Previously Uncharacterised Aliphatic Amino Acid Positions Modulate the Apparent Catalytic Activity of the EAL Domain of ZMO_1055 and Other Cyclic Di-GMP-Specific EAL Phosphodiesterases.

The nearly ubiquitous second messenger cyclic di-GMP signalling system decides about the bacterial lifestyle transition between sessility and motility. GGDEF diguanylate cyclase and EAL phosphodiesterase domains conventionally conduct the turnover of the signalling molecule being subject to micro- and macroevolution. While highly conserved signature amino acids involved in divalent cation binding and catalysis have readily been identified, recognition of single amino acid substitutions that modulate the catalytic activity has been rare. Associated with development towards cellulose-mediated self-flocculation in Zymomonas mobilis ZM401, the A526V substitution and substitutions with amino acids with longer aliphatic side chains gradually revert the apparent catalytic activity of the EAL domain in the PAS-GGDEF-EAL ZMO1055 phosphodiesterase as monitored by flocculation, biofilm and motility assays. Remarkably, the effect of the A526V substitution equivalent is observed among other investigated GGDEF-EAL proteins. Furthermore, substitutions of aliphatic side chain amino acids at distinct alternative positions affect ZOM1055 activity, while the M525L substitution has a context-dependent effect. Thus, single amino acid substitutions outside of signature amino acid positions can even revert the target output and thus significantly contribute to the flexibility and adaptability of the cyclic di-GMP signalling network. At a phylogenetic scale, ZMO1055 homologues seem to be a current evolutionary target.