Membrane topology and roles of Pseudomonas aeruginosa Alg8 and Alg44 in alginate polymerization

Abstract

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 bGTs 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 cdi-GMP [bis-(39,59)-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.

Cyclic di-GMP: the First 25 Years of a Universal Bacterial Second Messenger

“…However, the membrane fusion protein domain present in the periplasmic fragment of Alg44 suggests that it is involved in protein-protein interactions within the alginate translocation complex. Deletion of a short C-terminal sequence abolished alginate polymerization (157), suggesting that c-di-GMP binding to the cytoplasmic PilZ domain of Alg44 can be transferred via an inside-out signaling mechanism (67,68) to the periplasmic domain to activate alginate polymerization.…”

“…7). Alg44 is localized to the inner membrane, most likely as part of the large alginate synthase complex (149,157). The N-terminal cytoplasmic PilZ domain of Alg44 is separated from the long periplasmic C terminus by a single transmembrane domain.…”

“…Furthermore, PilZ domain proteins were found to adopt different oligomeric states (Fig. 1), from monomeric to tetrameric (82,83,87,152) (51,52,148,(154)(155)(156), polysaccharide synthesis and translocation (18,149,157), and DNA binding (120,158), are described in this review. It is clear that the PilZ domain functions as a versatile module that can regulate diverse activities in a c-di-GMP-dependent manner.…”

Revamping the role of biofilm regulating operons in device-associated Staphylococci and Pseudomonas aeruginosa

“…Post-transcriptional regulation of membrane anchoring protein Alg44 and the glycosyl transferase Alg8 together plays a vital role in polymerization leading to alginate production. [39] Attenuated total reflection/Fourier transform-infrared spectrometry (ATR/FT-IR) and scanning confocal laser microscopy (SCLM) have revealed that alginate is not a major component of P. aeruginosa biofilm and also for the interfacial adhesion or growth. [40] In the same study, in vitro mutagenesis of algD and algJ, expressed an improper alginate and an alginate without O-acetylation respectively.…”

Structural basis for alginate secretion across the bacterial outer membrane

“…Alg8, which contains multiple transmembrane domains and a large cytosolic domain that shares homology with family 2 glycosyl transferases, is the rate-limiting step in alginate production (12). In contrast, Alg44 possesses a single transmembrane domain that separates a cytoplasmic bis-(3′-5′)-cyclic-di-guanidine monophosphate (c-di-GMP) binding PilZ domain and a periplasmic domain that is predicted to resemble the membrane-fusion protein MexA from the MexAB-OprM multidrug efflux pump (10). Regulation of alginate polymerization requires the binding of the bacterial secondary messenger c-di-GMP to Alg44, thus it is speculated that the coordinated activities of Alg8 and Alg44 facilitate polymerization and export of alginate across the inner membrane (13).…”