-
BMC Genomics Jan 2024Pseudomonas putida S12 is a gram-negative bacterium renowned for its high tolerance to organic solvents and metabolic versatility, making it attractive for various...
BACKGROUND
Pseudomonas putida S12 is a gram-negative bacterium renowned for its high tolerance to organic solvents and metabolic versatility, making it attractive for various applications, including bioremediation and the production of aromatic compounds, bioplastics, biofuels, and value-added compounds. However, a metabolic model of S12 has yet to be developed.
RESULTS
In this study, we present a comprehensive and highly curated genome-scale metabolic network model of S12 (iSH1474), containing 1,474 genes, 1,436 unique metabolites, and 2,938 metabolic reactions. The model was constructed by leveraging existing metabolic models and conducting comparative analyses of genomes and phenomes. Approximately 2,000 different phenotypes were measured for S12 and its closely related KT2440 strain under various nutritional and environmental conditions. These phenotypic data, combined with the reported experimental data, were used to refine and validate the reconstruction. Model predictions quantitatively agreed well with in vivo flux measurements and the batch cultivation of S12, which demonstrated that iSH1474 accurately represents the metabolic capabilities of S12. Furthermore, the model was simulated to investigate the maximum theoretical metabolic capacity of S12 growing on toxic organic solvents.
CONCLUSIONS
iSH1474 represents a significant advancement in our understanding of the cellular metabolism of P. putida S12. The combined results of metabolic simulation and comparative genome and phenome analyses identified the genetic and metabolic determinants of the characteristic phenotypes of S12. This study could accelerate the development of this versatile organism as an efficient cell factory for various biotechnological applications.
Topics: Solvents; Pseudomonas putida; Genome, Bacterial; Genomics; Metabolic Networks and Pathways
PubMed: 38229031
DOI: 10.1186/s12864-023-09940-y -
Biology Jun 2024KT2440 is a popular platform for bioremediation due to its robust tolerance to environmental stress and strong biodegradation capacity. Limited research on the salt...
KT2440 is a popular platform for bioremediation due to its robust tolerance to environmental stress and strong biodegradation capacity. Limited research on the salt tolerance of KT2440 has hindered its application. In this study, the strain KT2440 was tested to tolerate a maximum of 4% / NaCl cultured with minimal salts medium. Transcriptomic data in a high-salinity environment showed significant expression changes in genes in membrane components, redox processes, chemotaxis, and cellular catabolic processes. -encoding betaine-aldehyde dehydrogenase was identified from the transcriptome data to overexpress and enhance growth profile of the strain KT2440 in minimal salts medium containing 4% / NaCl. Meanwhile, screening for exogenous salt-tolerant genes revealed that the Na/H antiporter from significantly increased the growth of the strain KT2440 in 4% / NaCl. Then, co-expression of and (KT2440-) increased the maximum salt tolerance of strain KT2440 to 5% / NaCl. Further addition of betaine and proline improved the salt tolerance of the engineered strain to 6% / NaCl. Finally, the engineered strain KT2440- was able to degrade 56.70% of benzoic acid and 95.64% of protocatechuic acid in minimal salt medium containing 4% / NaCl in 48 h, while no biodegradation was observed in the normal strain KT2440 in the same conditions. However, the strain KT2440- failed to degrade catechol in minimal salt medium containing 3% / NaCl. This study illustrated the improvement in the salt tolerance performance of KT2440 and the feasibility of engineered strain KT2440 as a potential salt-tolerant bioremediation platform.
PubMed: 38927284
DOI: 10.3390/biology13060404 -
Proteins Nov 2023The hypoxia-inducible factor (HIF) prolyl-hydroxylases (human PHD1-3) catalyze prolyl hydroxylation in oxygen-dependent degradation (ODD) domains of HIFα isoforms,...
The hypoxia-inducible factor (HIF) prolyl-hydroxylases (human PHD1-3) catalyze prolyl hydroxylation in oxygen-dependent degradation (ODD) domains of HIFα isoforms, modifications that signal for HIFα proteasomal degradation in an oxygen-dependent manner. PHD inhibitors are used for treatment of anemia in kidney disease. Increased erythropoietin (EPO) in patients with familial/idiopathic erythrocytosis and pulmonary hypertension is associated with mutations in EGLN1 (PHD2) and EPAS1 (HIF2α); a drug inhibiting HIF2α activity is used for clear cell renal cell carcinoma (ccRCC) treatment. We report crystal structures of PHD2 complexed with the C-terminal HIF2α-ODD in the presence of its 2-oxoglutarate cosubstrate or N-oxalylglycine inhibitor. Combined with the reported PHD2.HIFα-ODD structures and biochemical studies, the results inform on the different PHD.HIFα-ODD binding modes and the potential effects of clinically observed mutations in HIFα and PHD2 genes. They may help enable new therapeutic avenues, including PHD isoform-selective inhibitors and sequestration of HIF2α by the PHDs for ccRCC treatment.
Topics: Humans; Carcinoma, Renal Cell; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Inducible Factor-Proline Dioxygenases; Kidney Neoplasms; Oxygen; Procollagen-Proline Dioxygenase; Prolyl Hydroxylases; Protein Isoforms
PubMed: 37449559
DOI: 10.1002/prot.26541 -
Bioorganic & Medicinal Chemistry Letters Oct 2023Promysalin is an amphipathic antibiotic isolated from Pseudomonas promysalinigenes (previously Pseudomonas putida RW10S1) which shows potent antibacterial activities...
Promysalin is an amphipathic antibiotic isolated from Pseudomonas promysalinigenes (previously Pseudomonas putida RW10S1) which shows potent antibacterial activities against Gram-negative pathogens by inactivating succinate dehydrogenase. Based on the in-vivo studies, promysalin is hypothesized to be assembled from three building blocks: salicylic acid, proline, and myristic acid via a proposed but uncharacterized hybrid NRPS-PKS biosynthetic pathway. So far, no in-vitro biosynthetic studies have been reported for this promising antibiotic. Here, we report the first in-vitro reconstitution and biochemical characterization of two early enzymes on the pathway: PpgH, an isochorismate synthase (IS), and PpgG, an isochorismate pyruvate lyase (IPL) which are involved in the biosynthesis of salicylic acid, the polar fragment of promysalin. We also report a secondary chorismate mutase (CM) activity for PpgG. Based on our biochemical experiments, preliminary mechanistic proposals have been postulated for PpgH and PpgG. We believe this study will lay a strong foundation for elucidating the functions and mechanisms of other intriguing enzymes of the promysalin biosynthesis pathway, which may potentially unravel interesting enzyme chemistries and promote pathway engineering in the future.
Topics: Pyrrolidines; Salicylamides; Salicylic Acid; Anti-Bacterial Agents
PubMed: 37567320
DOI: 10.1016/j.bmcl.2023.129440 -
BMC Veterinary Research Apr 2024The present study aimed to determine the major cause of the high mortality affecting farmed gilthead seabream (Sparus aurata) and controlling this disease condition....
The present study aimed to determine the major cause of the high mortality affecting farmed gilthead seabream (Sparus aurata) and controlling this disease condition. Fifteen diseased S. aurata were sampled from a private fish farm located at Eldeba Triangle, Damietta, fish showed external skin hemorrhages, and ulceration. Bacterial isolates retrieved from the diseased fish were identified biochemically as Pseudomonas putida and then confirmed by phylogenetic analysis of the 16 S rRNA gene sequence. P. putida was also isolated from three batches of tilapia-trash feed given to S. aurata. Biofilm and hemolytic assay indicated that all P. putida isolates produced biofilm, but 61.11% can haemolyse red blood cells. Based on the antibiotic susceptibility test results, P. putida was sensitive to florfenicol with minimum inhibitory concentrations ranging between 0.25 and 1.0 µg mL, but all isolates were resistant to ampicillin and sulfamethoxazole-trimethoprim. Pathogenicity test revealed that P. putida isolate (recovered from the tilapia-trash feed) was virulent for S. aurata with LD equal to 4.67 × 10 colony forming unit (CFU) fish. After intraperitoneal (IP) challenge, fish treated with 10 mg kg of florfenicol showed 16.7% mortality, while no mortality was recorded for the fish group that received 20 mg kg. The non-treated fish group showed 46.7% mortality after bacterial challenge. HPLC analysis of serum florfenicol levels reached 1.07 and 2.52 µg mL at the 5th -day post-drug administration in the fish groups received 10 and 20 mg kg, respectively. In conclusion, P. putida was responsible for the high mortality affecting cultured S. aurata, in-feed administration of florfenicol (20 mg kg) effectively protected the challenged fish.
Topics: Animals; Thiamphenicol; Fish Diseases; Pseudomonas putida; Anti-Bacterial Agents; Animal Feed; Sea Bream; Pseudomonas Infections; Microbial Sensitivity Tests; Tilapia; Phylogeny; RNA, Ribosomal, 16S; Biofilms
PubMed: 38664683
DOI: 10.1186/s12917-024-04004-z -
Plant Science : An International... May 2024Iron (Fe) and phosphate (Pi) are two essential nutrients that are poorly available in the soil and should be supplemented either as fertilizers or organic amendments to...
Iron (Fe) and phosphate (Pi) are two essential nutrients that are poorly available in the soil and should be supplemented either as fertilizers or organic amendments to sustain crop production. Currently, determining how rhizosphere bacteria contribute to plant mineral nutrient acquisition is an area of growing interest regarding its potential application in agriculture. The aim of this study was to investigate the influence of root colonization by Pseudomonas putida for Arabidopsis growth through Fe and Pi nutritional signaling. We found that root colonization by the bacterium inhibits primary root elongation and promotes the formation of lateral roots. These effects could be related to higher expression of two Pi starvation-induced genes and AtPT1, the major Pi transporter in root tips. In addition, P. putida influenced the accumulation of Fe in the root and the expression of different elements of the Fe uptake pathway. The loss of function of the protein ligase BRUTUS (BTS), and the bHLH transcription factors POPEYE (PYE) and IAA-LEUCINE RESISTANT3 (ILR3) compromised the root branching stimulation triggered by bacterial inoculation while the leaf chlorosis in the fit1 and irt1-1 mutant plants grown under standard conditions could be bypassed by P. putida inoculation. The WT and both mutant lines showed similar Fe accumulation in roots. P. putida repressed the expression of the IRON-REGULATED TRANSPORTER 1 (IRT1) gene suggesting that the bacterium promotes an alternative Fe uptake mechanism. These results open the door for the use of P. putida to enhance nutrient uptake and optimize fertilizer usage by plants.
Topics: Arabidopsis; Pseudomonas putida; Phosphates; Arabidopsis Proteins; Basic Helix-Loop-Helix Transcription Factors; Plant Roots; Gene Expression Regulation, Plant
PubMed: 38360401
DOI: 10.1016/j.plantsci.2024.112028 -
International Journal of Molecular... Aug 2023A novel group of conjugative plasmids of is characterized. The prototype plasmid pPPUT-Tik1-1 (153,663 bp), isolated from a permafrost strain of Tik1, carries a...
A novel group of conjugative plasmids of is characterized. The prototype plasmid pPPUT-Tik1-1 (153,663 bp), isolated from a permafrost strain of Tik1, carries a defective mercury transposon, Tn, and a streptomycin resistance transposon, Tn Ten plasmids and 34 contigs with backbone regions closely related to pPPUT-Tik1-1 have been found in GenBank. Two of these plasmids from clinical strains of and are almost identical to the ancient plasmid. A characteristic feature of this group of plasmids is the presence of two genes encoding the initiators of replication ( and ). None of these genes have high similarity with plasmid replication genes belonging to known incompatibility groups. It has been demonstrated that while pPPUT-Tik1-1-like plasmids have homologous backbone regions, they significantly differ by the molecular structure and the predicted functions of their accessory regions. Some of the pPPUT-Tik1-1-related plasmids carry determinants of antibiotic resistance and/or heavy metal salts. Some plasmids are characterized by the ability to degrade xenobiotics. Plasmids related to pPPUT-Tik1-1 are characterized by a narrow host range and are found in various species of the genus. Interestingly, we also found shorter plasmid variants containing the same replication module, but lacking conjugation genes and containing other structural changes that strongly distinguish them from plasmids related to pPPUT-Tik1-1, indicating that the structure of the replication module cannot be used as the sole criterion for classifying plasmids. Overall, the results suggest that the plasmids of the novel group can be spread using conjugation in environmental and clinical strains of and may play diverse adaptive functions due to the presence of various accessory regions.
Topics: Pseudomonas putida; Permafrost; Pseudomonas; Databases, Nucleic Acid; Host Specificity
PubMed: 37686323
DOI: 10.3390/ijms241713518 -
International Journal of Biological... Dec 2023Bio-based plastics polyhydroxyalkanoates (PHAs) are considered as a good substitutive to traditional fossil-based plastics because PHAs outcompete chemical plastics in...
Bio-based plastics polyhydroxyalkanoates (PHAs) are considered as a good substitutive to traditional fossil-based plastics because PHAs outcompete chemical plastics in several important properties, such as biodegradability, biocompatibility, and renewability. However, the industrial production of PHA (especially medium-chain-length PHA, mcl-PHA) is greatly restricted by the cost of carbon sources. Currently, xylose and cellobiose derived from lignocellulose are potential substrates for mcl-PHA production. In this study, Pseudomonas putida KTU-U27, a genome-streamlined strain derived from a mcl-PHA producer P. putida KT2440, was used as the optimal chassis for the construction of microbial cell factories with the capacity to efficiently produce mcl-PHA from xylose and cellobiose by introducing the xylose and cellobiose metabolism modules and enhancing the transport of xylose and cellobiose. The lag phases of the xylose- and cellobiose-grown engineered strains were almost completely eliminated and the xylose- and cellobiose-utilizing performance was greatly improved via adaptive laboratory evolution. In shake-flask fermentation, the engineered strain 27A-P13-xylABE-P-tt and 27A-P13-bglC-P13-gts had a mcl-PHA content of 41.67 wt% and 45.18 wt%, respectively, and were able to efficiently utilize xylose or cellobiose as the sole carbon source for cell growth. Herein, microbial production of mcl-PHA using xylose as the sole carbon source has been demonstrated for the first time. Meanwhile, the highest yield of mcl-PHA produced from cellobiose has been obtained in this study. Interestingly, the engineered strains derived from genome-reduced P. putida strains showed higher xylose- and cellobiose-utilizing performance and higher PHA yield than those derived from P. putida KT2440. This study highlights enormous potential of the engineered strains as promising platforms for low-cost production of mcl-PHA from xylose- and cellobiose-rich substrates.
Topics: Metabolic Engineering; Pseudomonas putida; Xylose; Cellobiose; Polyhydroxyalkanoates; Carbon
PubMed: 37678685
DOI: 10.1016/j.ijbiomac.2023.126732 -
Genes Sep 2023strain U can be grown using, as sole carbon sources, the biogenic amines putrescine or cadaverine, as well as their catabolic intermediates, ɣ-aminobutyrate or...
strain U can be grown using, as sole carbon sources, the biogenic amines putrescine or cadaverine, as well as their catabolic intermediates, ɣ-aminobutyrate or δ-aminovalerate, respectively. Several paralogs for the genes that encode some of the activities involved in the catabolism of these compounds, such as a putrescine-pyruvate aminotransferase ( and genes) and a ɣ-aminobutyrate aminotransferase ( and genes) have been identified in this bacterium. When the expression pattern of these genes is analyzed by qPCR, it is drastically conditioned by supplying the carbon sources. Thus, is upregulated by putrescine, whereas seems to be exclusively induced by cadaverine. However, increases its expression in response to different polyamines or aminated catabolic derivatives from them (i.e., ɣ-aminobutyrate or δ-aminovalerate), although does not change its expression level concerning no-amine unrelated carbon sources (citrate). These results reveal differences between the mechanisms proposed for polyamine catabolism in and concerning strain U, as well as allow a deeper understanding of the enzymatic systems used by this last strain during polyamine metabolism.
Topics: Cadaverine; Putrescine; Pseudomonas putida; Polyamines; Pseudomonas aeruginosa; Escherichia coli; Aminobutyrates; Carbon; Gene Expression
PubMed: 37895246
DOI: 10.3390/genes14101897 -
International Journal of... 2024Heavy metal pollution threatens food security by accumulating in crops and soils, posing a significant challenge to modern agriculture due to its high toxicity. Urgent...
Heavy metal pollution threatens food security by accumulating in crops and soils, posing a significant challenge to modern agriculture due to its high toxicity. Urgent action is needed to restore affected agricultural fields. An efficient way to remove toxins is by bioremediation, which uses microorganisms. With the purpose of restoring soil in agriculture, this research attempts to assemble a consortium of microorganisms isolated from techno-genic soil. A number of promising strains, including , sp., , , and were chosen based on their capacity to eliminate heavy metals from tests. Heavy metal removal (Cd, Hg, As, Pb, and Ni) and phytohormone production have been shown to be effective using consortiums (, , and in a 1:1:2). In instances with mixed heavy-metal contamination, demonstrated efficacy because of its notable ability to absorb substantial quantities of heavy metals. The capacity of the cooperation to improve phytoremediation was investigated, with an emphasis on soil cleanup in agricultural areas. When combined with L., it was able to remove roughly 16% As, 14% Hg, 32% Ni, 26% Cd, and 33% Pb from the soil.
Topics: Biodegradation, Environmental; Metals, Heavy; Soil Pollutants; Soil Microbiology; Sorghum; Soil; Crops, Agricultural
PubMed: 38494751
DOI: 10.1080/15226514.2024.2327611