scholarly journals Time-Course Proteomic Analysis of Pseudomonas putida KT2440 during Mcl-Polyhydroxyalkanoate Synthesis under Nitrogen Deficiency

Polymers ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 748 ◽  
Author(s):  
Justyna Możejko-Ciesielska ◽  
Agnieszka Mostek
Keyword(s):  
Pseudomonas Putida ◽  
Proteomic Analysis ◽  
Cellular Response ◽  
Time Course ◽  
Molecular Mechanisms ◽  
Nitrogen Deficiency ◽  
Growth Stages ◽  
Bacterial Cells ◽  
Pseudomonas Putida Kt2440 ◽  
Wide Range

Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) have gained great attention as a new green alternative to petrochemical-derived polymers. Due to their outstanding material properties they can be used in a wide range of applications. Pseudomonas putida KT2440 is a metabolically versatile producer of mcl-polyhydroxyalkanoates. Although the metabolism of polyhydroxyalkanoate synthesis by this bacterium has been extensively studied, the comparative proteome analysis from three growth stages of Pseudomonas putida KT2440 cultured with oleic acid during mcl-PHA synthesis has not yet been reported. Therefore; the aim of the study was to compare the proteome of Pseudomonas putida KT2440 at different time points of its cultivation using the 2D difference gel electrophoresis (2D-DIGE) technique. The analyses showed that low levels of a nitrogen source were beneficial for mcl-PHA synthesis. Proteomic analysis revealed that the proteins associated with carbon metabolism were affected by nitrogen starvation and mcl-PHA synthesis. Furthermore, the induction of proteins involved in nitrogen metabolism, ribosome synthesis, and transport was observed, which may be the cellular response to stress related to nitrogen deficiency and mcl-PHA content in bacterial cells. To sum up; this study enabled the investigators to acquire a better knowledge of the molecular mechanisms underlying the induction of polyhydroxyalkanoate synthesis and accumulation in Pseudomonas putida KT2440 that could lead to improved strategies for PHAs in industrial production.

scholarly journals Proteomic Response of Pseudomonas putida KT2440 to Dual Carbon-Phosphorus Limitation during mcl-PHAs Synthesis

Biomolecules ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 796 ◽  
Author(s):  
Justyna Możejko-Ciesielska ◽  
Luísa S. Serafim
Keyword(s):  
Pseudomonas Putida ◽  
Time Course ◽  
Molecular Mechanisms ◽  
Phosphorus Limitation ◽  
Growth Conditions ◽  
Pseudomonas Putida Kt2440 ◽  
Medium Chain Length ◽  
Metabolic Activities ◽  
Proteomic Responses ◽  
Phosphate Regulon

Pseudomonas putida KT2440, one of the best characterized pseudomonads, is a metabolically versatile producer of medium-chain-length polyhydroxyalkanoates (mcl-PHAs) that serves as a model bacterium for molecular studies. The synthesis of mcl-PHAs is of great interest due to their commercial potential. Carbon and phosphorus are the essential nutrients for growth and their limitation can trigger mcl-PHAs’ production in microorganisms. However, the specific molecular mechanisms that drive this synthesis in Pseudomonas species under unfavorable growth conditions remain poorly understood. Therefore, the proteomic responses of Pseudomonas putida KT2440 to the limited carbon and phosphorus levels in the different growth phases during mcl-PHAs synthesis were investigated. The data indicated that biopolymers’ production was associated with the cell growth of P. putida KT2440 under carbon- and phosphorus-limiting conditions. The protein expression pattern changed during mcl-PHAs synthesis and accumulation, and during the different physiological states of the microorganism. The data suggested that the majority of metabolic activities ceased under carbon and phosphorus limitation. The abundance of polyhydroxyalkanoate granule-associated protein (PhaF) involved in PHA synthesis increased significantly at 24 and 48 h of the cultivations. The activation of proteins belonging to the phosphate regulon was also detected. Moreover, these results indicated changes in the protein profiles related to amino acids metabolism, replication, transcription, translation, stress response mechanisms, transport or signal transduction. The presented data allowed the investigation of time-course proteome alterations in response to carbon and phosphorus limitation, and PHAs synthesis. This study provided information about proteins that can be potential targets in improving the efficiency of mcl-PHAs synthesis.

Crosstalk between Platelet and Bacteria: A Therapeutic Prospect

2019 ◽  
Vol 25 (38) ◽  
pp. 4041-4052
Author(s):  
Vivek K. Yadav ◽  
Pradeep K. Singh ◽  
Vishnu Agarwal ◽  
Sunil K. Singh
Keyword(s):  
Platelet Activation ◽  
Molecular Mechanisms ◽  
Thrombus Formation ◽  
Critical Role ◽  
Direct Interaction ◽  
Vascular Wall ◽  
Cardiovascular Complications ◽  
Bacterial Cells ◽  
Platelet Surface ◽  
Wide Range

Platelets are typically recognized for their roles in the maintenance of hemostasis and vascular wall repair to reduce blood loss. Beyond hemostasis, platelets also play a critical role in pathophysiological conditions like atherosclerosis, stroke, thrombosis, and infections. During infection, platelets interact directly and indirectly with bacteria through a wide range of cellular and molecular mechanisms. Platelet surface receptors such as GPIbα, FcγRIIA, GPIIbIIIa, and TLRs, etc. facilitate direct interaction with bacterial cells. Besides, the indirect interaction between platelet and bacteria involves host plasma proteins such as von Willebrand Factor (vWF), fibronectin, IgG, and fibrinogen. Bacterial cells induce platelet activation, aggregation, and thrombus formation in the microvasculature. The activated platelets induce the Neutrophil Extracellular Traps (NETs) formation, which further contribute to thrombosis. Thus, platelets are extensively anticipated as vital immune modulator cells during infection, which may further lead to cardiovascular complications. In this review, we cover the interaction mechanisms between platelets and bacteria that may lead to the development of thrombotic disorders. Platelet receptors and other host molecules involved in such interactions can be used to develop new therapeutic strategies to combat against infection-induced cardiovascular complications. In addition, we highlight other receptor and enzyme targets that may further reduce infection-induced platelet activation and various pathological conditions.

scholarly journals Label-Free Quantitative Proteomic Analysis of the Global Response to Indole-3-Acetic Acid in Newly Isolated Pseudomonas sp. Strain LY1

2021 ◽  
Vol 12 ◽  
Author(s):  
Shuxue Zhao ◽  
Xi Chen ◽  
Qianshu Sun ◽  
Fei Wang ◽  
Chunhui Hu ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Proteomic Analysis ◽  
Cellular Response ◽  
Molecular Mechanisms ◽  
Degradation Mechanism ◽  
Label Free ◽  
Ros Scavenging ◽  
Protein Repair ◽  
Quantitative Proteomic Analysis ◽  
Indole 3 Acetic Acid

Indole-3-acetic acid (IAA), known as a common plant hormone, is one of the most distributed indole derivatives in the environment, but the degradation mechanism and cellular response network to IAA degradation are still not very clear. The objective of this study was to elucidate the molecular mechanisms of IAA degradation at the protein level by a newly isolated strain Pseudomonas sp. LY1. Label-free quantitative proteomic analysis of strain LY1 cultivated with IAA or citrate/NH4Cl was applied. A total of 2,604 proteins were identified, and 227 proteins have differential abundances in the presence of IAA, including 97 highly abundant proteins and 130 less abundant proteins. Based on the proteomic analysis an IAA degrading (iad) gene cluster in strain LY1 containing IAA transformation genes (organized as iadHABICDEFG), genes of the β-ketoadipate pathway for catechol and protocatechuate degradation (catBCA and pcaABCDEF) were identified. The iadA, iadB, and iadE-disrupted mutants lost the ability to grow on IAA, which confirmed the role of the iad cluster in IAA degradation. Degradation intermediates were analyzed by HPLC, LC-MS, and GC-MS analysis. Proteomic analysis and identified products suggested that multiple degradation pathways existed in strain LY1. IAA was initially transformed to dioxindole-3-acetic acid, which was further transformed to isatin. Isatin was then transformed to isatinic acid or catechol. An in-depth data analysis suggested oxidative stress in strain LY1 during IAA degradation, and the abundance of a series of proteins was upregulated to respond to the stress, including reaction oxygen species (ROS) scavenging, protein repair, fatty acid synthesis, RNA protection, signal transduction, chemotaxis, and several membrane transporters. The findings firstly explained the adaptation mechanism of bacteria to IAA degradation.

scholarly journals Inducible and tunable gene expression systems for Pseudomonas putida KT2440

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chandran Sathesh-Prabu ◽  
Rameshwar Tiwari ◽  
Doyun Kim ◽  
Sung Kuk Lee
Keyword(s):  
Pseudomonas Putida ◽  
Target Genes ◽  
Fluorescent Protein ◽  
Low Cost ◽  
Inducible Promoter ◽  
Expression Systems ◽  
Reporter Protein ◽  
Pseudomonas Putida Kt2440 ◽  
Wide Range ◽  
Green Fluorescent

AbstractInducible and tunable expression systems are essential for the microbial production of biochemicals. Five different carbon source- and substrate-inducible promoter systems were developed and further evaluated in Pseudomonas putida KT2440 by analyzing the expression of green fluorescent protein (GFP) as a reporter protein. These systems can be induced by low-cost compounds such as glucose, 3-hydroxypropionic acid (3HP), levulinic acid (LA), and xylose. 3HP-inducible HpdR/PhpdH was also efficiently induced by LA. LvaR/PlvaA and XutR/PxutA systems were induced even at low concentrations of LA (0.1 mM) and xylose (0.5 mM), respectively. Glucose-inducible HexR/Pzwf1 showed weak GFP expression. These inducer agents can be used as potent starting materials for both cell growth and the production of a wide range of biochemicals. The efficiency of the reported systems was comparable to that of conventional chemical-inducible systems. Hence, the newly investigated promoter systems are highly useful for the expression of target genes in the widely used synthetic biology chassis P. putida KT2440 for industrial and medical applications.

scholarly journals The Glycerol-Dependent Metabolic Persistence of Pseudomonas putida KT2440 Reflects the Regulatory Logic of the GlpR Repressor

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Pablo I. Nikel ◽  
Francisco J. Romero-Campero ◽  
Joshua A. Zeidman ◽  
Ángel Goñi-Moreno ◽  
Víctor de Lorenzo
Keyword(s):  
Carbon Source ◽  
Pseudomonas Putida ◽  
Molecular Mechanisms ◽  
Transcriptional Response ◽  
Time Dependent ◽  
Lag Phase ◽  
Gradual Change ◽  
Pseudomonas Putida Kt2440 ◽  
Genetic Changes ◽  
Content Type

ABSTRACTThe growth of the soil bacteriumPseudomonas putidaKT2440 on glycerol as the sole carbon source is characterized by a prolonged lag phase, not observed with other carbon substrates. We examined the bacterial growth in glycerol cultures while monitoring the metabolic activity of individual cells. Fluorescence microscopy and flow cytometry, as well as the analysis of the temporal start of growth in single-cell cultures, revealed that adoption of a glycerol-metabolizing regime was not the result of a gradual change in the whole population but rather reflected a time-dependent bimodal switch between metabolically inactive (i.e., nongrowing) and fully active (i.e., growing) bacteria. A transcriptional Φ(glpD-gfp) fusion (a proxy of the glycerol-3-phosphate [G3P] dehydrogenase activity) linked the macroscopic phenotype to the expression of theglpgenes. Either deletingglpR(encoding the G3P-responsive transcriptional repressor that controls the expression of theglpFKRDgene cluster) or altering G3P formation (by overexpressingglpK, encoding glycerol kinase) abolished the bimodalglpDexpression. These manipulations eliminated the stochastic growth start by shortening the otherwise long lag phase. Provision ofglpRintransrestored the phenotypes lost in theΔglpRmutant. The prolonged nongrowth regime ofP. putidaon glycerol could thus be traced to the regulatory device controlling the transcription of theglpgenes. Since the physiological agonist of GlpR is G3P, the arrangement of metabolic and regulatory components at this checkpoint merges a positive feedback loop with a nonlinear transcriptional response, a layout fostering the observed time-dependent shift between two alternative physiological states.IMPORTANCEPhenotypic variation is a widespread attribute of prokaryotes that leads,inter alia, to the emergence of persistent bacteria, i.e., live but nongrowing members within a genetically clonal population. Persistence allows a fraction of cells to avoid the killing caused by conditions or agents that destroy most growing bacteria (e.g., some antibiotics). Known molecular mechanisms underlying the phenomenon include genetic changes, epigenetic variations, and feedback-based multistability. We show that a prolonged nongrowing state of the bacterial population can be brought about by a distinct regulatory architecture of metabolic genes when cells face specific nutrients (e.g., glycerol).Pseudomonas putidamay have adopted the resulting carbon source-dependent metabolic bet hedging as an advantageous trait for exploring new chemical and nutritional landscapes. Defeating such naturally occurring adaptive features of environmental bacteria is instrumental in improving the performance of these microorganisms as whole-cell catalysts in a bioreactor setup.

scholarly journals Untargeted Metabolomics Investigation on Selenite Reduction to Elemental Selenium by Bacillus mycoides SeITE01

2021 ◽  
Vol 12 ◽  
Author(s):  
Greta Baggio ◽  
Ryan A. Groves ◽  
Roberto Chignola ◽  
Elena Piacenza ◽  
Alessandro Presentato ◽  
...  
Keyword(s):  
Time Course ◽  
Membrane Lipids ◽  
Untargeted Metabolomics ◽  
Elemental Selenium ◽  
Growth Stages ◽  
Bacterial Cells ◽  
Bacillus Mycoides ◽  
Thiol Compounds ◽  
Selenite Reduction ◽  
Signaling Strategies

Bacillus mycoides SeITE01 is an environmental isolate that transforms the oxyanion selenite (SeO32−) into the less bioavailable elemental selenium (Se0) forming biogenic selenium nanoparticles (Bio-SeNPs). In the present study, the reduction of sodium selenite (Na2SeO3) by SeITE01 strain and the effect of SeO32− exposure on the bacterial cells was examined through untargeted metabolomics. A time-course approach was used to monitor both cell pellet and cell free spent medium (referred as intracellular and extracellular, respectively) metabolites in SeITE01 cells treated or not with SeO32−. The results show substantial biochemical changes in SeITE01 cells when exposed to SeO32−. The initial uptake of SeO32− by SeITE01 cells (3h after inoculation) shows both an increase in intracellular levels of 4-hydroxybenzoate and indole-3-acetic acid, and an extracellular accumulation of guanosine, which are metabolites involved in general stress response adapting strategies. Proactive and defensive mechanisms against SeO32− are observed between the end of lag (12h) and beginning of exponential (18h) phases. Glutathione and N-acetyl-L-cysteine are thiol compounds that would be mainly involved in Painter-type reaction for the reduction and detoxification of SeO32− to Se0. In these growth stages, thiol metabolites perform a dual role, both acting against the toxic and harmful presence of the oxyanion and as substrate or reducing sources to scavenge ROS production. Moreover, detection of the amino acids L-threonine and ornithine suggests changes in membrane lipids. Starting from stationary phase (24 and 48h), metabolites related to the formation and release of SeNPs in the extracellular environment begin to be observed. 5-hydroxyindole acetate, D-[+]-glucosamine, 4-methyl-2-oxo pentanoic acid, and ethanolamine phosphate may represent signaling strategies following SeNPs release from the cytoplasmic compartment, with consequent damage to SeITE01 cell membranes. This is also accompanied by intracellular accumulation of trans-4-hydroxyproline and L-proline, which likely represent osmoprotectant activity. The identification of these metabolites suggests the activation of signaling strategies that would protect the bacterial cells from SeO32− toxicity while it is converting into SeNPs.

Proteome Analysis of Cellular Response of Pseudomonas putida KT2440 to Tetracycline Stress

2006 ◽  
Vol 53 (2) ◽  
pp. 95-101 ◽  
Author(s):  
Sung-Ho Yun ◽  
Young Hwan Kim ◽  
Eun Jin Joo ◽  
Jong-Soon Choi ◽  
Jung-Hoon Sohn ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Cellular Response ◽  
Proteome Analysis ◽  
Pseudomonas Putida Kt2440

Propanil Hydrolysis : Inhibition in Rice Plants by Diazinon and Carbaryl Translocated from the Soil

1973 ◽  
Vol 56 (5) ◽  
pp. 1178-1182
Author(s):  
Ali El-Refai ◽  
M M Mowafy
Keyword(s):  
Time Course ◽  
Growth Stages ◽  
Slow Increase ◽  
Rice Plants ◽  
Stages Of Growth ◽  
Early Stages ◽  
Tissue Extracts ◽  
Wide Range ◽  
Rate Of Hydrolysis ◽  
Time Course Study

Abstract Greenhouse and laboratory experiments were designed to determine the effect of soil-applied carbaryl and diazinon on the phytotoxicity of foliage-sprayed propanil. A time-course study of the activity of propanil-hydrolyzing enzyme in rice indicated a slow increase in the rate of hydrolysis during early stages of growth, followed by a sharp increase for extracts of the 7-week-old samples. The rate of hydrolysis reached its maximum at harvest time (approximately 17 weeks). The enzyme activity of barnyard grass was not detected at different growth stages. The enzyme was inhibited by diazinon and strongly inhibited by carbaryl absorbed and translocated from the soil over a wide range of growth stages of rice. Rice plants were more susceptible at early stages of growth and this confirms the significant correlation between growth stage and enzyme activities. Analyses of tissue extracts by paper chromatography and an enzyme inhibition method agreed closely at all intervals. It seems probable that the inhibition rate of enzyme is related to the persistence and rate of translocation of carbaryl.

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