Use of in-biofilm expression technology to identify genes involved in Pseudomonas aeruginosa biofilm development

Abstract

Mature Pseudomonas aeruginosa biofilms form complex three-dimensional architecture and are tolerant of antibiotics and other antimicrobial compounds. In this work, an in vivo expression technology system, originally designed to study virulence-associated genes in complex mammalian environments, was used to identify genes up-regulated in P. aeruginosa grown to a mature (5-day) biofilm. Five unique cloned promoters unable to promote in vitro growth in the absence of purines after recovery from the biofilm environment were identified. The open reading frames downstream of the cloned promoter regions were identified, and knockout mutants were generated. Insertional mutation of PA5065, a homologue of Escherichia coli ubiB, was lethal, while inactivation of PA0240 (a porin homologue), PA3710 (a putative alcohol dehydrogenase), and PA3782 (a homologue of the Streptomyces griseus developmental regulator adpA) had no effect on planktonic growth but caused defects in biofilm formation in static and flowing systems. In competition experiments, mutants demonstrated reduced fitness compared with the parent strain, comprising less than 0.0001% of total biofilm cells after 5 days. Therefore, using in-biofilm expression technology, we have identified novel genes that do not affect planktonic growth but are important for biofilm formation, development, and fitness.

FIG. 1.

Growth curves of recovered IVET clones. Planktonic growth of PAK, PAK-AR2, and IVET clones (PAK-AR2 with integrated IVET plasmids) recovered after 5 days in a biofilm was compared in DMM and DMM+A. Since strains 9.27B and C were identical, only 9.27B is shown. Note that strain 9.29 has a growth curve similar to that of AR2.

FIG. 2.

Analysis of plasmids isolated from recombinant PA5065::Gm^r insertional mutants. (A) Plasmid DNA was isolated from the recombinant PA5065::Gm^r/pUCP26-PA5065 strain after transformation with pUCP20 (lane 1) or pUCP20-ubiB_Ec (lane 2) and digested with EcoRI and HindIII to release the inserts. pUCP26-PA5065 was isolated in tandem with pUCP20 (lane 1) despite counterselection, but it was lost if pUCP20-ubiB_Ec was present in the cell (lane 2). (B) The plasmid DNA isolated from PA5065::Gm^r/pUCP26-PA5065 was used to transform E. coli JM109, which was grown on plates containing either 15 μg of tetracycline/ml or 50 μg of ampicillin/ml. Analysis of plasmid DNA isolated from the resulting Tet^r (lanes 1 and 2) and Amp^r (lanes 3 and 4) recombinant E. coli strains by digestion with EcoRI and HindIII confirmed that both plasmids were originally present in the recombinant P. aeruginosa mutant despite counterselection against pUCP26-PA5065.

FIG. 3.

Static biofilm formation assay. The abilities of PAO1 and its isogenic mutants to form biofilms in a static assay were compared. The amount of planktonic growth in each well as measured by the turbidity (reported here as optical density [O.D.]) at 600 nm is shown in white bars, while the amount of biofilm is shown as the absorbance of bound CV at 600 nm in black bars. Pilin (pilA) and flagellin (fliC) mutants were used as negative controls.

FIG. 4.

Biofilm competition assay. Equal quantities of PAO1 and its isogenic mutants were grown together as a biofilm for 5 days. A section of tubing was removed daily, and the total number of cells, as well as the number of Gm^r cells, was enumerated by serial dilution and plate count. All three mutants were less fit than the wild type and were reduced to 0.0001% or less of total cells after 5 days.

FIG. 5.

Biofilm morphology of mutants. (A) SEM of P. aeruginosa grown in silicone tubing. Each individual strain was grown under flow in DMM+ media for 24 h (longer growth times resulted in thicker biofilms that collapsed during dehydration, making structure difficult to see). Magnification, 1,000×; bars, 10 μm. (B) CSLM of BacLight live/dead stained 48-h biofilms. Sagittal (x-z) sections through the biofilm are indicated by white arrows. Note that defects in biofilm formation are consistent despite differences in imaging techniques (SEM versus CSLM), substrata (silicone versus glass), and length of incubation (24 versus 48 h), showing that these defects are not specific to cultivation conditions. Magnification, 100×; bar, 100 μm.