Master regulator NtrC controls the utilization of alternative nitrogen sources in Pseudomonas stutzeri A1501.
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ABSTRACT:
SUBMITTER: Yang Z
PROVIDER: S-EPMC8443478 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
| S-EPMC2820453 | biostudies-literature
Project description:The capacity to fix nitrogen is widely distributed in phyla of Bacteria and Archaea but has long been considered to be absent from the Pseudomonas genus. We report here the complete genome sequencing of nitrogen-fixing root-associated Pseudomonas stutzeri A1501. The genome consists of a single circular chromosome with 4,567,418 bp. Comparative genomics revealed that, among 4,146 protein-encoding genes, 1,977 have orthologs in each of the five other Pseudomonas representative species sequenced to date. The genome contains genes involved in broad utilization of carbon sources, nitrogen fixation, denitrification, degradation of aromatic compounds, biosynthesis of polyhydroxybutyrate, multiple pathways of protection against environmental stress, and other functions that presumably give A1501 an advantage in root colonization. Genetic information on synthesis, maturation, and functioning of nitrogenase is clustered in a 49-kb island, suggesting that this property was acquired by lateral gene transfer. New genes required for the nitrogen fixation process have been identified within the nif island. The genome sequence offers the genetic basis for further study of the evolution of the nitrogen fixation property and identification of rhizosphere competence traits required in the interaction with host plants; moreover, it opens up new perspectives for wider application of root-associated diazotrophs in sustainable agriculture.
| S-EPMC2396677 | biostudies-literature
Project description:NAD-independent L-lactate dehydrogenases (l-iLDHs) play important roles in L-lactate utilization of different organisms. All of the previously reported L-iLDHs were flavoproteins that catalyze the oxidation of L-lactate by the flavin mononucleotide (FMN)-dependent mechanism. Based on comparative genomic analysis, a gene cluster with three genes (lldA, lldB, and lldC) encoding a novel type of L-iLDH was identified in Pseudomonas stutzeri A1501. When the gene cluster was expressed in Escherichia coli, distinctive L-iLDH activity was detected. The expressed L-iLDH was purified by ammonium sulfate precipitation, ion-exchange chromatography, and affinity chromatography. SDS-PAGE and successive matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the purified L-iLDH indicated that it is a complex of LldA, LldB, and LldC (encoded by lldA, lldB, and lldC, respectively). Purified L-iLDH (LldABC) is a dimer of three subunits (LldA, LldB, and LldC), and the ratio between LldA, LldB, and LldC is 1:1:1. Different from the FMN-containing L-iLDH, absorption spectra and elemental analysis suggested that LldABC might use the iron-sulfur cluster for the L-lactate oxidation. LldABC has narrow substrate specificity, and only L-lactate and DL-2-hydrobutyrate were rapidly oxidized. Mg(2+) could activate L-iLDH activity effectively (6.6-fold). Steady-state kinetics indicated a ping-pong mechanism of LldABC for the L-lactate oxidation. Based on the gene knockout results, LldABC was confirmed to be required for the L-lactate metabolism of P. stutzeri A1501. LldABC is the first purified and characterized L-iLDH with different subunits that uses the iron-sulfur cluster as the cofactor.Providing new insights into the diversity of microbial lactate utilization could assist in the production of valuable chemicals and understanding microbial pathogenesis. An NAD-independent L-lactate dehydrogenase (L-iLDH) encoded by the gene cluster lldABC is indispensable for the L-lactate metabolism in Pseudomonas stutzeri A1501. This novel type of enzyme was purified and characterized in this study. Different from the well-characterized FMN-containing L-iLDH in other microbes, LldABC in P. stutzeri A1501 is a dimer of three subunits (LldA, LldB, and LldC) and uses the iron-sulfur cluster as a cofactor.
| S-EPMC4455268 | biostudies-other
Project description:The compatible solutes ectoine and hydroxyectoine are widely produced by bacteria as protectants against osmotic and temperature stress. l-Aspartate-beta-semialdehyde is used as the precursor molecule for ectoine/hydroxyectoine biosynthesis that is catalyzed by the EctABCD enzymes. l-Aspartate-beta-semialdehyde is a central intermediate in different biosynthetic pathways and is produced from l-aspartate by aspartokinase (Ask) and aspartate-semialdehyde-dehydrogenase (Asd). Ask activity is typically stringently regulated by allosteric control to avoid gratuitous synthesis of aspartylphosphate. Many organisms have evolved multiple forms of aspartokinase, and feedback regulation of these specialized Ask enzymes is often adapted to the cognate biochemical pathways. The ectoine/hydroxyectoine biosynthetic genes (ectABCD) are followed in a considerable number of microorganisms by an askgene (ask_ect), suggesting that Ask_Ect is a specialized enzyme for this osmoadaptive biosynthetic pathway. However, none of these Ask_Ect enzymes have been functionally characterized. Pseudomonas stutzeri A1501 synthesizes both ectoine and hydroxyectoine in response to increased salinity, and it possesses two Ask enzymes: Ask_Lys and Ask_Ect. We purified both Ask enzymes and found significant differences with regard to their allosteric control: Ask_LysC was inhibited by threonine and in a concerted fashion by threonine and lysine, whereas Ask_Ect showed inhibition only by threonine. The ectABCD_askgenes from P. stutzeri A1501 were cloned and functionally expressed in Escherichia coli, and this led to osmostress protection. An E. colistrain carrying the plasmid-based ectABCD_askgene cluster produced significantly more ectoine/hydroxyectoine than a strain expressing the ectABCDgene cluster alone. This finding suggests a specialized role for Ask_Ect in ectoine/hydroxyectoine biosynthesis.
| S-EPMC3165526 | biostudies-literature
Project description:BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.
| S-EPMC2907835 | biostudies-other
Project description:Pseudomonas stutzeri A1501, a plant-associated diazotrophic bacterium, prefers to conform to a nitrogen-fixing biofilm state under nitrogen-deficient conditions. The extracytoplasmic function (ECF) sigma factor AlgU is reported to play key roles in exopolysaccharide (EPS) production and biofilm formation in the Pseudomonas genus; however, the function of AlgU in P. stutzeri A1501 is still unclear. In this work, we mainly investigated the role of algU in EPS production, biofilm formation and nitrogenase activity in A1501. The algU mutant ΔalgU showed a dramatic decrease both in the EPS production and the biofilm formation capabilities. In addition, the biofilm-based nitrogenase activity was reduced by 81.4% in the ΔalgU mutant. The transcriptional level of pslA, a key Psl-like (a major EPS in A1501) synthesis-related gene, was almost completely inhibited in the algU mutant and was upregulated by 2.8-fold in the algU-overexpressing strain. A predicted AlgU-binding site was identified in the promoter region of pslA. The DNase I footprinting assays indicated that AlgU could directly bind to the pslA promoter, and β-galactosidase activity analysis further revealed mutations of the AlgU-binding boxes drastically reduced the transcriptional activity of the pslA promoter; moreover, we also demonstrated that AlgU was positively regulated by RpoN at the transcriptional level and negatively regulated by the RNA-binding protein RsmA at the posttranscriptional level. Taken together, these data suggest that AlgU promotes EPS production and nitrogen-fixing biofilm formation by directly activating the transcription of pslA, and the expression of AlgU is controlled by RpoN and RsmA at different regulatory levels.
| S-EPMC9141998 | biostudies-literature
Project description:Pseudomonas stutzeri A1501 possesses an extraordinary number of transporters which confer this rhizosphere bacterium with the sophisticated ability to metabolize various carbon sources. However, sugars are not a preferred carbon source for P. stutzeri A1501. The P. stutzeri A1501 genome has been sequenced, allowing for the homology-based in silico identification of genes potentially encoding sugar-transport systems by using established microbial sugar transporters as a template sequence. Genomic analysis revealed that there were 10 sugar transporters in P. stutzeri A1501, most of which belong to the ATP-binding cassette (ABC) family (5/10); the others belong to the phosphotransferase system (PTS), major intrinsic protein (MIP) family, major facilitator superfamily (MFS) and the sodium solute superfamily (SSS). These systems might serve for the import of glucose, galactose, fructose and other types of sugar. Growth analysis showed that the only effective medium was glucose and its corresponding metabolic system was relatively complete. Notably, the loci of glucose metabolism regulatory systems HexR, GltR/GtrS, and GntR were adjacent to the transporters ABCMalEFGK, ABCGtsABCD, and ABCMtlEFGK, respectively. Only the ABCGtsABCD expression was significantly upregulated under both glucose-sufficient and -limited conditions. The predicted structure and mutant phenotype data of the key protein GtsA provided biochemical evidence that P. stutzeri A1501 predominantly utilized the ABCGtsABCD transporter for glucose uptake. We speculate that gene absence and gene diversity in P. stutzeri A1501 was caused by sugar-deficient environmental factors and hope that this report can provide guidance for further analysis of similar bacterial lifestyles.
| S-EPMC7232493 | biostudies-literature
Project description:BackgroundStudies on membrane proteins are often hampered by insufficient yields of the protein of interest. Several prokaryotic hosts have been tested for their applicability as production platform but still Escherichia coli by far is the one most commonly used. Nevertheless, it has been demonstrated that in some cases hosts other than E. coli are more appropriate for certain target proteins.ResultsHere we have developed an expression system for the heterologous production of membrane proteins using a single plasmid-based approach. The gammaproteobacterium Pseudomonas stutzeri was employed as a new production host. We investigated several basic microbiological features crucial for its handling in the laboratory. The organism belonging to bio-safety level one is a close relative of the human pathogen Pseudomonas aeruginosa. Pseudomonas stutzeri is comparable to E. coli regarding its growth and cultivation conditions. Several effective antibiotics were identified and a protocol for plasmid transformation was established. We present a workflow including cloning of the target proteins, small-scale screening for the best production conditions and finally large-scale production in the milligram range. The GFP folding assay was used for the rapid analysis of protein folding states. In summary, out of 36 heterologous target proteins, 20 were produced at high yields. Additionally, eight transporters derived from P. aeruginosa could be obtained with high yields. Upscaling of protein production and purification of a Gluconate:H+ Symporter (GntP) family transporter (STM2913) from Salmonella enterica to high purity was demonstrated.ConclusionsPseudomonas stutzeri is an alternative production host for membrane proteins with success rates comparable to E. coli. However, some proteins were produced with high yields in P. stutzeri but not in E. coli and vice versa. Therefore, P. stutzeri extends the spectrum of useful production hosts for membrane proteins and increases the success rate for highly produced proteins. Using the new pL2020 vector no additional cloning is required to test both hosts in parallel.
| S-EPMC5607611 | biostudies-literature
Project description:The regulatory mechanisms underlying the uptake and utilization of multiple types of carbohydrates in actinomycetes remain poorly understood. In this study, we show that GlnR (central regulator of nitrogen metabolism) serves as a universal regulator of nitrogen metabolism and plays an important, previously unknown role in controlling the transport of non-phosphotransferase-system (PTS) carbon sources in actinomycetes. It was observed that GlnR can directly interact with the promoters of most (13 of 20) carbohydrate ATP-binding cassette (ABC) transporter loci and can activate the transcription of these genes in response to nitrogen availability in industrial, erythromycin-producing Saccharopolyspora erythraea. Deletion of the glnR gene resulted in severe growth retardation under the culture conditions used, with select ABC-transported carbohydrates (maltose, sorbitol, mannitol, cellobiose, trehalose, or mannose) used as the sole carbon source. Furthermore, we found that GlnR-mediated regulation of carbohydrate transport was highly conserved in actinomycetes. These results demonstrate that GlnR serves a role beyond nitrogen metabolism, mediating critical functions in carbon metabolism and crosstalk of nitrogen- and carbon-metabolism pathways in response to the nutritional states of cells. These findings provide insights into the molecular regulation of transport and metabolism of non-PTS carbohydrates and reveal potential applications for the cofermentation of biomass-derived sugars in the production of biofuels and bio-based chemicals.
| S-EPMC4697395 | biostudies-literature
Project description:Microbial colonization of the mammalian gut is largely ascribed to the ability to utilize nutrients available in that environment. To understand how beneficial microbes establish a relationship with their hosts, it is crucial to determine what other abilities promote gut colonization. We now report that colonization of the murine gut by the beneficial microbe Bacteroides thetaiotaomicron requires activation of a putative translation factor by the major transcriptional regulator of gut colonization and carbohydrate utilization. To ascertain how this regulator-called BT4338-promotes gut colonization, we identified BT4338-regulated genes and BT4338-bound DNA sequences. Unexpectedly, the gene whose expression was most reduced upon BT4338 inactivation was fusA2, specifying a putative translation factor. We determined that fusA2 activation by BT4338 is conserved in another Bacteroides species and essential for gut colonization in B. thetaiotaomicron because a mutant lacking the BT4338 binding site in the fusA2 promoter exhibited a colonization defect similar to that of a mutant lacking the fusA2 gene. Furthermore, we demonstrated that BT4338 promotes gut colonization independently of its role in carbohydrate utilization because the fusA2 gene was dispensable for utilization of carbohydrates that depend on BT4338 Our findings suggest that microbial gut colonization requires the use of alternative protein synthesis factors.IMPORTANCE The bacteria occupying the mammalian gut have evolved unique strategies to thrive in their environment. Bacteroides organisms, which often comprise 25 to 50% of the human gut microbiota, derive nutrients from structurally diverse complex polysaccharides, commonly called dietary fibers. This ability requires an expansive genetic repertoire that is coordinately regulated to achieve expression of those genes dedicated to utilizing only those dietary fibers present in the environment. Here we identify the global regulon of a transcriptional regulator necessary for dietary fiber utilization and gut colonization. We demonstrate that this transcription factor regulates hundreds of genes putatively involved in dietary fiber utilization as well as a putative translation factor dispensable for growth on such nutrients but necessary for survival in the gut. These findings suggest that gut bacteria coordinate cellular metabolism with protein synthesis via specialized translation factors to promote survival in the mammalian gut.
| S-EPMC6989115 | biostudies-literature