Membrane topology and roles of Pseudomonas aeruginosa Alg8 and Alg44 in alginate polymerization.
Mucoid strains of Pseudomonas aeruginosa that overproduce alginate are associated with chronic pulmonary disease (e.g. cystic fibrosis). Mutants defective in one of several periplasmic proteins (AlgKGX) for alginate secretion release alginate fragments due to the activity of an alginate lyase (AlgL) in the periplasm, which cleaves the newly formed polymers. However, mutants defective in Alg8 or Alg44 did not secrete polymer or alginate fragments, suggesting that both these membrane proteins have a role in the polymerization reaction. A model for the membrane topology of Alg8, a glycosyltransferase (GT), was constructed using PhoA fusions. This provided evidence for a large cytoplasmic loop containing the active domains predicted for beta-GTs such as Alg8 and five transmembrane (TM) domains, one of which resembles a cleavable signal peptide. The C-terminal TM domain of Alg8 was critical for the polymerization reaction in vivo. Alanine substitution mutagenesis showed that all of the predicted active site residues in the widely spaced D, DxD, D, LxxRW motif were required for polymerization activity in vivo, and two of these substitutions also affected Alg8 protein stability. A membrane topology model for Alg44 was also constructed using PhoA fusions, and this showed a central TM domain and predicted an N-terminal TM domain that may be a membrane anchor. An N-terminal PilZ domain in Alg44 for c-di-GMP [bis-(3',5')-cyclic dimeric GMP] binding, which is required for alginate synthesis, was localized to the cytoplasmic loop. The long periplasmic C terminus of Alg44 contains a region similar to membrane fusion proteins (MFPs) of multi-drug efflux systems, which predicts the possibility of its interaction with another protein in this compartment. A Western blot analysis of the outer-membrane porin AlgE showed reduced AlgE levels in the alg44 mutant, whereas expression of Alg44 in trans restored AlgE within the cell. C-terminal truncations of Alg44 as small as 24 amino acids blocked alginate polymerization in vivo, indicating a critical role for the MFP domain. These studies suggest that Alg44 may act as a co-polymerase in concert with Alg8, the major GT, and that both inner-membrane proteins are required in vivo for the polymerization reaction leading to alginate production.
McGuire Veterans Affairs Medical Center, Richmond, VA 23249, USA.|Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, Richmond, VA 23298-0678, USA.
Lashanda L
LL
Oglesby
McGuire Veterans Affairs Medical Center, Richmond, VA 23249, USA.|Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, Richmond, VA 23298-0678, USA.
Sumita
S
Jain
McGuire Veterans Affairs Medical Center, Richmond, VA 23249, USA.|Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, Richmond, VA 23298-0678, USA.
Dennis E
DE
Ohman
D000464:Alginates / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D020723:Glucuronic Acid / Q000378:metabolism; D006603:Hexuronic Acids / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D011108:Polymers / Q000378:metabolism; D018272:Porins / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
C085500:Alg44 protein, Pseudomonas aeruginosa; C085499:Alg8 protein, Pseudomonas aeruginosa; D000464:Alginates; D001426:Bacterial Proteins; D006603:Hexuronic Acids; D008565:Membrane Proteins; D011108:Polymers; D018272:Porins; D011993:Recombinant Fusion Proteins; D020723:Glucuronic Acid; D000469:Alkaline Phosphatase