The second messenger bis-(3'-5')-cyclic-GMP and its PilZ domain-containing receptor Alg44 are required for alginate biosynthesis in Pseudomonas aeruginosa.
The ubiquitous bacterial second messenger c-di-GMP regulates the expression of various virulence determinants in a wide range of bacterial pathogens. Several studies have suggested that proteins with a PilZ domain function as c-di-GMP receptors. We have identified in the Pseudomonas aeruginosa genome eight genes encoding for PilZ orhologues and demonstrated binding of c-di-GMP to all but one of these proteins in a direct ligand binding assay. One protein with the PilZ domain, Alg44, is involved in biosynthesis of the extracellular polysaccharide alginate. We have shown that increasing c-di-GMP levels by overexpression of highly active diguanylate cyclases, or hydrolysis of c-di-GMP by phosphodiesterases, enhanced or reduced formation of alginate in mucoid strains, respectively. We have engineered substitutions in several conserved residues of the PilZ domain of Alg44 determined that they resulted in simultaneous loss of c-di-GMP binding and the ability to support production of alginate in P. aeruginosa. A 6xHis-tagged Alg44 fusion was also shown to localize in the membrane fraction of P. aeruginosa independently from its ability to bind c-di-GMP. Alg44 appears to be an essential component of the alginate biosynthetic apparatus, where, following binding of c-di-GMP, it controls polymerization or transport of the polysaccharide.
Merighi
Massimo
M
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA.
Lee
Vincent T
VT
Hyodo
Mamoru
M
Hayakawa
Yoshihiro
Y
Lory
Stephen
S
D000464:Alginates; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D017124:Conserved Sequence; D003432:Cross-Linking Reagents / Q000494:pharmacology; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism; D017353:Gene Deletion; D016680:Genome, Bacterial; D020723:Glucuronic Acid / Q000096:biosynthesis; D006603:Hexuronic Acids; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010641:Phenotype; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D011550:Pseudomonas aeruginosa / Q000166:cytology / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015290:Second Messenger Systems; D013347:Subcellular Fractions / Q000378:metabolism; D013379:Substrate Specificity / Q000187:drug effects
C085500:Alg44 protein, Pseudomonas aeruginosa; D000464:Alginates; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D006603:Hexuronic Acids; D008565:Membrane Proteins; C062025:bis(3',5')-cyclic diguanylic acid; D020723:Glucuronic Acid; D010727:Phosphoric Diester Hydrolases; D006152:Cyclic GMP