PilS and PilR, a two-component transcriptional regulatory system controlling expression of type 4 fimbriae in Pseudomonas aeruginosa
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M Hobbs1, E S Collie, P D Free
1Centre for Molecular Biology and Biotechnology, University of Queensland, Brisbane, Australia.
Abstract:
Transposon mutagenesis was used to identify genes necessary for the expression of Pseudomonas aeruginosa type 4 fimbriae. In a library of 12,700 mutants, 147 were observed to have lost the spreading colony morphology associated with the presence of functional fimbriae. Of these, 28 had also acquired resistance to the fimbrial-specific bacteriophage PO4. The mutations conferring this phage resistance were found to have occurred at at least six different loci, including the three that had been previously shown to be required for fimbrial biosynthesis or function: the structural subunit (pilA) and adjacent genes (pilB,C,D), the twitching motility gene (pilT), and the sigma 54 RNA polymerase initiation factor gene (rpoN). One novel group of phage-resistant mutants was identified in which the transposon had inserted near sequences that cross-hybridized to an oligonucleotide probe designed against conserved domains in regulators of RpoN-dependent promoters. These mutants had no detectable transcription of pilA and did not produce fimbriae. A probe derived from inverse polymerase chain reaction was used to isolate the corresponding wild-type sequences from a P. aeruginosa PAO cosmid reference library, and two adjacent genes affected by transposon insertions, pilS and pilR, were located and sequenced. These genes were shown to be capable of complementing the corresponding mutants, both at the level of restoring the phenotypes associated with functional fimbriae and by the restoration of pilA transcription. The pilSR operon was physically mapped to Spel fragment 5 (corresponding to about 72-75/0 min on the genetic map), and shown to be located approximately 25 kb from pilA-D. PilS and PilR clearly belong to the family of two-component transcriptional regulatory systems which have been described in many bacterial species. PilS is predicted to be a sensor protein which when stimulated by the appropriate environmental signals activates PilR through kinase activity. PilR then activates transcription of pilA, probably by interacting with RNA polymerase containing RpoN. The identification of pilS and pilR makes possible a more thorough examination of the signal transduction systems controlling expression of virulence factors in P. aeruginosa.
Insights
Researchers identified new genes, pilS and pilR, essential for Pseudomonas aeruginosa type 4 fimbriae expression. These genes form a two-component regulatory system controlling fimbrial production and virulence factors.
Area of Science:
- Microbiology
- Bacterial Genetics
- Molecular Biology
Background:
- Pseudomonas aeruginosa utilizes type 4 fimbriae for various functions, including adhesion and motility.
- Expression of type 4 fimbriae is a complex process involving multiple genes and regulatory pathways.
- Understanding fimbrial gene regulation is crucial for controlling P. aeruginosa virulence.
Purpose of the Study:
- To identify novel genes involved in the regulation of Pseudomonas aeruginosa type 4 fimbriae expression.
- To elucidate the function and mechanism of newly discovered regulatory genes.
- To investigate the signal transduction pathways controlling fimbrial gene expression.
Main Methods:
- Transposon mutagenesis library screening to identify mutants with altered fimbrial phenotypes.
- Bacteriophage resistance screening to detect mutations affecting fimbrial function.
- Inverse polymerase chain reaction (PCR) and DNA sequencing to identify and characterize novel genes.
- Complementation studies to confirm gene function.
- Physical mapping of identified genes on the bacterial chromosome.
Main Results:
- Identified 147 mutants lacking fimbriae, with 28 exhibiting phage resistance.
- Discovered two novel genes, pilS and pilR, essential for pilA transcription and fimbrial production.
- Characterized pilS and pilR as a two-component regulatory system controlling RpoN-dependent transcription of pilA.
- Mapped the pilSR operon to a specific region of the P. aeruginosa genome.
Conclusions:
- PilS and PilR constitute a novel two-component system regulating type 4 fimbriae expression in P. aeruginosa.
- This regulatory system likely senses environmental signals to control virulence factor production.
- The discovery of pilSR provides new targets for understanding and potentially manipulating P. aeruginosa pathogenesis.