INDUCTION AND MULTI-SENSITIVE END-PRODUCT REPRESSION IN THE ENZYMIC PATHWAY DEGRADING MANDELATE IN PSEUDOMONAS FLUORESCENS.
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ABSTRACT:
SUBMITTER: MANDELSTAM J
PROVIDER: S-EPMC1206590 | biostudies-other | 1965 Mar
REPOSITORIES: biostudies-other
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Project description:1. Control of enzyme formation has been examined in the pathways degrading mandelate and p-hydroxymandelate in Pseudomonas fluorescens. 2. The first three enzymes form a group which is common to both pathways and which is co-ordinately induced or repressed. The genes controlling these enzymes are assumed to form a ;regulon'. This group of enzymes is induced by mandelate or p-hydroxymandelate and repressed by benzoate and by p-hydroxybenzoate (the immediate end products resulting from the action of this group of enzymes). 3. Repression is independently exerted by end products of enzymes controlled by succeeding regulons, i.e. by catechol, by protocatechuate and finally by succinate and acetate. 4. The pattern is repeated further along the pathway, so that benzoate oxidase (controlled by the second regulon) is repressed by its immediate end product, catechol, and again by succinate and acetate. 5. Pyrocatechase, an enzyme controlled by the third regulon, is repressed by succinate and acetate. 6. There is a parallel system of multi-sensitive repression mechanisms controlling production of the enzymes that degrade the hydroxy compounds. Again, the enzymes of each regulon are repressed by the immediate end product of their action and by the end products of each succeeding group of enzymes. 7. Repressor activity appears to be exerted by compounds that are likely to occur as such in the external environment or that occur at points of convergence of the degradative pathways of the cell. 8. The net effect of this control system, involving both induction and end-product repression, appears to be that cells will not form inducible degradative enzymes if the end products are already being supplied from without or are being produced by degradation of some alternative source of carbon and energy.
| S-EPMC1207047 | biostudies-other
Project description:1. l-Mandelate dehydrogenase and mandelate racemase were partially purified from extracts of Pseudomonas fluorescens A-312 grown in media containing d-mandelate. 2. The activity of mandelate racemase, but not that of l-mandelate dehydrogenase, is greatly stimulated by Mg(2+), Mn(2+), Co(2+) and, though less effectively, by Ni(2+). Other metal ions are inactive or inhibitory. 3. Racemase activity is inhibited by phosphate, fluoride, pyrophosphate and EDTA. The inhibitions by pyrophosphate and EDTA are competitive with respect to the metal ion activator; those by phosphate and fluoride are competitive with respect to the substrate. 4. The addition of Mg(2+) diminishes the Michaelis constant of racemase. 5. The pH optimum of the racemase is at 7.8. The pH-activity curve of the dehydrogenase complex of enzymes has two peaks, at 7.0 and 8.2. 6. The enzymic racemization of d-mandelate is initially faster than that of l-mandelate. 7. The rates of oxidation of related substrates, catalysed by l-mandelate dehydrogenase, are in the decreasing order: l-p-hydroxymandelate; l-3,4-dihydroxymandelate; l-4-hydroxy-3-methoxymandelate. The racemase is active towards d-p-hydroxymandelate but inactive towards d-3,4-dihydroxymandelate and d-4-hydroxy-3-methoxymandelate. Since 4-hydroxy-3-methoxymandelate, and presumably also 3,4-dihydroxymandelate, arising from the metabolism of catechol-amines, have the d-configuration, the enzymes studied cannot be utilized for estimation of the last two acids in urine.
| S-EPMC1270079 | biostudies-other
Project description:BackgroundCyanide is one of the most toxic chemicals produced by anthropogenic activities like mining and jewelry industries, which generate wastewater residues with high concentrations of this compound. Pseudomonas pseudoalcaligenes CECT5344 is a model microorganism to be used in detoxification of industrial wastewaters containing not only free cyanide (CN(-)) but also cyano-derivatives, such as cyanate, nitriles and metal-cyanide complexes. Previous in silico analyses suggested the existence of genes putatively involved in metabolism of short chain length (scl-) and medium chain length (mcl-) polyhydroxyalkanoates (PHAs) located in three different clusters in the genome of this bacterium. PHAs are polyesters considered as an alternative of petroleum-based plastics. Strategies to optimize the bioremediation process in terms of reducing the cost of the production medium are required.ResultsIn this work, a biological treatment of the jewelry industry cyanide-rich wastewater coupled to PHAs production as by-product has been considered. The functionality of the pha genes from P. pseudoalcaligenes CECT5344 has been demonstrated. Mutant strains defective in each proposed PHA synthases coding genes (Mpha(-), deleted in putative mcl-PHA synthases; Spha(-), deleted in the putative scl-PHA synthase) were generated. The accumulation and monomer composition of scl- or mcl-PHAs in wild type and mutant strains were confirmed by gas chromatography-mass spectrometry (GC-MS). The production of PHAs as by-product while degrading cyanide from the jewelry industry wastewater was analyzed in batch reactor in each strain. The wild type and the mutant strains grew at similar rates when using octanoate as the carbon source and cyanide as the sole nitrogen source. When cyanide was depleted from the medium, both scl-PHAs and mcl-PHAs were detected in the wild-type strain, whereas scl-PHAs or mcl-PHAs were accumulated in Mpha(-) and Spha(-), respectively. The scl-PHAs were identified as homopolymers of 3-hydroxybutyrate and the mcl-PHAs were composed of 3-hydroxyoctanoate and 3-hydroxyhexanoate monomers.ConclusionsThese results demonstrated, as proof of concept, that talented strains such as P. pseudoalcaligenes might be applied in bioremediation of industrial residues containing cyanide, while concomitantly generate by-products like polyhydroxyalkanoates. A customized optimization of the target bioremediation process is required to gain benefits of this type of approaches.
| S-EPMC4460949 | biostudies-literature
Project description:The relationship between the number of cells colonizing a new environment and time for resumption of growth is a subject of long-standing interest. In microbiology this is known as the "inoculum effect." Its mechanistic basis is unclear with possible explanations ranging from the independent actions of individual cells, to collective actions of populations of cells. Here, we use a millifluidic droplet device in which the growth dynamics of hundreds of populations founded by controlled numbers of Pseudomonas fluorescens cells, ranging from a single cell, to one thousand cells, were followed in real time. Our data show that lag phase decreases with inoculum size. The decrease of average lag time and its variance across droplets, as well as lag time distribution shapes, follow predictions of extreme value theory, where the inoculum lag time is determined by the minimum value sampled from the single-cell distribution. Our experimental results show that exit from lag phase depends on strong interactions among cells, consistent with a "leader cell" triggering end of lag phase for the entire population.
| S-EPMC10117806 | biostudies-literature
Project description:2-Hydroxy-6-oxo-7-methylocta-2,4-dienoate hydrolase (CumD) from Pseudomonas fluorescens IP01 hydrolyzes a meta-cleavage product generated in the cumene (isopropylbenzene) degradation pathway. The crystal structures of the inactive S103A mutant of the CumD enzyme complexed with isobutyrate and acetate ions were determined at 1.6 and 2.0 A resolution, respectively. The isobutyrate and acetate ions were located at the same position in the active site, and occupied the site for a part of the hydrolysis product with CumD, which has the key determinant group for the substrate specificity of related hydrolases. One of the oxygen atoms of the carboxyl group of the isobutyrate ion was hydrogen bonded with a water molecule and His252. Another oxygen atom of the carboxyl group was situated in an oxyanion hole formed by the two main-chain N atoms. The isopropyl group of the isobutyric acid was recognized by the side-chains of the hydrophobic residues. The substrate-binding pocket of CumD was long, and the inhibition constants of various organic acids corresponded well to it. In comparison with the structure of BphD from Rhodococcus sp. RHA1, the structural basis for the substrate specificity of related hydrolases, is revealed.
| S-EPMC2373588 | biostudies-literature
Project description:Massetolide A is a cyclic lipopeptide (CLP) antibiotic produced by various Pseudomonas strains from diverse environments. Cloning, sequencing, site-directed mutagenesis, and complementation showed that massetolide A biosynthesis in P. fluorescens SS101 is governed by three nonribosomal peptide synthetase (NRPS) genes, designated massA, massB, and massC, spanning approximately 30 kb. Prediction of the nature and configuration of the amino acids by in silico analysis of adenylation and condensation domains of the NRPSs was consistent with the chemically determined structure of the peptide moiety of massetolide A. Structural analysis of massetolide A derivatives produced by SS101 indicated that most of the variations in the peptide moiety occur at amino acid positions 4 and 9. Regions flanking the mass genes contained several genes found in other Pseudomonas CLP biosynthesis clusters, which encode LuxR-type transcriptional regulators, ABC transporters, and an RND-like outer membrane protein. In contrast to most Pseudomonas CLP gene clusters known to date, the mass genes are not physically linked but are organized in two separate clusters, with massA disconnected from massB and massC. Quantitative real-time PCR analysis indicated that transcription of massC is strongly reduced when massB is mutated, suggesting that these two genes function in an operon, whereas transcription of massA is independent of massBC and vice versa. Massetolide A is produced in the early exponential growth phase, and biosynthesis appears not to be regulated by N-acylhomoserine lactone-based quorum sensing. Massetolide A production is essential in swarming motility of P. fluorescens SS101 and plays an important role in biofilm formation.
| S-EPMC2293227 | biostudies-literature
Project description:In recent years, evolutionary biologists have developed an increasing interest in the use of barcoding strategies to study eco-evolutionary dynamics of lineages within evolving populations and communities. Although barcoded populations can deliver unprecedented insight into evolutionary change, barcoding microbes presents specific technical challenges. Here, strategies are described for barcoding populations of the model bacterium Pseudomonas fluorescens SBW25, including the design and cloning of barcoded regions, preparation of libraries for amplicon sequencing, and quantification of resulting barcoded lineages. In so doing, we hope to aid the design and implementation of barcoding methodologies in a broad range of model and non-model organisms.
| S-EPMC10275814 | biostudies-literature
Project description:The gene pcp, encoding pyrrolidone carboxyl peptidase (Pcp), from Pseudomonas fluorescens MFO was cloned and its nucleotide sequence was determined. This sequence contains a unique open reading frame (pcp) coding for a polypeptide of 213 amino acids (M(r) 22,441) which has significant homology to the Pcps from Streptococcus pyogenes, Bacillus subtilis, and Bacillus amyloliquefaciens. Comparison of the four Pcp sequences revealed two highly conserved motifs which may be involved in the active site of these enzymes. The cloned Pcp from P. fluorescens was purified to homogeneity and appears to exist as a dimer. This enzyme displays a Michaelis constant of 0.21 mM with L-pyroglutamyl-beta-naphthylamide as the substrate and an absolute substrate specificity towards N-terminal pyroglutamyl residues. Studies of inhibition by chemical compounds revealed that the cysteine and histidine residues are essential for enzyme activity. From their conservation in the four enzyme sequences, the Cys-144 and His-166 amino acids are proposed to form a part of the active site of these enzymes.
| S-EPMC205394 | biostudies-other
Project description:The goal of the study was to examine the influence of flowback water exposure on bacterial survival and biocide tolerance. The transcriptome of P. fluorescens was analyzed using RNA-seq to determine the gene rxpression profile changes occuring upon flowback water exposure. The results indicate that that P. fluorescens induces a well-coordinated genetic response that aids in its survival in flowback water as well as imparts enhanced tolerance against typically used slow acting biocides such as gluteraldehyde but increased susceptibity towards sodium hypochlorite. RNA-seq data demonstrated significant induction of genes involved in osmotic stress, energy production and conversion, membrane integrity, protein transport among others.
2014-12-23 | GSE55388 | GEO
Project description:The antimicrobial metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) contributes to the capacity of Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soilborne pathogens. A 2, 4-DAPG-negative Tn5 insertion mutant of strain CHA0 was isolated, and the nucleotide sequence of the 4-kb genomic DNA region adjacent to the Tn5 insertion site was determined. Four open reading frames were identified, two of which were homologous to phlA, the first gene of the 2,4-DAPG biosynthetic operon, and to the phlF gene encoding a pathway-specific transcriptional repressor. The Tn5 insertion was located in an open reading frame, tentatively named phlH, which is not related to known phl genes. In wild-type CHA0, 2, 4-DAPG production paralleled expression of a phlA'-'lacZ translational fusion, reaching a maximum in the late exponential growth phase. Thereafter, the compound appeared to be degraded to monoacetylphloroglucinol by the bacterium. 2,4-DAPG was identified as the active compound in extracts from culture supernatants of strain CHA0 specifically inducing phlA'-'lacZ expression about sixfold during exponential growth. Induction by exogenous 2,4-DAPG was most conspicuous in a phlA mutant, which was unable to produce 2, 4-DAPG. In a phlF mutant, 2,4-DAPG production was enhanced severalfold and phlA'-'lacZ was expressed at a level corresponding to that in the wild type with 2,4-DAPG added. The phlF mutant was insensitive to 2,4-DAPG addition. A transcriptional phlA-lacZ fusion was used to demonstrate that the repressor PhlF acts at the level of transcription. Expression of phlA'-'lacZ and 2,4-DAPG synthesis in strain CHA0 was strongly repressed by the bacterial extracellular metabolites salicylate and pyoluteorin as well as by fusaric acid, a toxin produced by the pythopathogenic fungus Fusarium. In the phlF mutant, these compounds did not affect phlA'-'lacZ expression and 2, 4-DAPG production. PhlF-mediated induction by 2,4-DAPG and repression by salicylate of phlA'-'lacZ expression was confirmed by using Escherichia coli as a heterologous host. In conclusion, our results show that autoinduction of 2,4-DAPG biosynthesis can be countered by certain bacterial (and fungal) metabolites. This mechanism, which depends on phlF function, may help P. fluorescens to produce homeostatically balanced amounts of extracellular metabolites.
| S-EPMC94405 | biostudies-literature