The exopolysaccharide, alginate, produced by the opportunistic human pathogen 28 Pseudomonas aeruginosa represents a survival advantage by contributing to formation of 29 characteristic biofilms during infection. Membrane anchored proteins Alg8 (catalytic subunit) 30 and Alg44 (co-polymerase) constitute the alginate polymerase which is being activated by the 31 second messenger molecule c-di-GMP, but the mechanism of activation remains elusive. To 32 shed light on the c-di-GMP mediated activation of alginate polymerization in vivo, an in 33 silico structural model of Alg8 fused to the c-di-GMP binding PilZ domain informed by the 34 structure of cellulose synthase, BcsA, was developed. This structural model was ... More
The exopolysaccharide, alginate, produced by the opportunistic human pathogen 28 Pseudomonas aeruginosa represents a survival advantage by contributing to formation of 29 characteristic biofilms during infection. Membrane anchored proteins Alg8 (catalytic subunit) 30 and Alg44 (co-polymerase) constitute the alginate polymerase which is being activated by the 31 second messenger molecule c-di-GMP, but the mechanism of activation remains elusive. To 32 shed light on the c-di-GMP mediated activation of alginate polymerization in vivo, an in 33 silico structural model of Alg8 fused to the c-di-GMP binding PilZ domain informed by the 34 structure of cellulose synthase, BcsA, was developed. This structural model was probed by 35 site-specific mutagenesis and different cellular levels of c-di-GMP. Results suggested that c- 36 di-GMP-mediated activation of alginate polymerization involves amino acids residing at two 37 loops including H323 (loop A), T457 and E460 (loop B) surrounding the catalytic site in the 38 predicted model. Activity of respective Alg8 variants suggested that c-di-GMP-mediated 39 control of substrate access to the catalytic site of Alg8 is dissimilar to the known activation 40 mechanism of BcsA. Alg8 variants responded differently to various c-di-GMP levels while 41 MucR imparted c-di-GMP for activation of alginate polymerase. Furthermore, we showed 42 that Alg44 co-polymerase constituted a stable dimer, with its periplasmic domains required 43 for protein localization, alginate polymerization and modification. Superfolder GFP fusions 44 of Alg8 and Alg44 showed a non-uniform, punctuate and patchy arrangement of both 45 proteins surrounding the cell. Overall, this study provides insights into the c-di-GMP 46 mediated activation of alginate polymerization while assigning functional roles to Alg8 and 47 Alg44 including their subcellular localization and distribution
