Nitrobenzoates and aminobenzoates are chemoattractants for Pseudomonas strains

Abstract

Three Pseudomonas strains were tested for the ability to sense and respond to nitrobenzoate and aminobenzoate isomers in chemotaxis assays. Pseudomonas putida PRS2000, a strain that grows on benzoate and 4-hydroxybenzoate by using the beta-ketoadipate pathway, has a well-characterized beta-ketoadipate-inducible chemotactic response to aromatic acids. PRS2000 was chemotactic to 3- and 4-nitrobenzoate and all three isomers of aminobenzoate when grown under conditions that induce the benzoate chemotactic response. P. putida TW3 and Pseudomonas sp. strain 4NT grow on 4-nitrotoluene and 4-nitrobenzoate by using the ortho (beta-ketoadipate) and meta pathways, respectively, to complete the degradation of protocatechuate derived from 4-nitrotoluene and 4-nitrobenzoate. However, based on results of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase assays, both strains were found to use the beta-ketoadipate pathway for the degradation of benzoate. Both strains were chemotactic to benzoate, 3- and 4-nitrobenzoate, and all three aminobenzoate isomers after growth with benzoate but not succinate. Strain TW3 was chemotactic to the same set of aromatic compounds after growth with 4-nitrotoluene or 4-nitrobenzoate. In contrast, strain 4NT did not respond to any aromatic acids when grown with 4-nitrotoluene or 4-nitrobenzoate, apparently because these substrates are not metabolized to the inducer (beta-ketoadipate) of the chemotaxis system. The results suggest that strains TW3 and 4NT have a beta-ketoadipate-inducible chemotaxis system that responds to a wide range of aromatic acids and is quite similar to that present in PRS2000. The broad specificity of this chemotaxis system works as an advantage in strains TW3 and 4NT because it functions to detect diverse carbon sources, including 4-nitrobenzoate.

FIG. 1.

Chemotactic responses of Pseudomonas sp. strain 4NT and P. putida TW3 to 1 mM benzoate (A) and 0.5 mM 4-nitrobenzoate (B) in swarm plates.

FIG. 2.

Chemotactic responses of P. putida TW3 (A) and Pseudomonas sp. strain 4NT (B) in smooth swimming response assays after growth with 10 mM succinate, 5 mM benzoate, or saturating amounts of 4-nitrobenzoate (4NB) or 4-nitrotoluene (4NT). The response in this assay is the amount of time in seconds needed for approximately 50% of the cells to adapt to the added attractant. The abbreviations for the attractants in the chemotaxis buffer are as follows: 0.2% CAA, 0.2% Casamino Acids; Ben, benzoate; 4NB, 4-nitrobenzoate; 3NB, 3-nitrobenzoate; 4AB, 4-aminobenzoate; 3AB, 3-aminobenzoate; 2AB, 2-aminobenzoate. No response was seen with chemotaxis buffer alone or with 1 mM 2-nitrobenzoate (data not shown).

FIG. 3.

Time courses of the chemotactic responses of the three Pseudomonas strains to 4-nitrobenzoate in modified capillary assays (magnification, ×40). Capillaries (1 μl) contained crystals of 4-nitrobenzoate in chemotaxis buffer. (A) Benzoate-grown PRS2000; (B) benzoate-grown TW3; (C) 4-nitrobenzoate-grown TW3; (D) 4-nitrotoluene-grown TW3; (E) 4-hydroxybenzoate-grown strain 4NT; (F) 4-nitrobenzoate-grown strain 4NT. No responses were seen when capillaries contained only agarose and chemotaxis buffer or silica in chemotaxis buffer (data not shown).

FIG. 4.

Pathways for 4-nitrotoluene and 4-nitrobenzoate degradation in P. putida TW3 and Pseudomonas sp. strain 4NT. Note that P. putida TW3 utilizes ortho cleavage and Pseudomonas sp. strain 4NT carries out meta cleavage of protocatechuate.