Global regulation of food supply by Pseudomonas putida DOT-T1E

Abstract

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.

FIG. 1.

Growth of P. putida DOT-T1E and its isogenic Δcrc, ΔcyoB, and ΔptsN strains with different C sources. The wild-type and mutant strains were assayed for their ability to grow on 35 defined carbon sources at 5 mM over a 24-h period. Results represent the average of at least three different experiments. Final turbidities of less than 0.2 are considered to indicate a lack of growth ability on that compound. The Δcrp mutant is absent from this analysis because it does not grow in NH₄Cl, which is the nitrogen source used in these assays.

FIG. 2.

Growth of P. putida DOT-T1E and its isogenic Δcrp mutant with different C sources. The wild-type and Δcrp mutant strains were assayed for their ability to grow in defined carbon sources at 5 mM over a period of 24 h, using Ala-Thr as a nitrogen source. Results presented are the average of three assays standard deviations.

FIG. 3.

Growth with different N sources of P. putida DOT-T1E and isogenic mutants deficient in a global regulator. The wild-type and mutant strains were assayed for their ability to grow on 40 defined nitrogen sources at 5 mM over a period of 24 h. Results presented are the averages and standard deviations of at least three assays. Interestingly the Δcrp mutant could not utilize NH₄Cl as a nitrogen source. This observation led us to use Ala-Thr the nitrogen source which was preferred by the Δcrp mutant strain in future assays for with this mutant.

FIG. 4.

(A) The wild-type and mutant strains were assayed for their ability to grow on 15 defined sulfur sources at 5 mM over a 24-h period. An average from three independent experiments is represented. The Δcrp mutant is absent from this analysis because it does not grow in NH₄Cl, which is the nitrogen source used in these assays. (B) The wild-type and Δcrp mutant strains were assayed for their ability to grow in defined sulfur sources at 5 mM over a period of 24 h, using Ala-Thr as a nitrogen source. Results presented are the average of three assays and standard deviations.

FIG. 5.

Effect on growth of P. putida DOT-T1E and its isogenic mutants in the presence of organic and inorganic stressors. The wild-type and all mutant strains were assayed for their ability to grow in the presence of 45 different stress compounds over a period of 24 h. Results presented are the average of three assays and standard deviations.