scholarly journals Nitrogen metabolism in Pseudomonas putida: functional analysis using random barcode transposon sequencing

2021 ◽  
Author(s):  
Matthias Schmidt ◽  
Allison N. Pearson ◽  
Matthew R. Incha ◽  
Mitchell G. Thompson ◽  
Edward E. K. Baidoo ◽  
...  
Keyword(s):  
Amino Acid ◽  
Pseudomonas Putida ◽  
Transcriptional Regulators ◽  
Drop Out ◽  
Visualization Tool ◽  
Acid Biosynthesis ◽  
Pseudomonas Putida Kt2440 ◽  
Related Proteins ◽  
Nitrogen Containing Compounds

Pseudomonas putida KT2440 has long been studied for its diverse and robust metabolisms, yet many genes and proteins imparting these growth capacities remain uncharacterized. Using pooled mutant fitness assays, we identified genes and proteins involved in the assimilation of 52 different nitrogen containing compounds. To assay amino acid biosynthesis, 19 amino acid drop-out conditions were also tested. From these 71 conditions, significant fitness phenotypes were elicited in 672 different genes including 100 transcriptional regulators and 112 transport-related proteins. We divide these conditions into 6 classes, and propose assimilatory pathways for the compounds based on this wealth of genetic data. To complement these data, we characterize the substrate range of three promiscuous aminotransferases relevant to metabolic engineering efforts in vitro. Furthermore, we examine the specificity of five transcriptional regulators, explaining some fitness data results and exploring their potential to be developed into useful synthetic biology tools. In addition, we use manifold learning to create an interactive visualization tool for interpreting our BarSeq data, which will improve the accessibility and utility of this work to other researchers.

scholarly journals Transcription factor GCN4 for control of amino acid biosynthesis also regulates the expression of the gene for lipoamide dehydrogenase

1999 ◽  
Vol 340 (3) ◽  
pp. 855-862 ◽  
Author(s):  
Zafar ZAMAN ◽  
Susan B. BOWMAN ◽  
Geoff D. KORNFELD ◽  
Alistair J. P. BROWN ◽  
Ian W. DAWES
Keyword(s):  
Transcription Factor ◽  
Amino Acid ◽  
Amino Acid Biosynthesis ◽  
Upstream Region ◽  
Northern Analysis ◽  
General Control ◽  
Acid Biosynthesis ◽  
Gene Encoding ◽  
Lipoamide Dehydrogenase

The yeast LPD1 gene encoding lipoamide dehydrogenase is subject to the general control of amino acid biosynthesis mediated by the GCN4 transcription factor. This is striking in that it demonstrates that GCN4-mediated regulation extends much farther upstream than simply to the direct pathways for amino acid and purine biosynthesis. In yeast, lipoamide dehydrogenase functions in at least three multienzyme complexes: pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase (which function in the entry of pyruvate into, and metabolism via, the citric acid cycle) and glycine decarboxylase. When wild-type cells were shifted from growth on amino acid-rich to amino acid-deficient medium, the expression of lipoamide dehydrogenase was induced approx. 2-fold. In a similar experiment no such induction was observed in isogenic gcn4 mutant cells. Northern analysis indicated that amino acid starvation affected levels of the LPD1 transcript. In the upstream region of LPD1 are three matches to the consensus for control mediated by GCN4. Directed mutagenesis of each site, and of all combinations of sites, suggests that only one site might be important for the general control response under the conditions tested. Gel-retardation analysis with GCN4 protein synthesized in vitro has indicated that GCN4 can bind in vitro to at least two of the consensus motifs.

scholarly journals Analysis of Pseudomonas putida KT2440 Gene Expression in the Maize Rhizosphere: In Vitro Expression Technology Capture and Identification of Root-Activated Promoters

2005 ◽  
Vol 187 (12) ◽  
pp. 4033-4041 ◽  
Author(s):  
María Isabel Ramos-González ◽  
María Jesús Campos ◽  
Juan L. Ramos
Keyword(s):  
Pseudomonas Putida ◽  
Defense Mechanism ◽  
Essential Gene ◽  
Cell Envelope ◽  
Original Position ◽  
Pseudomonas Putida Kt2440 ◽  
Maize Rhizosphere ◽  
Semialdehyde Dehydrogenase

ABSTRACT Pseudomonas putida KT2440, a paradigm organism in biodegradation and a good competitive colonizer of the maize rhizosphere, was the subject of studies undertaken to establish the genetic determinants important for its rhizospheric lifestyle. By using in vivo expression technology (IVET) to positively select single cell survival, we identified 28 rap genes (root-activated promoters) preferentially expressed in the maize rhizosphere. The IVET system had two components: a mutant affected in aspartate-β-semialdehyde dehydrogenase (asd), which was unable to survive in the rhizosphere, and plasmid pOR1, which carries a promoterless asd gene. pOR1-borne transcriptional fusions of the rap promoters to the essential gene asd, which were integrated into the chromosome at the original position of the corresponding rap gene, were active and allowed growth of the asd strain in the rhizosphere. The fact that five of the rap genes identified in the course of this work had been formerly characterized as being related to root colonization reinforced the IVET approach. Up to nine rap genes encoded proteins either of unknown function or that had been assigned an unspecific role based on conservation of the protein family domains. Rhizosphere-induced fusions included genes with probable functions in the cell envelope, chemotaxis and motility, transport, secretion, DNA metabolism and defense mechanism, regulation, energy metabolism, stress, detoxification, and protein synthesis.

scholarly journals Outer membrane vesicles catabolize lignin-derived aromatic compounds in Pseudomonas putida KT2440

2020 ◽  
Vol 117 (17) ◽  
pp. 9302-9310 ◽  
Author(s):  
Davinia Salvachúa ◽  
Allison Z. Werner ◽  
Isabel Pardo ◽  
Martyna Michalska ◽  
Brenna A. Black ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Outer Membrane ◽  
Aromatic Compounds ◽  
Value Added ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Pseudomonas Putida Kt2440 ◽  
Rich Media

Lignin is an abundant and recalcitrant component of plant cell walls. While lignin degradation in nature is typically attributed to fungi, growing evidence suggests that bacteria also catabolize this complex biopolymer. However, the spatiotemporal mechanisms for lignin catabolism remain unclear. Improved understanding of this biological process would aid in our collective knowledge of both carbon cycling and microbial strategies to valorize lignin to value-added compounds. Here, we examine lignin modifications and the exoproteome of three aromatic–catabolic bacteria: Pseudomonas putida KT2440, Rhodoccocus jostii RHA1, and Amycolatopsis sp. ATCC 39116. P. putida cultivation in lignin-rich media is characterized by an abundant exoproteome that is dynamically and selectively packaged into outer membrane vesicles (OMVs). Interestingly, many enzymes known to exhibit activity toward lignin-derived aromatic compounds are enriched in OMVs from early to late stationary phase, corresponding to the shift from bioavailable carbon to oligomeric lignin as a carbon source. In vivo and in vitro experiments demonstrate that enzymes contained in the OMVs are active and catabolize aromatic compounds. Taken together, this work supports OMV-mediated catabolism of lignin-derived aromatic compounds as an extracellular strategy for nutrient acquisition by soil bacteria and suggests that OMVs could potentially be useful tools for synthetic biology and biotechnological applications.

scholarly journals Enhanced Tolerance to Naphthalene and Enhanced Rhizoremediation Performance for Pseudomonas putida KT2440 via the NAH7 Catabolic Plasmid

2012 ◽  
Vol 78 (15) ◽  
pp. 5104-5110 ◽  
Author(s):  
Matilde Fernández ◽  
José Luis Niqui-Arroyo ◽  
Susana Conde ◽  
Juan Luis Ramos ◽  
Estrella Duque
Keyword(s):  
Pseudomonas Putida ◽  
Cellular Damage ◽  
Pseudomonas Putida Kt2440 ◽  
Content Type ◽  
Naphthalene Degradation ◽  
Repair Enzymes ◽  
Damage Repair ◽  
Catabolic Plasmid

ABSTRACTIn this work, we explore the potential use of thePseudomonas putidaKT2440 strain for bioremediation of naphthalene-polluted soils.Pseudomonas putidastrain KT2440 thrives in naphthalene-saturated medium, establishing a complex response that activates genes coding for extrusion pumps and cellular damage repair enzymes, as well as genes involved in the oxidative stress response. The transfer of the NAH7 plasmid enables naphthalene degradation byP. putidaKT2440 while alleviating the cellular stress brought about by this toxic compound, without affecting key functions necessary for survival and colonization of the rhizosphere.Pseudomonas putidaKT2440(NAH7) efficiently expresses the Nah catabolic pathwayin vitroandin situ, leading to the complete mineralization of [14C]naphthalene, measured as the evolution of14CO2, while the rate of mineralization was at least 2-fold higher in the rhizosphere than in bulk soil.

scholarly journals Molecular characterization of FinR, a novel redox-sensing transcriptional regulator in Pseudomonas putida KT2440

Microbiology ◽  
2010 ◽  
Vol 156 (5) ◽  
pp. 1487-1496 ◽  
Author(s):  
Sujin Yeom ◽  
Jinki Yeom ◽  
Woojun Park
Keyword(s):  
Dna Binding ◽  
Pseudomonas Putida ◽  
Stress Conditions ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Cysteine Residues ◽  
Functional Domain ◽  
Pseudomonas Putida Kt2440 ◽  
Redox Sensing

FinR is required for the induction of fpr (ferredoxin-NADP+ reductase) under superoxide stress conditions in Pseudomonas putida. Many proteobacteria harbour FinR homologues in their genome as a putative LysR-type protein. Three cysteine residues (at positions 150, 239 and 289 in P. putida FinR) are conserved in all FinR homologues. When these conserved cysteines, along with two other cysteine residues present in FinR, were individually mutated to serines, the FinR remained active, unlike SoxR and OxyR in Escherichia coli. The results of our in vitro DNA-binding assay with cellular extracts showed that FinR binds directly to the fpr promoter region. In order to identify the FinR functional domain for sensing superoxide stress, we employed random and site-directed mutagenesis of FinR. Among 18 single amino acid mutants, three mutants (T39A, R194A and E225A) abolished fpr induction without any alteration of their DNA-binding ability, whereas other mutants also abrogated their DNA-binding abilities. Interestingly, two mutants (L215P and D51A) appeared to be constitutively active, regardless of superoxide stress conditions. Ferrous iron depletion, ferric iron addition and fdxA (ferredoxin) gene deletion also participate in the regulation of fpr. These data indicate that FinR has unusual residues for redox sensing and that the redox-sensing mechanism of FinR differs from the well-known mechanisms of OxyR and SoxR.

scholarly journals Rare earth element (REE)-dependent growth of Pseudomonas putida KT2440 depends on the ABC-transporter PedA1A2BC and is influenced by iron availability

2019 ◽  
Author(s):  
Matthias Wehrmann ◽  
Charlotte Berthelot ◽  
Patrick Billard ◽  
Janosch Klebensberger
Keyword(s):  
Rare Earth ◽  
Pseudomonas Putida ◽  
Rare Earth Element ◽  
Earth Element ◽  
Iron Availability ◽  
Pseudomonas Putida Kt2440 ◽  
Independent Manner ◽  
Metal Interactions ◽  
Dependent Growth

AbstractIn the soil-dwelling organism Pseudomonas putida KT2440, the rare earth element (REE)-utilizing and pyrroloquinoline quinone (PQQ)-dependent ethanol dehydrogenase PedH is part of a periplasmic oxidation system that is vital for growth on various alcoholic volatiles. Expression of PedH and its Ca2+-dependent counterpart PedE is inversely regulated in response to lanthanide (Ln3+) bioavailability, a mechanism termed the REE-switch. In the present study, we demonstrate that copper, zinc, and in particular, iron availability influences this regulation in a pyoverdine-independent manner by increasing the minimal Ln3+ concentration required for the REE-switch to occur by several orders of magnitude. A combined genetic- and physiological approach reveals that an ABC-type transporter system encoded by the gene cluster pedA1A2BC is essential for efficient growth with low (nanomolar) Ln3+ concentrations. In the absence of pedA1A2BC, a ~100-fold higher La3+ concentration is needed for PedH-dependent growth but not for the ability to repress growth based on PedE activity. From these results, we conclude that cytoplasmic uptake of lanthanides through PedA1A2BC is essential to facilitate REE-dependent growth under environmental conditions with poor REE bioavailability. Our data further suggest that the La3+/Fe3+ ratio impacts the REE-switch through the mismetallation of putative La3+-binding proteins, such as the sensor histidine kinase PedS2, in the presence of high iron concentrations. As such, this study provides an example for the complexity of bacteria-metal interactions and highlights the importance of medium compositions when studying physiological traits in vitro in particular in regards to REE-dependent phenomena.

Electron probe x-ray microanalysis and electron microscopy of amino acid absorption and transport by the small intestine: inhibition by chemical poisons and correlation to the elemental composition of the epithelial cells

1986 ◽  
Vol 44 ◽  
pp. 134-135
Author(s):  
A. J. Tousimis
Keyword(s):  
Small Intestine ◽  
Amino Acid ◽  
Epithelial Cells ◽  
Elemental Composition ◽  
Isotope Labeling ◽  
Structural Components ◽  
X Ray ◽  
Human Small Intestine ◽  
Transport Studies

The elemental composition of amino acids is similar to that of the major structural components of the epithelial cells of the small intestine and other tissues. Therefore, their subcellular localization and concentration measurements are not possible by x-ray microanalysis. Radioactive isotope labeling: I131-tyrosine, Se75-methionine and S35-methionine have been successfully employed in numerous absorption and transport studies. The latter two have been utilized both in vitro and vivo, with similar results in the hamster and human small intestine. Non-radioactive Selenomethionine, since its absorption/transport behavior is assumed to be the same as that of Se75- methionine and S75-methionine could serve as a compound tracer for this amino acid.

Sign in / Sign up

Export Citation Format

Share Document