Quorum sensing in Pseudomonas aeruginosa controls expression of catalase and superoxide dismutase genes and mediates biofilm susceptibility to hydrogen peroxide

Quorum sensing (QS) governs the production of virulence factors and the architecture and sodium dodecyl sulphate (SDS) resistance of biofilm‐grown Pseudomonas aeruginosa. P. aeruginosa QS requires two transcriptional activator proteins known as LasR and RhlR and their cognate autoinducers PAI‐1 (N‐(3‐oxododecanoyl)‐l‐homoserine lactone) and PAI‐2 (N‐butyryl‐l‐homoserine lactone) respectively. This study provides evidence of QS control of genes essential for relieving oxidative stress. Mutants devoid of one or both autoinducers were more sensitive to hydrogen peroxide and phenazine methosulphate, and some PAI mutant strains also demonstrated decreased expression of two superoxide dismutases (SODs), Mn‐SOD and Fe‐SOD, and the major catalase, KatA. The expression of sodA (encoding Mn‐SOD) was particularly dependent on PAI‐1, whereas the influence of autoinducers on Fe‐SOD and KatA levels was also apparent but not to the degree observed with Mn‐SOD. β‐Galactosidase reporter fusion results were in agreement with these findings. Also, the addition of both PAIs to suspensions of the PAI‐1/2‐deficient double mutant partially restored KatA activity, while the addition of PAI‐1 only was sufficient for full restoration of Mn‐SOD activity. In biofilm studies, catalase activity in wild‐type bacteria was significantly reduced relative to planktonic bacteria; catalase activity in the PAI mutants was reduced even further and consistent with relative differences observed between each strain grown planktonically. While wild‐type and mutant biofilms contained less catalase activity, they were more resistant to hydrogen peroxide treatment than their respective planktonic counterparts. Also, while catalase was implicated as an important factor in biofilm resistance to hydrogen peroxide insult, other unknown factors seemed potentially important, as PAI mutant biofilm sensitivity appeared not to be incrementally correlated to catalase levels.

[1]  D. Hassett,et al.  Response of Pseudomonas aeruginosa to pyocyanin: mechanisms of resistance, antioxidant defenses, and demonstration of a manganese-cofactored superoxide dismutase , 1992, Infection and immunity.

[2]  G. Salmond,et al.  Multiple N-acyl-L-homoserine lactone signal molecules regulate production of virulence determinants and secondary metabolites in Pseudomonas aeruginosa. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[3]  I W SIZER,et al.  A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. , 1952, The Journal of biological chemistry.

[4]  P. Stewart,et al.  Spatial Patterns of Alkaline Phosphatase Expression within Bacterial Colonies and Biofilms in Response to Phosphate Starvation , 1998, Applied and Environmental Microbiology.

[5]  B. Holloway,et al.  Chromosomal genetics of Pseudomonas. , 1979, Microbiological reviews.

[6]  Z Lewandowski,et al.  Biofilms, the customized microniche , 1994, Journal of bacteriology.

[7]  S. Marklund,et al.  Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. , 1974, European journal of biochemistry.

[8]  D. Hassett,et al.  Ferric uptake regulator (Fur) mutants of Pseudomonas aeruginosa demonstrate defective siderophore-mediated iron uptake, altered aerobic growth, and decreased superoxide dismutase and catalase activities , 1996, Journal of bacteriology.

[9]  S. Kjelleberg,et al.  Global analysis of the carbon starvation response of a marine Vibrio species with disruptions in genes homologous to relA and spoT , 1996, Journal of bacteriology.

[10]  C. Reimmann,et al.  The global activator GacA of Pseudomonas aeruginosa PAO positively controls the production of the autoinducer N‐butyryl‐homoserine lactone and the formation of the virulence factors pyocyanin, cyanide, and lipase , 1997, Molecular microbiology.

[11]  B. Iglewski,et al.  Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes , 1997, Journal of bacteriology.

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

[13]  D. Hassett,et al.  Pseudomonas aeruginosa biofilm sensitivity to biocides: use of hydrogen peroxide as model antimicrobial agent for examining resistance mechanisms. , 1999, Methods in enzymology.

[14]  J. Govan,et al.  Pseudomonas aeruginosa and cystic fibrosis: unusual bacterial adaptation and pathogenesis. , 1986, Microbiological sciences.

[15]  H. Steinman Bacteriocuprein superoxide dismutases in pseudomonads , 1985, Journal of bacteriology.

[16]  H. Schweizer,et al.  Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa. , 1994, Gene.

[17]  E. Greenberg,et al.  Census and consensus in bacterial ecosystems: the LuxR-LuxI family of quorum-sensing transcriptional regulators. , 1996, Annual review of microbiology.

[18]  A. Kropinski,et al.  Construction of broad-host-range plasmid vectors for easy visible selection and analysis of promoters , 1990, Journal of bacteriology.

[19]  G. Diaz,et al.  A double staining method for differentiating between two classes of mycobacterial catalase in polyacrylamide electrophoresis gels. , 1986, Analytical biochemistry.

[20]  D. Ohman,et al.  Synthesis of multiple exoproducts in Pseudomonas aeruginosa is under the control of RhlR-RhlI, another set of regulators in strain PAO1 with homology to the autoinducer-responsive LuxR-LuxI family , 1995, Journal of bacteriology.

[21]  M L Howell,et al.  An operon containing fumC and sodA encoding fumarase C and manganese superoxide dismutase is controlled by the ferric uptake regulator in Pseudomonas aeruginosa: fur mutants produce elevated alginate levels , 1997, Journal of bacteriology.

[22]  R. Kolter,et al.  Sensing starvation: a homoserine lactone--dependent signaling pathway in Escherichia coli. , 1994, Science.

[23]  Michiel Kleerebezem,et al.  Quorum sensing by peptide pheromones and two‐component signal‐transduction systems in Gram‐positive bacteria , 1997, Molecular microbiology.

[24]  P. Seed,et al.  Regulation of las and rhl quorum sensing in Pseudomonas aeruginosa , 1997, Journal of bacteriology.

[25]  D. Hassett,et al.  Fumarase C activity is elevated in response to iron deprivation and in mucoid, alginate-producing Pseudomonas aeruginosa: cloning and characterization of fumC and purification of native fumC , 1997, Journal of bacteriology.

[26]  E. Greenberg,et al.  Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[27]  M. Vasil,et al.  Coordinate regulation of siderophore and exotoxin A production: molecular cloning and sequencing of the Pseudomonas aeruginosa fur gene , 1993, Journal of bacteriology.

[28]  K. Nealson,et al.  Cellular Control of the Synthesis and Activity of the Bacterial Luminescent System , 1970, Journal of bacteriology.

[29]  D. Wheeler,et al.  The Pseudomonas aeruginosaQuorum-Sensing Signal MoleculeN-(3-Oxododecanoyl)-l-Homoserine Lactone Has Immunomodulatory Activity , 1998, Infection and Immunity.

[30]  E. Vijgenboom,et al.  In vivo studies disprove an obligatory role of azurin in denitrification in Pseudomonas aeruginosa and show that azu expression is under control of rpoS and ANR. , 1997, Microbiology.

[31]  M. Akrim,et al.  Regulation of the xcp secretion pathway by multiple quorum‐sensing modulons in Pseudomonas aeruginosa , 1997, Molecular microbiology.

[32]  D. Hassett,et al.  Cloning and characterization of the katB gene of Pseudomonas aeruginosa encoding a hydrogen peroxide-inducible catalase: purification of KatB, cellular localization, and demonstration that it is essential for optimal resistance to hydrogen peroxide , 1995, Journal of bacteriology.

[33]  D. Hassett,et al.  Cloning and characterization of the Pseudomonas aeruginosa sodA and sodB genes encoding manganese- and iron-cofactored superoxide dismutase: demonstration of increased manganese superoxide dismutase activity in alginate-producing bacteria , 1993, Journal of bacteriology.

[34]  G. Salmond,et al.  Characterization of the Erwinia chrysanthemi expI–expR locus directing the synthesis of two N‐acyl‐homoserine lactone signal molecules , 1998, Molecular microbiology.

[35]  E. Greenberg,et al.  A second N-acylhomoserine lactone signal produced by Pseudomonas aeruginosa. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[36]  I. Fridovich,et al.  Effects of molecular oxygen on detection of superoxide radical with nitroblue tetrazolium and on activity stains for catalase. , 1984, Analytical biochemistry.

[37]  M. Gambello,et al.  LasR of Pseudomonas aeruginosa is a transcriptional activator of the alkaline protease gene (apr) and an enhancer of exotoxin A expression , 1993, Infection and immunity.

[38]  B. Holloway Genetics of Pseudomonas. , 1969, Bacteriological reviews.

[39]  D. Hassett,et al.  Bacterioferritin A Modulates Catalase A (KatA) Activity and Resistance to Hydrogen Peroxide in Pseudomonas aeruginosa , 1999, Journal of bacteriology.

[40]  Jeffrey H. Miller,et al.  A short course in bacterial genetics , 1992 .

[41]  S. Kjelleberg,et al.  Extracellular Signal Molecule(s) Involved in the Carbon Starvation Response of Marine Vibrio sp. Strain S14 , 1998, Journal of bacteriology.

[42]  C. Yanisch-Perron,et al.  Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. , 1985, Gene.

[43]  B. Iglewski,et al.  Functional analysis of the Pseudomonas aeruginosa autoinducer PAI , 1996, Journal of bacteriology.

[44]  D. Hassett,et al.  Effect of rpoS Mutation on the Stress Response and Expression of Virulence Factors in Pseudomonas aeruginosa , 1999, Journal of bacteriology.

[45]  B. Iglewski,et al.  Starvation Selection Restores Elastase and Rhamnolipid Production in a Pseudomonas aeruginosaQuorum-Sensing Mutant , 1998, Infection and Immunity.

[46]  G. Salmond,et al.  Integration of the quorum‐sensing system in the regulatory networks controlling virulence factor synthesis in Erwinia chrysanthemi , 1998, Molecular microbiology.

[47]  M. Gambello,et al.  Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication. , 1993, Science.

[48]  Roger S Smith,et al.  Roles of Pseudomonas aeruginosa las andrhl Quorum-Sensing Systems in Control of Twitching Motility , 1999, Journal of bacteriology.

[49]  H. Schweizer,et al.  Improved broad-host-range lac-based plasmid vectors for the isolation and characterization of protein fusions in Pseudomonas aeruginosa. , 1991, Gene.

[50]  C. D. Cox,et al.  Pseudomonas aeruginosa mutants altered in their sensitivity to the effect of iron on toxin A or elastase yields , 1982, Journal of bacteriology.

[51]  M. M. Bradford A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[52]  H. Schweizer,et al.  Two plasmids, X1918 and Z1918, for easy recovery of the xylE and lacZ reporter genes. , 1993, Gene.

[53]  G. Salmond,et al.  In vitro biosynthesis of the Pseudomonas aeruginosa quorum‐sensing signal molecule N‐butanoyl‐L‐homoserine lactone , 1998, Molecular microbiology.

[54]  K. Tanaka,et al.  A hierarchical quorum‐sensing cascade in Pseudomonas aeruginosa links the transcriptional activators LasR and RhIR (VsmR) to expression of the stationary‐phase sigma factor RpoS , 1996, Molecular microbiology.

[55]  P. Stewart,et al.  Protective Role of Catalase in Pseudomonas aeruginosa Biofilm Resistance to Hydrogen Peroxide , 1999, Applied and Environmental Microbiology.

[56]  J. Costerton,et al.  The involvement of cell-to-cell signals in the development of a bacterial biofilm. , 1998, Science.

[57]  E. Geldreich,et al.  A new medium for the enumeration and subculture of bacteria from potable water , 1985, Applied and environmental microbiology.

[58]  D. Hassett,et al.  Pseudomonas aeruginosa sodA and sodB mutants defective in manganese- and iron-cofactored superoxide dismutase activity demonstrate the importance of the iron-cofactored form in aerobic metabolism , 1995, Journal of bacteriology.

[59]  J. Reiser,et al.  Autoinducer-mediated regulation of rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[60]  N. A. Whitehead,et al.  Quorum-sensing in Gram-negative bacteria. , 2001, FEMS microbiology reviews.

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

[62]  S. E. West,et al.  Copyright © 1997, American Society for Microbiology Vfr Controls Quorum Sensing in Pseudomonas aeruginosa , 1997 .