Expression of the psl Operon in Pseudomonas aeruginosa PAO1 Biofilms: PslA Performs an Essential Function in Biofilm Formation

ABSTRACT The psl gene cluster, comprising 15 cotranscribed genes from Pseudomonas aeruginosa, was recently identified as being involved in exopolysaccharide biosynthesis and biofilm formation. In this study, we investigated the regulation of the psl gene cluster and the function of the first gene in this cluster, the pslA gene. PslA shows strong similarities to UDP-glucose lipid carriers. An isogenic marker-free pslA deletion mutant of P. aeruginosa PAO1 deficient in attachment and biofilm formation was used for complementation studies. The expression of only the pslA gene, comprising a coding region of 1,437 bp, restored the biofilm-forming phenotype of the wild type, indicating that PslA is required for biofilm formation by nonmucoid P. aeruginosa. The promoter region of the psl gene cluster, which encodes PslA-PslO, was identified by rapid amplification of cDNA 5′ ends. Promoter assays using transcriptional fusions to lacZ and gfp indicated a constitutive expression of the psl cluster in planktonic cells and a highly regulated and localized expression in biofilms, respectively. Expression of the psl cluster in biofilms was almost exclusively found in the centers of microcolonies, as revealed by confocal laser scanning microscopy. These data suggest that constitutive expression of the psl operon enables efficient attachment to surfaces and that regulated localized psl operon expression is required for biofilm differentiation.

[1]  B. Rehm,et al.  The role of polyhydroxyalkanoate biosynthesis by Pseudomonas aeruginosa in rhamnolipid and alginate production as well as stress tolerance and biofilm formation. , 2004, Microbiology.

[2]  L. Eberl,et al.  Pseudomonas aeruginosa and Burkholderia cepacia in cystic fibrosis: genome evolution, interactions and adaptation. , 2004, International journal of medical microbiology : IJMM.

[3]  S. Brunak,et al.  Improved prediction of signal peptides: SignalP 3.0. , 2004, Journal of molecular biology.

[4]  R. Kolter,et al.  Two Genetic Loci Produce Distinct Carbohydrate-Rich Structural Components of the Pseudomonas aeruginosa Biofilm Matrix , 2004, Journal of bacteriology.

[5]  M. Parsek,et al.  Identification of psl, a Locus Encoding a Potential Exopolysaccharide That Is Essential for Pseudomonas aeruginosa PAO1 Biofilm Formation , 2004, Journal of bacteriology.

[6]  E. Greenberg,et al.  Putative Exopolysaccharide Synthesis Genes Influence Pseudomonas aeruginosa Biofilm Development , 2004, Journal of bacteriology.

[7]  S. Häussler.  Biofilm formation by the small colony variant phenotype of Pseudomonas aeruginosa. , 2004, Environmental microbiology.

[8]  Roberto Kolter,et al.  Genes involved in matrix formation in Pseudomonas aeruginosa PA14 biofilms , 2003, Molecular microbiology.

[9]  Matthew R. Parsek,et al.  Alginate is not a significant component of the extracellular polysaccharide matrix of PA14 and PAO1 Pseudomonas aeruginosa biofilms , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[10]  B. Rehm.  Alginates from Bacteria , 2002 .

[11]  H. Schweizer,et al.  Small broad-host-range lacZ operon fusion vector with low background activity. , 2001, BioTechniques.

[12]  S. Molin,et al.  Alginate Overproduction Affects Pseudomonas aeruginosa Biofilm Structure and Function , 2001, Journal of bacteriology.

[13]  D. Nivens,et al.  Role of Alginate and Its O Acetylation in Formation of Pseudomonas aeruginosa Microcolonies and Biofilms , 2001, Journal of bacteriology.

[14]  A. Krogh,et al.  Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. , 2001, Journal of molecular biology.

[15]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[16]  J. Leveau,et al.  Improved gfp and inaZ broad-host-range promoter-probe vectors. , 2000, Molecular plant-microbe interactions : MPMI.

[17]  S. Lory,et al.  Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen , 2000, Nature.

[18]  B. Tümmler,et al.  Cystic fibrosis: an inherited susceptibility to bacterial respiratory infections. , 1999, Molecular medicine today.

[19]  D A Turner,et al.  Use of intrinsic optical signals to monitor physiological changes in brain tissue slices. , 1999, Methods.

[20]  J. Costerton,et al.  Bacterial biofilms: a common cause of persistent infections. , 1999, Science.

[21]  R. Kolter,et al.  Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development , 1998, Molecular microbiology.

[22]  Erik L. L. Sonnhammer,et al.  A Hidden Markov Model for Predicting Transmembrane Helices in Protein Sequences , 1998, ISMB.

[23]  H. Schweizer,et al.  A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. , 1998, Gene.

[24]  C. Goodwin,et al.  Burn Wound Infections: Current Status , 1998, World Journal of Surgery.

[25]  S. Valla,et al.  Bacterial alginates: biosynthesis and applications , 1997, Applied Microbiology and Biotechnology.

[26]  V. Deretic,et al.  Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. , 1996, Microbiological reviews.

[27]  D. Roop,et al.  Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. , 1995, Gene.

[28]  F. Collins,et al.  Cystic fibrosis: molecular biology and therapeutic implications. , 1992, Science.

[29]  M. de Braekeleer,et al.  Spatial distribution of the DF508 mutation in cystic fibrosis: a review. , 1992, Human biology.

[30]  C. Nast,et al.  Functional role of mucoid exopolysaccharide (alginate) in antibiotic-induced and polymorphonuclear leukocyte-mediated killing of Pseudomonas aeruginosa , 1991, Infection and immunity.

[31]  D. Hanahan.  Studies on transformation of Escherichia coli with plasmids. , 1983, Journal of molecular biology.

[32]  B. Friedrich,et al.  Naturally occurring genetic transfer of hydrogen-oxidizing ability between strains of Alcaligenes eutrophus , 1981, Journal of bacteriology.

[33]  A. Linker,et al.  Production and Characterization of the Slime Polysaccharide of Pseudomonas aeruginosa , 1973, Journal of bacteriology.

[34]  B. Eberhart,et al.  Induction of β-Glucosidases in Neurospora crassa , 1973 .

[35]  B. Cunha.  Nosocomial pneumonia. Diagnostic and therapeutic considerations. , 2001, The Medical clinics of North America.

[36]  S. Lory,et al.  Complete genome sequence of Pseudomonas aeruginosa PAO 1 , an opportunistic pathogen , 2000 .

[37]  P. Watnick,et al.  Genetic approaches to study of biofilms. , 1999, Methods in enzymology.

[38]  C. Osborne,et al.  Diagnostic and Therapeutic Considerations , 1996 .

[39]  H. Schweizer,et al.  An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa. , 1995, Gene.

[40]  N. Høiby.  Antibiotic therapy for chronic infection of pseudomonas in the lung. , 1993, Annual review of medicine.

[41]  W. Bullock.  XL1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with beta-galactosidase selection. , 1987 .

[42]  A. Pühler,et al.  A Broad Host Range Mobilization System for In Vivo Genetic Engineering: Transposon Mutagenesis in Gram Negative Bacteria , 1983, Bio/Technology.

[43]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .

[44]  J. Gustafson,et al.  Cystic Fibrosis , 2009, Journal of the Iowa Medical Society.