Microarray analysis of Pseudomonas aeruginosa quorum-sensing regulons: effects of growth phase and environment

Abstract

Bacterial communication via quorum sensing (QS) has been reported to be important in the production of virulence factors, antibiotic sensitivity, and biofilm development. Two QS systems, known as the las and rhl systems, have been identified previously in the opportunistic pathogen Pseudomonas aeruginosa. High-density oligonucleotide microarrays for the P. aeruginosa PAO1 genome were used to investigate global gene expression patterns modulated by QS regulons. In the initial experiments we focused on identifying las and/or rhl QS-regulated genes using a QS signal generation-deficient mutant (PAO-JP2) that was cultured with and without added exogenous autoinducers [N-(3-oxododecanoyl) homoserine lactone and N-butyryl homoserine lactone]. Conservatively, 616 genes showed statistically significant differential expression (P </= 0.05) in response to the exogenous autoinducers and were classified as QS regulated. A total of 244 genes were identified as being QS regulated at the mid-logarithmic phase, and 450 genes were identified as being QS regulated at the early stationary phase. Most of the previously reported QS-promoted genes were confirmed, and a large number of additional QS-promoted genes were identified. Importantly, 222 genes were identified as being QS repressed. Environmental factors, such as medium composition and oxygen availability, eliminated detection of transcripts of many genes that were identified as being QS regulated.

FIG. 1.

Changes in the distribution of QS-regulated genes. The numbers of QS-regulated genes displaying specific magnitudes of differential expression (PAO-JP2 induced versus uninduced) are shown. Genes were placed in groups according to the magnitude of differential expression (1.3- to 2.0-, 2.0- to 4.0-, 4.0- to 8.0-, 8.0- to 16-, 16- to 32-, 32- to 64-, and more than 64-fold changes).

FIG. 2.

Alignment of las boxes upstream of QS-regulated genes. Previously identified las boxes were aligned to form a consensus sequence (11, 29, 43). This sequence was used to search all upstream regions in front of the translational start site of all QS-regulated genes. The consensus sequence NHCTRNSNNDHNDKNNAGNB was derived from inspection, where H = C, T, or A; R = A or G; S = C or G; D = G, A, or T; K = G or T; B = G or T; and N = A, C, G, or T. Sequences that matched the consensus sequence were identified, and the position of the las box is shown in Table 1.

FIG. 3.

Functional classes of QS-regulated genes. QS-regulated genes were grouped according to a functional classification. The number of genes either promoted or repressed for each category is shown. LPS, lipopolysaccharide.

FIG. 4.

Effect of media and oxygen conditions on lasR, lasI, rhlR, and rhlI expression. The levels of transcript expression for mRNA encoding the known key elements of the las and rhl regulatory system are shown for PAO1 grown to the early stationary phase. Statistically significant differences between the transcript levels for PAO1 grown in modified FAB and the transcript levels for PAO1 grown under other conditions are indicated by an asterisk (P ≤ 0.05, as determined by one-way analysis of variance with the Tukey post-hoc test).