Alginate Polymerization and Modification Are Linked in Pseudomonas aeruginosa

Abstract

The molecular mechanisms of alginate polymerization/modification/secretion by a proposed envelope-spanning multiprotein complex are unknown. Here, bacterial two-hybrid assays and pulldown experiments showed that the catalytic subunit Alg8 directly interacts with the proposed copolymerase Alg44 while embedded in the cytoplasmic membrane. Alg44 additionally interacts with the lipoprotein AlgK bridging the periplasmic space. Site-specific mutagenesis of Alg44 showed that protein-protein interactions and stability were independent of conserved amino acid residues R17 and R21, which are involved in c-di-GMP binding, the N-terminal PilZ domain, and the C-terminal 26 amino acids. Site-specific mutagenesis was employed to investigate the c-di-GMP-mediated activation of alginate polymerization by the PilZAlg44 domain and Alg8. Activation was found to be different from the proposed activation mechanism for cellulose synthesis. The interactive role of Alg8, Alg44, AlgG (epimerase), and AlgX (acetyltransferase) on alginate polymerization and modification was studied by using site-specific deletion mutants, inactive variants, and overproduction of subunits. The compositions, molecular masses, and material properties of resulting novel alginates were analyzed. The molecular mass was reduced by epimerization, while it was increased by acetylation. Interestingly, when overproduced, Alg44, AlgG, and the nonepimerizing variant AlgG(D324A) increased the degree of acetylation, while epimerization was enhanced by AlgX and its nonacetylating variant AlgX(S269A). Biofilm architecture analysis showed that acetyl groups promoted cell aggregation while nonacetylated polymannuronate alginate promoted stigmergy. Overall, this study sheds new light on the arrangement of the multiprotein complex involved in alginate production. Furthermore, the activation mechanism and the interplay between polymerization and modification of alginate were elucidated.

Discovery of a Novel Alginate Lyase from Nitratiruptor sp. SB155-2 Thriving at Deep-sea Hydrothermal Vents and Identification of the Residues Responsible for Its Heat Stability

“…Yet the candidate genes for AlgK and AlgX, which are involved in alginate polymerization, were not found. These proteins are needed to form the alginate biosynthesis machinery complex (46), whereby AlgK interacts with AlgE, Alg44, and AlgX. Furthermore, AlgX interacts with Alg44 and the specifically interacting serine protease MucD (47).…”

Biological function of a polysaccharide degrading enzyme in the periplasm

“…This precursor is polymerized by the alginate polymerase Alg8, forming poly-mannuronate (poly-M) which is transported across the inner membrane (IM)101112. This requires binding of bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP), a bacterial secondary messenger, to the PilZ domain of the alginate co-polymerase Alg44131415. After crossing the IM, poly-M is transported through the periplasm, the space between the inner and outer membranes (OM), by a membrane spanning multi-protein machine (Alg44, X, G, K)141617 to the OM protein, AlgE, for secretion181920.…”

Alginate Biosynthesis Factories in Pseudomonas fluorescens: Localization and Correlation with Alginate Production Level

“…It has been shown that the alginate biosynthesis machinery components interact with each other, span the entire periplasm, and probably form a multiprotein complex guiding the newly synthesized alginate chain to the AlgE porin (22). In the present study, we developed an immunogold labeling method to detect FLAG-tagged AlgE protein as a representative of the alginate biosynthesis factory by using TEM.…”

“…AlgK is a periplasmic lipoprotein bound to the outer membrane and is important, but not essential, for the localization of the porin AlgE. Figure 1 represents the proposed model for alginate biosynthesis machinery based on recent studies (13,21,22). The interactions between different Alg proteins, assumed to contribute to the formation of the mentioned protein scaffold, have been extensively studied by mutual stability and pulldown analyses.…”

“…Several studies have indicated that the newly synthesized polymannuronate chain needs to be translocated across the periplasm with the aid of a protein scaffold composed of Alg8, Alg44, AlgG, AlgX, AlgK, and AlgE (17)(18)(19)(20)(21)(22)(23). AlgK is a periplasmic lipoprotein bound to the outer membrane and is important, but not essential, for the localization of the porin AlgE.…”