80 °C. Through whole-genome sequencing and genome annotation analysis of the strain, the gene functions of G. stearothermophilus H6 were predicted and the thermophilic enzyme in the strain was mined. The G. stearothermophilus H6 draft genome consisted of 3,054,993 bp, with a genome GC content of 51.66%, and it was predicted to contain 3750 coding genes. The analysis showed that strain H6 contained a variety of enzyme-coding genes, including protease, glycoside hydrolase, xylanase, amylase and lipase genes. A skimmed milk plate experiment showed that G. stearothermophilus H6 could produce extracellular protease that functioned at 60 °C, and the genome predictions included 18 secreted proteases with signal peptides. By analyzing the sequence of the strain genome, a protease gene gs-sp1 was successfully screened. The gene sequence was analyzed and heterologously expressed, and the protease was successfully expressed in Escherichia coli. These results could provide a theoretical basis for the development and application of industrial strains.