Chemotaxis to pyrimidines and identification of a cytosine chemoreceptor in Pseudomonas putida

Abstract

We developed a high-throughput quantitative capillary assay and demonstrated that Pseudomonas putida strains F1 and PRS2000 were attracted to cytosine, but not thymine or uracil. In contrast, Pseudomonas aeruginosa PAO1 was not chemotactic to any pyrimidines. Chemotaxis assays with a mutant strain of F1 in which the putative methyl-accepting chemotaxis protein-encoding gene Pput_0623 was deleted revealed that this gene (designated mcpC) encodes a chemoreceptor for positive chemotaxis to cytosine. P. putida F1 also responded weakly to cytidine, uridine, and thymidine, but these responses were not mediated by mcpC. Complementation of the F1 DeltamcpC mutant XLF004 with the wild-type gene restored chemotaxis to cytosine. In addition, introduction of this gene into P. aeruginosa PAO1 conferred the ability to respond to cytosine. To our knowledge, this is the first report of a chemoreceptor for cytosine.

FIG. 1.

Concentration-response curves for chemotaxis to pyrimidines by P. putida strains F1 (A) and PRS2000 (B). Cells were grown at 30°C in MSB containing 10 mM succinate. Assays were performed at room temperature with various concentrations of each pyrimidine, up to its limit of solubility. Results are the averages of at least 15 capillaries from at least two independent experiments; error bars indicate standard errors. The background accumulations in capillaries containing buffer only were 450 and 200 cells for P. putida F1 and PRS2000, respectively.

FIG. 2.

Chemotactic responses of wild-type P. putida F1, the mcpC mutant (strain XLF004), and the complemented strain [XLF004 (pXLF204)] in qualitative and quantitative capillary assays. Cells were grown as described in the legend to Fig. 1, except that tetracycline was used at 20 μg/ml to maintain the plasmid in the complemented strain. (A) Chemotactic responses in modified capillary assays. Capillaries contained chemotaxis buffer or 50 mM cytosine in chemotaxis buffer solidified with 2% low-melting-temperature agarose. Assays were carried out at room temperature for 30 min. (B) Chemotactic responses in high-throughput quantitative capillary assays. Results are the averages of at least 15 capillaries from at least two independent experiments; error bars indicate standard errors. XLF004(pRK415) responded to cytosine at a level similar to that of XLF004 (data not shown).

FIG. 3.

Chemotactic responses of wild-type P. putida F1 and the mcpC mutant (strain XLF004) to pyrimidine nucleosides in high-throughput quantitative capillary assays. Cells were grown in MSB with 10 mM succinate. Results are the averages of at least 12 capillaries from at least two independent experiments; error bars indicate standard errors.

FIG. 4.

Chemotactic responses of P. aeruginosa PAO1(pRK415) (wild-type vector control) and PAO1(pXLF204) (carrying mcpC) to cytosine in high-throughput quantitative capillary assays. Cells were grown in MSB with 27.5 mM glucose. Results are the averages of at least 15 capillaries from at least two independent experiments; error bars indicate standard errors.