Redefining HexR regulatory landscape in Pseudomonas putida through integrative systems biology [ChIP-exo]
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ABSTRACT:
ORGANISM(S): Pseudomonas putida
PROVIDER: GSE275523 | GEO | 2026/08/20
REPOSITORIES: GEO
Similar Datasets
Project description:Pseudomonas putida strains are highly valued biocatalysts due to their abilities to degrade various chemicals, tolerate organic solvents, and resist environmental stressors. Fine–tuning the regulation of primary carbon metabolism is essential for their successful adaptation, with HexR emerging as a key regulator. While previous studies have assessed HexR binding using in vitro assays and comparative transcriptomics, the in vivo binding sites of HexR and its genome–scale regulon remain undefined. In this study, we performed comparative expression profiling of P. putida wild-type and hexR mutant strains on distinct growth substrates, including a glycolytic substrate (glucose), gluconeogenic substrates (acetate, succinate), and a substrate inducing both metabolic responses (glycerol). Our findings revealed extensive regulation of HexR in acetate metabolism, which simultaneously deactivates the glycolytic route and upregulates pyruvate metabolism, glyoxylate shunt, gluconeogenesis to support growth. Subsequently, ChIP–exo data integration identified 43 HexR binding locations in P. putida on acetate, controlling 68 direct regulon genes. Leveraging model–based in silico simulation provided contextual insight into metabolic flux states, enhancing our understanding of carbon metabolism orchestrated by this transcription factor. This study thus contributes to a holistic view of HexR regulatory landscape, highlighting its relevance in P. putida metabolism.
2026-08-20 | GSE275525 | GEO
Project description:We investigated the way global gene expression changed in E. coli correlated with the metabolism of seven carbon substrates chosen to trigger a large panel of metabolic pathways with features varying in: carbon influx flow rate; entry points in the metabolic network; modes of transport. Quantitative coarse-grained expression-pattern analysis demonstrated that the gene expression trend following immediately the reduction of carbon influx flow rate was determined by its initial expression level. Subsequent fine-grained expression-pattern analysis demonstrated that the Crp regulator, associated to a change in growth rate, governed the response of most carbon influx-dependent genes. By contrast, the Cra, Mlc and Fur regulators governed the expression of genes responding to non-glycolytic substrates, glycolytic substrates or PTS (phosphotransferase system) transported sugars following an idiosyncratic way, with the function of each of these regulators being titrated by the metabolism of the corresponding substrates. This work also allowed us to expand the extent of the gene complement regulated by each regulator with additional genes (27 genes in the Crp regulon, 49 genes in the Cra regulon, 1 gene in the Mlc regulon and 2 genes in Fur regulon) and to elucidate the regulatory functions of each regulator comprehensively. Strikingly, Crp and Cra worked in concert to coordinate central carbon metabolism with amino acids biosynthesis. Together, these results demonstrated the power of physiology-based interperation of global data in revealing regulatory networks of bacteria.
2021-03-02 | GSE156143 | GEO
Project description:Isoprenyl acetate, a volatile ester derived from isoprenol, is a key biosynthetic intermediate for a candidate for next-generation aviation fuel, 1,4-dimethylcyclooctane. Here, we engineered Pseudomonas putida KT2440 for the production of isoprenyl acetate from mixed sugar substrates. We first generated isoprenyl acetate by introducing a heterologous alcohol acetyltransferase (ATF1) and deleting three promiscuous native esterases to reduce product degradation. Then, we engineered efficient glucose and xylose co-utilization by integrating a heterologous xylose isomerase pathway and deleting global regulators crc and hexR to alleviate catabolite repression. Additionally, intracellular acetyl-CoA flux was reinforced through the expression of auxiliary carbon-conserving routes, including non-oxidative glycolysis and acetate assimilation. Culture conditions were systematically optimized by adjusting media composition, induction, and overlay solvent to maximize product yield. These cumulative efforts led to isoprenyl acetate titers of 1.5 g/L in shake flasks and 1.9 g/L in fed-batch fermentor cultures from mixed sugars, corresponding to a yield of 0.067 g/g of total sugar consumed. Our work demonstrates the potential of P. putida as a robust microbial chassis for scalable biosynthesis of ester-based biofuels from lignocellulosic feedstocks.
2026-03-23 | PXD067010 | Pride
Project description:<p>High-affinity iron (Fe) scavenging compounds, or siderophores, are widely employed by soil bacteria to survive scarcity in bioavailable Fe. Siderophore biosynthesis relies on cellular carbon metabolism, despite reported decrease in both carbon uptake and Fe-containing metabolic proteins in Fe-deficient cells. Given this paradox, the metabolic network required to sustain the Fe-scavenging strategy is poorly understood. Here, through multiple <sup>13</sup>C-metabolomics experiments with Fe-replete and Fe-limited cells, we uncover how soil <em>Pseudomonas</em> species reprogram their metabolic pathways to prioritize siderophore biosynthesis. Across the three species investigated (<em>Pseudomonas putida</em> KT2440, <em>Pseudomonas protegens</em> Pf-5, and <em>Pseudomonas putida</em> S12), siderophore secretion is higher during growth on gluconeogenic substrates than during growth on glycolytic substrates. In response to Fe limitation, we capture decreased flux toward the tricarboxylic acid (TCA) cycle during the metabolism of glycolytic substrates but, due to carbon recycling to the TCA cycle via enhanced anaplerosis, the metabolism of gluconeogenic substrates results in an increase in both siderophore secretion (up to threefold) and Fe extraction (up to sixfold) from soil minerals. During simultaneous feeding on the different substrate types, Fe deficiency triggers a hierarchy in substrate utilization, which is facilitated by changes in protein abundances for substrate uptake and initial catabolism. Rerouted metabolism further promotes favorable fluxes in the TCA cycle and the gluconeogenesis-anaplerosis nodes, despite decrease in several proteins in these pathways, to meet carbon and energy demands for siderophore precursors in accordance with increased proteins for siderophore biosynthesis. Hierarchical carbon metabolism thus serves as a critical survival strategy during the metal nutrient deficiency.</p><p><br></p><p><strong>Data availability:</strong></p><p>The proteomics data have been deposited into the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier <a href='https://www.ebi.ac.uk/pride/archive/projects/PXD013605' rel='noopener noreferrer' target='_blank'>PXD013605</a>.</p>
2020-12-05 | MTBLS1715 | MetaboLights
Project description:Background The catabolite control protein A (CcpA) is a member of the LacI/GalR family of transcriptional regulators controlling carbon-metabolism pathways in low-GC Gram positive bacteria. It functions as a catabolite repressor or activator, allowing the bacteria to utilize the preferred carbon source over secondary carbon sources. This study is the first CcpA-dependent transcriptome and proteome analysis in S. aureus wild type and ccpA-deleted mutant, focussing on short-time effects of glucose under stable pH conditions. Results The addition of glucose to exponentially growing S. aureus increased enzymes of glycolytic pathway, indicating a higher glycolytic activity, while proteins required for the complete oxidation in the TCA cycle were repressed via CcpA. Phosphotransacetylase and acetate kinase, converting acetylCoA to acetate with a concomitant substrate-level phosphorylation were neither regulated by glucose nor by CcpA. Most CcpA directly repressed genes were involved in utilization of amino acids as secondary carbon sources. More genes were found to be differentially expressed by CcpA in a glucose-independent manner than in the classical, glucose dependent way, suggesting that glucose-independent regulation by CcpA may be of particular importance in S. aureus. In the presence of glucose, CcpA was found to regulate expression of genes involved in metabolism, but that of genes coding for virulence determinants. Conclusions This study identified the CcpA regulon of exponentially growing S. aureus, for the first time. As in other bacteria, the CcpA-regulon of S. aureus comprised a large amount of metabolic genes but also some 50 genes associated with virulence. CcpA seemed to work in a glucose- as well as glucose-independent way.
2008-12-20 | GSE12614 | GEO
Project description:Background The catabolite control protein A (CcpA) is a member of the LacI/GalR family of transcriptional regulators controlling carbon-metabolism pathways in low-GC Gram positive bacteria. It functions as a catabolite repressor or activator, allowing the bacteria to utilize the preferred carbon source over secondary carbon sources. This study is the first CcpA-dependent transcriptome and proteome analysis in S. aureus wild type and ccpA-deleted mutant, focussing on short-time effects of glucose under stable pH conditions. Results The addition of glucose to exponentially growing S. aureus increased enzymes of glycolytic pathway, indicating a higher glycolytic activity, while proteins required for the complete oxidation in the TCA cycle were repressed via CcpA. Phosphotransacetylase and acetate kinase, converting acetylCoA to acetate with a concomitant substrate-level phosphorylation were neither regulated by glucose nor by CcpA. Most CcpA directly repressed genes were involved in utilization of amino acids as secondary carbon sources. More genes were found to be differentially expressed by CcpA in a glucose-independent manner than in the classical, glucose dependent way, suggesting that glucose-independent regulation by CcpA may be of particular importance in S. aureus. In the presence of glucose, CcpA was found to regulate expression of genes involved in metabolism, but that of genes coding for virulence determinants. Conclusions This study identified the CcpA regulon of exponentially growing S. aureus, for the first time. As in other bacteria, the CcpA-regulon of S. aureus comprised a large amount of metabolic genes but also some 50 genes associated with virulence. CcpA seemed to work in a glucose- as well as glucose-independent way. The transcriptomes of strain Newman and its isogenic ccpA-deleted mutant were determined in early exponential growth and 30 min after the addition of 10 mM glucose, under controlled pH conditions. In the absence of glucose, the wild type grew slightly faster than the mutant, reaching an OD600 of 1 approximately 20 min earlier than the mutant. Adding 10 mM glucose at OD600 1 increased the growth rate of the wild type but had only a minor effect on that of the mutant. 60 min after glucose addition, glucose was depleted down to 0.3 mM by the wild type, while still 3 mM glucose was left in the culture of the mutant. Despite increased glucose consumption rates in the wild type, acetate production was only slightly enhanced compared to the mutant. No lactate was excreted at any time point sampled. Acidification of the medium upon glucose metabolism was prevented by buffering, maintaining a pH of 7.5 for both strains and under both growth conditions for at least 2 h after glucose addition, allowing to rule out any pH effects.
2010-06-24 | E-GEOD-12614 | biostudies-arrayexpress
Project description:Caldicellulosiruptor saccharolyticus is an extremely thermophilic, Gram-positive anaerobe, which ferments cellulose-, hemicellulose- and pectin-containing biomass to acetate, CO2 and hydrogen. Its broad substrate range, high hydrogen-producing capacity, and ability to co-utilize glucose and xylose, make this bacterium an attractive candidate for microbial bioenergy production. Glycolytic pathways and an ABC-type sugar transporter were significantly up-regulated during growth on glucose and xylose, indicating that C. saccharolyticus co-ferments these sugars unimpeded by glucose-based catabolite repression. The capacity to simultaneously process and utilize a range of carbohydrates associated with biomass feedstocks represents a highly desirable feature of a lignocellulose-utilizing, biofuel-producing bacterium. Keywords: substrate response C. saccharolyticus was subcultured (overnight) 3 times on the substrate of interest in modified DSMZ 640 medium before inoculating a pH-controlled (pH = 7) 1-liter fermentor containing 4 gram substrate per liter. Cells were grown at 70 °C until mid-logarithmic phase (~OD660 = 0.3-0.4) and harvested by centrifugation and rapid cooling to 4 °C and stored at -80 °C. To elucidate the central carbon metabolic pathways and their regulation, transcriptome analysis was performed after growth on glucose, xylose and a 1:1 mixture of both substrates. L-Rhamnose, which was likely to follow another pathway, was used as a reference substrate.
2008-04-15 | E-GEOD-11153 | biostudies-arrayexpress
Project description:Isoprenyl acetate, a volatile ester derived from isoprenol, is a key biosynthetic intermediate for a candidate for next-generation aviation fuel, 1,4-dimethylcyclooctane. Here, we engineered Pseudomonas putida KT2440 for the production of isoprenyl acetate from mixed sugar substrates. We first generated isoprenyl acetate by introducing a heterologous alcohol acetyltransferase (ATF1) and deleting three promiscuous native esterases to reduce product degradation. Then, we engineered efficient glucose and xylose co-utilization by integrating a heterologous xylose isomerase pathway and deleting global regulators crc and hexR to alleviate catabolite repression. Additionally, intracellular acetyl-CoA flux was reinforced through the expression of auxiliary carbon-conserving routes, including non-oxidative glycolysis and acetate assimilation. Culture conditions were systematically optimized by adjusting media composition, induction, and overlay solvent to maximize product yield. These cumulative efforts led to isoprenyl acetate titers of 1.5 g/L in shake flasks and 1.9 g/L in fed-batch fermentor cultures from mixed sugars, corresponding to a yield of 0.067 g/g of total sugar consumed. Our work demonstrates the potential of P. putida as a robust microbial chassis for scalable biosynthesis of ester-based biofuels from lignocellulosic feedstocks.
| PXD067104 | panorama
Project description:Pseudomonas putida KT2440 is a metabolically versatile soil bacterium useful both as a model biodegradative organism and as a host of catalytic activities of biotechnological interest. In this report, we present the high-resolution transcriptome of P. putida grown in different carbon sources as revealed by deep sequencing of the corresponding RNA pools. Examination of the data from growth on glycolytic (glucose, fructose) and gluconeogenic (succinate or glycerol) substrates revealed that > 20% of the P. putida genome is differentially expressed depending on the ensuing metabolic regime. Changes affected not only metabolic genes but also a suite of global regulators, e.g. the rpoS sigma subunit of RNAP, various cold-shock proteins and the three HU histone-like proteins. Specifically, the genes encoding HU subunit variants hupA, hupB and hupN drastically altered their expression levels (and thus their ability to form heterodimeric combinations) under the different growth conditions. Furthermore, we found that the two small RNAs crcZ and crcY, known to inhibit the Crc protein that mediates catabolite repression in P. putida, were both down-regulated by glucose. cDNA libraries from Pseudomonas supplemented with different carbon sources (glucose, glycerol, fructose, succinate) were sequenced using HiSeq 2000 to yield 91 paired-end reads. Gene expression values were compared.
2013-07-23 | E-GEOD-46491 | biostudies-arrayexpress
Project description:In metabolically versatile bacteria, carbon catabolite repression (CCR) facilitates the preferential assimilation of the most efficient carbon sources, improving growth rate and fitness. In Pseudomonas putida, the Crc protein and the CrcZ and CrcY small RNAs (sRNAs), which are believed to antagonise Crc, are key players in CCR. Contrary to what occurs in other bacterial species, succinate or glucose elicit a weak CCR in this bacterium. We combined metabolic, transcriptomics and constraints-based metabolic flux analyses to clarify whether P. putida prefers succinate or glucose, and the role of the Crc/CrcZ-CrcY regulatory system in their metabolization. Succinate was consumed faster than glucose and allowed a higher growth rate. However, both compounds were co-metabolised when provided simultaneously. The levels of CrcZ and CrcY were lower when both substrates were present than when only one of them was provided, suggesting a role for Crc in coordinating metabolism. Metabolic reconstructions suggested that, when both substrates are present, Crc works to organize a metabolism in which carbon compounds flow in two opposite directions: from glucose to pyruvate, and from succinate to pyruvate. Therefore, Crc serves not only to favour the assimilation of preferred compounds, but also to balance the carbon fluxes, optimising metabolism and growth.
2015-03-06 | GSE63987 | GEO