Table 3 - uploaded by ZHIHONG XIE
Content may be subject to copyright.
Source publication
Pseudomonas stutzeri strain A1501 isolated from rice fixes nitrogen under microaerobic conditions in the free-living state. This paper describes the properties of nifL and nifA mutants as well as the physical interaction between NifL and NifA proteins. A nifL mutant strain that carried a mutation non-polar on nifA expression retained nitrogenase ac...
Contexts in source publication
Context 1
... orien- tation of the Km resistance gene was the opposite of that of the nifA gene. Plasmids were transferred into P. stutzeri recipients by conju- gation, using Escherichia coli S17-1 as the donor, as previously MATa trp1-109 leu2-3,112 ura3-52 his3-200 gal4D gal80 LYS2 : : GAL1-HIS3 GAL2-ADE2 met2 : : GAL7-lacZ *For pGAD and pGBD derivatives containing entire or portions of nifA and nifL, constructed in this work, see Fig. 2, Table 2 and Table 3. described (Desnoues et al., 2003). Recombination in the host genome at the correct location was checked for by PCR amplifica- tion with appropriate oligonucleotide primers. ...
Context 2
... cerevisiae competent cells were prepared using the method described in the Matchmaker II protocol and pGAD and pGBD derivatives were co-transformed into the yeast recipient. The interac- tion between NifA and NifL domains was screened for by growth on SD medium lacking Leu, His, Trp and Ade (Table 3). The filter and quantitative liquid b-galactosidase assay were done according to the protocol described by the Matchmaker II system (Clontech). ...
Context 3
... sites are underlined. DY2H: amplicons were cloned in pGAD and pGBD vectors for the yeast two-hybrid experiments; see Table 3 and Fig. 2. ...
Context 4
... co-transformation into yeast strain PJ69-4A, an interaction between the peptides fused to the pGAD and pGBD vectors is required so that the transcription from the promoters under the control of GAL4 can proceed (Table 3). As one of the reporter genes is lacZ, an enzymic assay for b-galactosidase gives an estimation of the relative strength of the interaction (James et al., 1996). ...
Context 5
... one of the reporter genes is lacZ, an enzymic assay for b-galactosidase gives an estimation of the relative strength of the interaction (James et al., 1996). Fig. 3 shows an example of the growth on selective media and Table 3 summarizes the data obtained for growth and b-galactosi- dase activity. From these data it is concluded that an interaction is detected between the entire NifA and NifL, and that the binding is limited to the C-terminal part of NifL and the central domain of NifA. ...
Context 6
... further confirm the interaction between NifLc and NifA, the corresponding DNA fragments were cloned into Table 3. expression vectors to obtain a GST-NifLc and a His 6 -NifA fusion protein as described in Methods. ...
Citations
... Despite its close relation to Geobacter, Geomonas was shown to possess a distinct nif gene cluster. Compared to facultative anaerobic or strict anaerobic diazotrophs like Anaeromyxobacter, Azotobacter, Pseudomo nas, Pelobacter (33)(34)(35), Geomonas exhibited a remarkably compact structure of a nif cluster and also contained the minimum required gene set for nitrogenase, nifHDKENBX. Furthermore, the essential residues of the active site of nitrogenase were also conserved (Fig. 1C). ...
Nitrogen gas (N 2 ) fixation driven by diazotrophs is a crucial process for supplying nitrogen to paddy soil ecosystems. The genus Geomonas has been considered to be an important potential diazotroph in paddy soils, but direct experimental evidence of the nitrogen-fixing ability of Geomonas in pure culture is still lacking. Hence, we aimed to demonstrate this nitrogen-fixing capability and shed light on how this process was regulated in response to ammonium (NH 4 ⁺ ) in Geomonas . In this study, we determined that a key nitrogenase gene ( nifH ) was present in 50 isolates from paddy soils. Members of Geomonas contained the minimum nitrogen fixation gene cluster ( nifBHDKEN ) based on genomic analysis, implying Geomonas species had the potential to fix nitrogen. Acetylene reduction assay (ARA), ¹⁵ N 2 isotope labeling, and total nitrogen accumulation assays validated that Geomonas was, indeed, able to fix nitrogen in pure culture. Under nitrogen-fixing conditions, the cell morphology of Geomonas changed from short rod-shaped (with NH 4 ⁺ ) to long rod-shaped and flagella became longer and thicker. The expression of genes correlated to nitrogen fixation in the Geomonas transcriptome was quantified in response to NH 4 ⁺ . Expression of genes associated with nitrogenase, flavin-based electron bifurcation complexes (such as the FixAB system), NH 4 ⁺ uptake, and transformation (e.g., glutamine and glutamate synthetases) were significantly upregulated under nitrogen-fixing conditions, suggesting these mechanisms might be involved in N 2 fixation in Geomonas . These results were verified by RT-qPCR. Taken together, our results demonstrate that Geomonas species possess the ability to fix N 2 and expand our understanding on the ecological significance and potential applications of Geomonas in paddy soil ecosystems.
IMPORTANCE
The ability of Geomonas species to fix nitrogen gas (N 2 ) is an important metabolic feature for its application as a plant growth-promoting rhizobacterium. This research is of great importance as it provides the first comprehensive direct experimental evidence of nitrogen fixation by the genus Geomonas in pure culture. We isolated a number of Geomonas strains from paddy soils and determined that nifH was present in these strains. This study demonstrated that these Geomonas species harbored genes encoding nitrogenase, as do Geobacter and Anaeromyxobacter in the same class of Deltaproteobacteria . We demonstrated N 2 -dependent growth of Geomonas and determined regulation of gene expression associated with nitrogen fixation. The research establishes and advances our understanding of nitrogen fixation in Geomonas .
... In addition, because the associated nitrogen-fixing bacteria and root system could not form root nodules and other special tissue structures, nif gene expression is also greatly affected by environmental factors. It is conceivable that the protein NtrC may participate in the expression of the regulator NifA in P. stutzeri (Xie et al. 2006). However, the role and mechanism of NtrC in the nitrogen metabolism of P. stutzeri A1501 have not been clarified clearly. ...
... In particular, nifA, which codes for the transcriptional activator of all nif operons (Chengtao et al. 2004;Demtröder et al. 2019), showed a 0.16-fold decrease, and glnK, which codes for a PII family protein (Xu et al. 1998;Blauwkamp and Ninfa 2002), showed a 0.14-fold decrease in the ntrC mutant. In P. stutzeri, GlnK is required for both NifA synthesis and activity, particularly by preventing the inhibitory effect of NifL on NifA activity (Xie et al. 2006;He et al. 2008). These data were consistent with the observation that inactivation of NtrC affected nitrogenase activity, suggesting a role in positive regulation of nif genes. ...
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.
... At the transcriptional level, oxygen repressed nitrogenase synthesis much more rapidly than ammonia did in Azotobacter (45). In most Proteobacteria, including P. stutzeri A1501, NifA is the transcriptional activator of other nif operons (46)(47)(48)(49)(50). In K. pneumoniae strains that can fix nitrogen anaerobically, NifL responds to oxygen and prevents NifA-mediated activation of nif gene expression (46,47). ...
... In K. pneumoniae strains that can fix nitrogen anaerobically, NifL responds to oxygen and prevents NifA-mediated activation of nif gene expression (46,47). In P. stutzeri, whose optimal nitrogenase activity was observed at an oxygen concentration of 1%, the expression of nifLA was also controlled by oxygen (22,49). Transcription of nif genes from Rhodobacter capsulatus, a bacterium that does not contain an nifL gene, is inhibited by oxygen, probably through direct inactivation of NifA (50). ...
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.
... This global regulation occurs at transcriptional and posttranscriptional levels, including a classical nitrogen regulatory cascade, an ADP-ribosyl-transferase/glycohydrolase (DraT/DraG) system, and a recently identified complex network of ncRNAs. The RpoN-NtrC-NifA regulatory cascade is a major and best-studied regulatory mechanism for nitrogen fixation at the transcriptional level (27,40). In addition, nitrogenase activity can be modulated by ADP ribosylation of NifH, the so-called "switch-off" effect. ...
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 n itrogen f ixation condition- i nducible nc R NA), 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.
... nov ( Yamane and Suzuki, 1988;Hazlewood et al., 1992;Huang et al., 2012;Menéndez et al., 2015). Some of the Pseudomonas species also play a role in nitrogen fixation and metabolism such as P. aeruginosa ( Wu et al., 2005), and P. stutzeri ( Xie et al., 2006). Thus, Pseudomonas could also play an important role in the nitrogen metabolism of the gut system. ...
The Eurasian or European beaver (Castor fiber) is the second-largest living rodent after the capybara. It is a semi-aquatic animal known for building dams and lodges. They strictly feed on lignocellulose-rich plants and correspondingly harbor cellulolytic microbial communities in their digestive tract. In this study, the bacterial community composition, diversity, and functional profile of different gut compartments ranging from stomach to colon have been explored. A total of 277 bacterial operational taxonomic units (OTUs) at species level were obtained from the gut systems of two males (juvenile and subadult) and one subadult female beaver. In general, cecum and colon are dominated by Firmicutes and Actinobacteria. High abundance of Bacteroidetes was observed only in male juvenile beaver cecum and colon, suggesting that the bacterial composition changes with age. Within the cecum and colon, members of known cellulase-producing bacterial taxa including the families Ruminococcaceae, Lachnospiraceae, and Clostridiaceae 1 were detected. The presence of putative genes encoding cellulolytic and carbohydrate-degrading enzymes indicated also the degradation of recalcitrant plant material in both gut compartments. The bacterial community in the gut systems of the Eurasian beaver differed from that of the North American beaver. Higher abundance of Actinobacteria and lower abundances of Bacteroidetes were recorded in the Eurasian beaver. Similar differences were obtained to bacterial communities of termites and herbivorous animals such as bovine. The data presented in this study provides the first insight into bacterial communities in the gut system of the Eurasian beaver.
... Pseudomonas stutzeri A1501 is a root-associated bacterium that exhibits an unusual feature, for a Pseudomonas strain, the ability to fix nitrogen [20][21][22][23][24] . The P. stutzeri A1501 genome contains a 49-kb nitrogen fixation island (NFI) that comprises the largest group of nif genes identified to date 25 . ...
... Within this island, a total of 52 nif-related genes are organized into 11 putative NifA-δ 54 -dependent operons 24 . nif gene expression in A1501 was revealed to be tightly regulated at both the transcriptional and post-transcriptional levels 22,23,26,27 . Given its natural integrity and well-studied regulation, the A1501 NFI is a promising model for studying the synthetic biology of nitrogen fixation systems. ...
... The nitrogen regulatory cascade of P. stutzeri A1501 comprises the AmtB-GlnK-NtrBC general nitrogen regulation proteins and the nif-specific regulatory protein NifLA 22,24,26,31 . E. coli has a similar nitrogen regulatory system to P. stutzeri but harbors an additional PII protein and a cascade regulation by the Nac regulatory protein 32,33 . ...
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.
... P. stutzeri A1501, which is isolated from the rice rhizosphere in China, is the best studied strain regarding nitrogen fixation. It possesses a general nitrogen regulatory system in the core genome (NtrBC and related genes) (23)(24)(25), but, during its evolution, it acquired a nif-specific regulatory system (NifLA) (25) from a diazotrophic ancestor. Thus, the regulatory network controlling nitrogen fixation in A1501 may result from regulatory systems of different evolutionary origins. ...
... No binding site for NtrC or NifA was identified. A band corresponding to the migration of a 250-nt molecule was detected by Northern blot analysis in the WT strain A1501, but it was not detected when the analysis was performed with RNA extracted from deletion mutants for rpoN, ntrC, or nifA under nitrogen fixation conditions (Fig. 1A, Inset, lanes 1-4), which are involved in the regulation of nitrogen fixation (17,18,24,25). The band intensity was decreased when the analysis was performed with RNA extracted from an rpoS deletion mutant, known in other systems as the RNA polymerase sigma factor of the general stress response (30) (Fig. 1A, Inset, lane 5). ...
... In contrast, their expression was remarkably enhanced by overproduction of NfiS. In particular, nifA, coding for the transcriptional activator of all nif operons (18,24,25), showed a threefold increase, and glnK, coding for a PII family protein (31,32), showed a sixfold increase (Fig. S4A). In P. stutzeri, GlnK is essential for both NifA synthesis and activity, in particular by preventing the inhibitory effect of NifL on NifA activity (26,32). ...
Significance
The biological nitrogen fixation process responsible for the reduction of atmospheric nitrogen to ammonia represents the primary source of nitrogen supporting extant life. We have identified a novel noncoding RNA (ncRNA) in the Pseudomonas stutzeri core genome, called NfiS, that is involved in the stress response and controls the expression of genes located in a genomic nitrogen-fixing ( nif ) island. NfiS was found to optimize nitrogen fixation by the direct posttranscriptional regulation of nitrogenase gene nifK mRNA. The acquisition of the nif island and the recruitment of NfiS by nifK mRNA are evolutionary events that seem to contribute to fine-tuned regulation of nitrogenase activity in P. stutzeri . This study provides a new regulatory pathway, mediated by an ncRNA for optimal nitrogen fixation, that may operate in other diazotrophs.
... Full assembly of the nitrogenase complex needs the products of at least twelve nif genes, especially for the processing of catalytic stability and nitrogenase metalloclusters (nifMZ, nifUS, and nifW) and for synthesis of a particular molybdenum cofactor (FeMo-co) 35 . The D5A genome contains all the above nif genes together with the NifA and NifL genes which are the positive/negative regulatory proteins for nif genes 36 . The rnfABCDEG operon, which encodes a membrane-bound protein complex related to electron transport to nitrogenase 37 , is also found in the D5A genome (Table S3). ...
This research undertook the systematic analysis of the Klebsiella sp. D5A genome and identification of genes that contribute to plant growth-promoting (PGP) traits, especially genes related to salt tolerance and wide pH adaptability. The genome sequence of isolate D5A was obtained using an Illumina HiSeq 2000 sequencing system with average coverages of 174.7× and 200.1× using the paired-end and mate-pair sequencing, respectively. Predicted and annotated gene sequences were analyzed for similarity with the Kyoto Encyclopedia of Genes and Genomes (KEGG) enzyme database followed by assignment of each gene into the KEGG pathway charts. The results show that the Klebsiella sp. D5A genome has a total of 5,540,009 bp with 57.15% G + C content. PGP conferring genes such as indole-3-acetic acid (IAA) biosynthesis, phosphate solubilization, siderophore production, acetoin and 2,3-butanediol synthesis, and N2 fixation were determined. Moreover, genes putatively responsible for resistance to high salinity including glycine-betaine synthesis, trehalose synthesis and a number of osmoregulation receptors and transport systems were also observed in the D5A genome together with numerous genes that contribute to pH homeostasis. These genes reveal the genetic adaptation of D5A to versatile environmental conditions and the effectiveness of the isolate to serve as a plant growth stimulator.
... 酸化或去磷酸化,进而引起氮代谢中重要基因和 操纵子的激活或抑制 [3] 。 NtrC 主要作用于氮代 谢调控系统,ntrC 基因突变会导致细菌对氮源的 利用和固氮作用发生改变。 最近的一些研究突出 显示了 NtrC 能够与碳代谢调控蛋白 CbrB 偶联作 用 [4,5] 。 另外,NtrC 也可以参与调节三聚氰酸降 解 [6] 和 抗 压 力 胁 迫 过 程 [7,8] [9] 。 随后发现其在各种细菌中存在并与氮代 谢相关,其编码产物为 NtrC [10] 。 不同物种 NtrC 另一侧是相互平行的 α2、α3 和 α4。 当天冬氨酸 -54 残基磷酸化,α3 与 β5 互换位置,从而使 N 端结构域磷酸化与中间结构域互作激活转录 [11] 。 中间结构域是所有已知 σ 54 RNA 聚合酶激活子的 区域 [12] ,其三维结构还不清楚,可根据已报道的 核苷酸结合蛋白( EF-Tu) 的结构进行预测。 NtrC 效应器结构域与倒位刺激因子( FIS) 结构域的序 列有很高的相似性,系统发育分析表明 FIS 来源 于祖先 α-变形菌 门 的 NtrC 蛋 白 的 C 末 端 结 构 域 [13] 。 NtrC 蛋白效应器结构域是一个二聚体,每 [17] ntrC 突变株能够利用硝 酸盐作为唯一氮源生长,但以铵盐为唯一氮源时 不能正常生长。 不同细菌中,NtrC 对氨基酸利用 情况的调控也有差异 [18] 。 近年来,Zhang 等 [5] [22,23] [25] [33,34] [4,5] ...
NtrBC is a two-component system involved in bacteria nitrogen metabolism regulation, and NtrC is a transcription activator protein in response to environmental signal and activates the transcription of target genes. The past studies have indicated that bacterial NtrC plays important roles in regulating diverse processes including nitrogenous compounds utilization, biological nitrogen fixation, the biosynthesis of biopolymer and maintenance on the carbon-nitrogen balance. Studies on NtrC's overall regulating function are one of the hotspot of microbe metabolic regulatory networks. This paper reviewed the research progress on NtrC in recent years, and described in detail the biological functions of bacterial NtrC, so as to better understand microbian environment adaptation, metabolic diversity, and the molecular mechanism of C-N coupling regulation.
... The bacterial strains and plasmids used in this work are listed inTable 1 . Bacterial strains were grown in Luria- Bertani (LB) and minimal lactate-containing medium (medium K), as previously described [43]. When required, carbon sources were supplemented at the following final concentrations: 4 mM glucose, 4 mM succinate , 4 mM lactate, 4 mM acetate, 4 mM benzoate, 0.4 mM catechol and 0.4 mM 4-hydroxybenzoate. ...
Soil microorganisms are mainly responsible for the complete mineralization of aromatic compounds that usually originate from plant products or environmental pollutants. In many cases, structurally diverse aromatic compounds can be converted to a small number of structurally simpler intermediates, which are metabolized to tricarboxylic acid intermediates via the beta-ketoadipate pathway. This strategy provides great metabolic flexibility and contributes to increased adaptation of bacteria to their environment. However, little is known about the evolution and regulation of the beta-ketoadipate pathway in root-associated diazotrophs.
In this report, we performed a genome-wide analysis of the benzoate and 4-hydroxybenzoate catabolic pathways of Pseudomonas stutzeri A1501, with a focus on the functional characterization of the beta-ketoadipate pathway. The P. stutzeri A1501 genome contains sets of catabolic genes involved in the peripheral pathways for catabolism of benzoate (ben) and 4-hydroxybenzoate (pob), and in the catechol (cat) and protocatechuate (pca) branches of the beta-ketoadipate pathway. A particular feature of the catabolic gene organization in A1501 is the absence of the catR and pcaK genes encoding a LysR family regulator and 4-hydroxybenzoate permease, respectively. Furthermore, the BenR protein functions as a transcriptional activator of the ben operon, while transcription from the catBC promoter can be activated in response to benzoate. Benzoate degradation is subject to carbon catabolite repression induced by glucose and acetate in A1501. The HPLC analysis of intracellular metabolites indicated that low concentrations of 4-hydroxybenzoate significantly enhance the ability of A1501 to degrade benzoate.
The expression of genes encoding proteins involved in the beta-ketoadipate pathway is tightly modulated by both pathway-specific and catabolite repression controls in A1501. This strain provides an ideal model system for further study of the evolution and regulation of aromatic catabolic pathways.
