MucR, a novel membrane-associated regulator of alginate biosynthesis in Pseudomonas aeruginosa.
Alginate biosynthesis by Pseudomonas aeruginosa was shown to be regulated by the intracellular second messenger bis-(3'-5')-cyclic-dimeric-GMP (c-di-GMP), and binding of c-di-GMP to the membrane protein Alg44 was required for alginate production. In this study, PA1727, a c-di-GMP-synthesizing enzyme was functionally analyzed and identified to be involved in regulation of alginate production. Deletion of the PA1727 gene in the mucoid alginate-overproducing P. aeruginosa strain PDO300 resulted in a nonmucoid phenotype and an about 38-fold decrease in alginate production; thus, this gene is designated mucR. The mucoid alginate-overproducing phenotype was restored by introducing the mucR gene into the isogenic DeltamucR mutant. Moreover, transfer of the MucR-encoding plasmid into strain PDO300 led to an about sevenfold increase in alginate production, wrinkly colony morphology, increased pellicle formation, auto-aggregation, and the formation of highly structured biofilms as well as the inhibition of swarming motility. Outer membrane protein profile analysis showed that overproduction of MucR mediates a strong reduction in the copy number of FliC (flagellin), required for flagellum-mediated motility. Translational reporter enzyme fusions with LacZ and PhoA suggested that MucR is located in the cytoplasmic membrane with a cytosolic C terminus. Deletion of the proposed C-terminal GGDEF domain abolished MucR function. MucR was purified and identified using tryptic peptide fingerprinting and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Overall, experimental evidence was provided suggesting that MucR specifically regulates alginate biosynthesis by activation of alginate production through generation of a localized c-di-GMP pool in the vicinity of Alg44.
Hay
Iain D
ID
Institute of Molecular Biosciences, Massey University, Private Bag 11222, Palmerston North, New Zealand.
Remminghorst
Uwe
U
Rehm
Bernd H A
BH
D000464:Alginates; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D002462:Cell Membrane / Q000737:chemistry; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D020723:Glucuronic Acid / Q000096:biosynthesis; D006603:Hexuronic Acids; D008124:Locomotion; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D000464:Alginates; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D006603:Hexuronic Acids; D011993:Recombinant Fusion Proteins; C062025:bis(3',5')-cyclic diguanylic acid; D020723:Glucuronic Acid; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase; D006152:Cyclic GMP