Identification of a periplasmic AlgK-AlgX-MucD multiprotein complex in Pseudomonas aeruginosa involved in biosynthesis and regulation of alginate

The opportunistic human pathogen Pseudomonas aeruginosa produces an extracellular polysaccharide called alginate. This is especially relevant in pulmonary infection of cystic fibrosis patients where it protects the bacteria from the hosts' immune system and the diffusion of antibiotics. Here a connection between the stability of a proposed alginate polymerisation/secretion complex and the regulation of the operon encoding these proteins was assessed. Experimental evidence was provided for a periplasmic multiprotein complex composed of AlgX, AlgK, and the regulatory protein MucD. Disruption of the alginate machinery in a mucoid strain, either by removal, or over production of various essential proteins resulted in an at least 2-fold increase in transcription of a lacZ reporter under the control of the algD promoter. Instability of the complex was indicated by an increase in secretion of alginate degradation products. This increase in transcription was found to be dependent on the negative regulatory protein MucD. Surprisingly, over production of MucD leads to a 3.3-fold increase in transcription from the alginate promoter and a 1.7-fold increase in the levels of alginate produced, suggesting an additional positive regulatory role for MucD in mucoid strains. Overall, this study provided experimental evidence for the proposed periplasmic multiprotein complex and established a link of a constituent of this complex, MucD, to transcriptional regulation of alginate biosynthesis genes.