Microarray analysis of the osmotic stress response in Pseudomonas aeruginosa

Abstract

Transcriptional profiling of Pseudomonas aeruginosa grown under steady-state hyperosmotic stress conditions showed an up-regulation of genes associated with osmoprotectant synthesis, putative hydrophilins, and the type III secretion system with associated cytotoxins. A large number of regulatory genes, including several two-component systems not previously known to be influenced by osmolarity, were differentially expressed by P. aeruginosa in immediate response to hyperosmotic shock.

FIG. 1.

Disruption of putative osmoprotectant synthesis genes impairs growth of P. aeruginosa PAO1 in a high-osmolarity medium. Cells were grown at 37°C in a glucose minimal medium containing 0.5 M NaCl. For complementation, a wild-type copy of the interrupted gene containing the native ribosome binding site was PCR generated and cloned (1) as an EcoRI-HindIII or EcoRI-BamHI fragment downstream of the lac promoter in pUCP20 (17). Plasmid constructs were introduced into P. aeruginosa by use of electroporation (18), and transformants were selected using Trypticase soy agar containing 100 μg/ml carbenicillin. FA015, PA3459::ISphoA/hah; FA011, PA3460::ISlacZ/hah. PA3459 and PA3460 encode a putative glutamine amidotransferase and an acetyltransferase, respectively. (A) Open circles, PAO1/pUCP20; open triangles, FA015/pUCP20::PA3459; closed triangles, FA015/pUCP20. (B) Open circles, PAO1/pUCP20; open diamonds, FA011/pUCP20::PA3460; closed diamonds, FA011/pUCP20. Values are representative of at least three independent trials.

FIG. 2.

The osmoprotectant betaine recovers growth rate in an osmotic stress-susceptible mutant. Cells were grown in the presence of 0.5 M NaCl under the same conditions described for Fig. 1. Open symbols, cells grown with 0.5 mM betaine; closed symbols, cells grown without betaine. Circles, PAO1; diamonds, FA011 (PA3460::ISlacZ/hah). Values are representative of two independent trials.