Subtilase SprP exerts pleiotropic effects in Pseudomonas aeruginosa

Abstract

The open reading frame PA1242 in the genome of Pseudomonas aeruginosa PAO1 encodes a putative protease belonging to the peptidase S8 family of subtilases. The respective enzyme termed SprP consists of an N-terminal signal peptide and a so-called S8 domain linked by a domain of unknown function (DUF). Presumably, this DUF domain defines a discrete class of Pseudomonas proteins as homologous domains can be identified almost exclusively in proteins of the genus Pseudomonas. The sprP gene was expressed in Escherichia coli and proteolytic activity was demonstrated. A P. aeruginosa ∆sprP mutant was constructed and its gene expression pattern compared to the wild-type strain by genome microarray analysis revealing altered expression levels of 218 genes. Apparently, SprP is involved in regulation of a variety of different cellular processes in P. aeruginosa including pyoverdine synthesis, denitrification, the formation of cell aggregates, and of biofilms.

Keywords: Biofilm; Pseudomonas aeruginosa; Pyoverdine.; microarray; motility; orf PA1242; protease.

Figure 1

SprP domain composition. SprP consists of 590 amino acids (aa) with an N-terminal signal sequence (I, SS), a domain of unknown function (II, DUF), a peptidase S8 domain (III), and a C-terminal extension (IV). Numbers indicate the amino acids flanking the respective domains; putative catalytic triad residues E₂₆₂, H₂₉₉, and S₄₉₉ are labeled.

Figure 2

SprP is a protease. (A) Escherichia coli DH5α was transformed with plasmid pBBRSP harboring sprP (right) or empty vector (left), plated on agar containing 3% skim milk and plates were incubated for 16 h at 37°C. (B) Cell extracts of strains shown in (A) were prepared after centrifugation and subsequent sonication and tested for proteolytic activity with Suc-Ala-Ala-Pro-Phe-p-nitroanilide as the substrate. Relative activity of 100% corresponds to 0.308; error bars indicate standard deviation.

Figure 3

Promoter activity of sprP. Pseudomonas aeruginosa PAO1 containing plasmid pTZsprP, which harbors a 556 bp DNA fragment upstream of sprP fused to lacZ was inoculated to an initial cell density of OD_580nm = 0.1 and incubated for 24 h at 37°C. Activity of β-galactosidase (bars) was determined as described by Miller (1972) and the corresponding growth of the culture (line) was determined as absorbance of the bacterial culture at 580 nm. Error bars indicate standard deviation.

Figure 4

SprP affects cell aggregation and biofilm formation. Pseudomonas aeruginosa wild-type, ΔsprP mutant and ΔsprP mutant complemented with plasmid pBBRSP were inoculated to an initial cell density of OD_580nm = 0.1 and incubated until the cultures reached an OD_580nm of 2.5. (A) Cell aggregation was examined, cells transferred to petri dishes, and photographed. (B) Biofilm formation was analyzed in microtiter-plates inoculated with different strains and cultivated for 16 h at 37°C. Biofilms were stained with crystal violet, photographed, and dye was extracted with 100% ethanol and quantified photometrically at 590 nm. Biofilm formation is indicated as bound dye (A_590nm) per cell density (OD_580nm). Error bars indicate standard deviation. wt = P. aeruginosa PAO1, ΔsprP = P. aeruginosa ΔsprP harboring either ev = empty vector or sprP = plasmid pBBRSP.

Figure 5

Deletion of sprP abolishes cell motility of Pseudomonas aeruginosa. Swarming and swimming agar plates contained M9 minimal medium with 0.5% agar for swarming and 0.3% agar for swimming. 5 μL of bacterial culture at OD_580nm = 3 were added. Twitching motility was assessed after stabbing cells through 3 mm-thick LB agar plates onto the ground of the petri dishes. All plates were incubated for 16 h at 37°C. wt = P. aeruginosa PAO1, ΔsprP = P. aeruginosa ΔsprP harboring either ev = empty vector or sprP = plasmid pBBRSP.

Figure 6

Deletion of sprP increases pyoverdine production by Pseudomonas aeruginosa. Strains were incubated at 37°C until cell growth reached an OD_580nm of 2.5 and pyoverdine in cell-free culture supernatants was determined by absorbance at 403 nm (Meyer and Abdallah 1978). Pyoverdine production is indicated as pyoverdine absorbance (A_403nm) per cell density (OD_580 nm). Error bars indicate standard deviation calculated from biological triplicates. Cuvettes containing culture supernatants are shown underneath the diagram. wt = P. aeruginosa PAO1, ΔsprP = P. aeruginosa ΔsprP harboring either ev = empty vector or sprP = plasmid pBBRSP.

Figure 7

SprP affects anaerobic growth of Pseudomonas aeruginosa. (A) Bacterial cultures were incubated at 37°C for 6 h and growth differences under anaerobic and aerobic conditions are illustrated. (B) The cell aggregation phenotype of P. aeruginosa ΔsprP after cultivation in LB-medium and LB-medium supplemented with the nitric oxide donor sodium nitroprusside (SNP). wt = P. aeruginosa PAO1, ΔsprP = P. aeruginosa ΔsprP harboring either ev = empty vector or sprP = plasmid pBBRSP.