salicylates and hexanoic-acid

Pseudomonas putida DOT-T1E was used as a model to develop a "phenomics" platform to investigate the ability of P. putida to grow using different carbon, nitrogen, and sulfur sources and in the presence of stress molecules. Results for growth of wild-type DOT-T1E on 90 different carbon sources revealed the existence of a number of previously uncharted catabolic pathways for compounds such as salicylate, quinate, phenylethanol, gallate, and hexanoate, among others. Subsequent screening on the subset of compounds on which wild-type DOT-TIE could grow with four knockout strains in the global regulatory genes Deltacrc, Deltacrp, DeltacyoB, and DeltaptsN allowed analysis of the global response to nutrient supply and stress. The data revealed that most global regulator mutants could grow in a wide variety of substrates, indicating that metabolic fluxes are physiologically balanced. It was found that the Crc mutant did not differ much from the wild-type regarding the use of carbon sources. However, certain pathways are under the preferential control of one global regulator, i.e., metabolism of succinate and d-fructose is influenced by CyoB, and l-arginine is influenced by PtsN. Other pathways can be influenced by more than one global regulator; i.e., l-valine catabolism can be influenced by CyoB and Crp (cyclic AMP receptor protein) while phenylethylamine is affected by Crp, CyoB, and PtsN. These results emphasize the cross talk required in order to ensure proper growth and survival. With respect to N sources, DOT-T1E can use a wide variety of inorganic and organic nitrogen sources. As with the carbon sources, more than one global regulator affected growth with some nitrogen sources; for instance, growth with nucleotides, dipeptides, d-amino acids, and ethanolamine is influenced by Crp, CyoB, and PtsN. A surprising finding was that the Crp mutant was unable to flourish on ammonium. Results for assayed sulfur sources revealed that CyoB controls multiple points in methionine/cysteine catabolism while PtsN and Crc are needed for N-acetyl-l-cysteamine utilization. Growth of global regulator mutants was also influenced by stressors of different types (antibiotics, oxidative agents, and metals). Overall and in combination with results for growth in the presence of various stressors, these phenomics assays provide multifaceted insights into the complex decision-making process involved in nutrient supply, optimization, and survival.

Topics: Bacterial Proteins; Caproates; Carbon; Fructose; Gene Expression Regulation, Bacterial; Nitrogen; Oligonucleotide Array Sequence Analysis; Phenethylamines; Phenotype; Phenylethyl Alcohol; Phosphoenolpyruvate Sugar Phosphotransferase System; Pseudomonas putida; Quaternary Ammonium Compounds; Quinic Acid; Salicylates; Succinic Acid; Sulfur; Valine

Molecular characteristics of carboxylic acids were investigated for the ability to inhibit a purified medium chain acyl-CoA synthetase, using hexanoic acid as a substrate. Salicylic acid, 4-methylsalicylic acid, 2-hydroxynaphtoic acid, and 2-hydroxyoctanoic acid, which do not act as substrates for the medium chain acyl-CoA synthetase, were potent as inhibitors. Valproic acid was not an inhibitor. Salicylic acid, 2-hydroxynaphthoic acid, and 2-hydroxyoctanoic acid inhibited the medium chain acyl-CoA synthetase with Ki values of 37, 5.2, and 500 microM, respectively. 4-Methylsalicylic acid was more potent than salicylic acid. The inhibitory carboxylic acids were competitive with respect to hexanoic acid. The distance of the hydroxyl group from the carboxylic acid group of the benzene ring influenced the inhibitory activity. The hydroxyl group on the carbon adjacent to the carboxylic acid group was required for inhibitory activity. In addition, there was a good correlation between the lipophilicity of the carboxylic acids and the Ki values, suggesting that the lipophilicity of the carboxylic acids is a major determinant for inhibition of the medium chain acyl-CoA synthetase.

Topics: Animals; Caproates; Caprylates; Carboxylic Acids; Cattle; Coenzyme A Ligases; Enzyme Inhibitors; Glycine; In Vitro Techniques; Kinetics; Liver; Salicylates; Structure-Activity Relationship; Substrate Specificity