MucR, a novel membrane-associated regulator of alginate biosynthesis in Pseudomonas aeruginosa

Abstract

Alginate biosynthesis by Pseudomonas aeruginosa was shown to be regulated by the intracellular second messenger bis-(3'-5')-cyclic-dimeric-GMP (c-di-GMP), and binding of c-di-GMP to the membrane protein Alg44 was required for alginate production. In this study, PA1727, a c-di-GMP-synthesizing enzyme was functionally analyzed and identified to be involved in regulation of alginate production. Deletion of the PA1727 gene in the mucoid alginate-overproducing P. aeruginosa strain PDO300 resulted in a nonmucoid phenotype and an about 38-fold decrease in alginate production; thus, this gene is designated mucR. The mucoid alginate-overproducing phenotype was restored by introducing the mucR gene into the isogenic DeltamucR mutant. Moreover, transfer of the MucR-encoding plasmid into strain PDO300 led to an about sevenfold increase in alginate production, wrinkly colony morphology, increased pellicle formation, auto-aggregation, and the formation of highly structured biofilms as well as the inhibition of swarming motility. Outer membrane protein profile analysis showed that overproduction of MucR mediates a strong reduction in the copy number of FliC (flagellin), required for flagellum-mediated motility. Translational reporter enzyme fusions with LacZ and PhoA suggested that MucR is located in the cytoplasmic membrane with a cytosolic C terminus. Deletion of the proposed C-terminal GGDEF domain abolished MucR function. MucR was purified and identified using tryptic peptide fingerprinting and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Overall, experimental evidence was provided suggesting that MucR specifically regulates alginate biosynthesis by activation of alginate production through generation of a localized c-di-GMP pool in the vicinity of Alg44.

FIG. 1.

MucR is essential for the mucoid colony morphology. Colonies of various P. aeruginosa strains were grown on PIA medium for 24 h. The top row shows the mucoid strain PDO300 and the strains derived from it; the bottom row shows the nonmucoid strain PAO1 and the plasmids derived from it. Plasmids harbored by certain strains are indicated in parentheses.

FIG. 2.

Multiple copies of mucR induce auto-aggregation and the formation of pellicles. Various strains were grown in LB for 16 h. The top row for each strain indicated on the left contains photographs of overnight cultures grown in LB overnight showing pellicle formation. The bottom row for each strain contains phase-contrast microscopic images showing the cell aggregation; the black bars represent 50 μm. Plasmids (where present) are indicated along the top.

FIG. 3.

Multiple copies of mucR lead to the formation of highly structured biofilms. (A) Phase-contrast images of 24-h-old biofilms grown in a continuous-culture flow cell. Black bar represents 200 μm. (B) Confocal laser scanning microscopy images of a 24-h-old biofilm of the strain PDO300(pBBR1MCS-5:mucR). Each frame represents a picture 10 μm away from the last. The white bar represents 150 μm; the white arrow shows the formation of bridge-like structures connecting adjacent microcolonies.

FIG. 4.

Loss of mucR does not influence swarming motility, but multiple copies of mucR inhibit swarming motility. (A) Swarming motility of the nonmucoid P. aeruginosa strain PAO1 and its isogenic mucR knockout mutant. (B) Swarming motility of the mucoid P. aeruginosa strain PDO300 and its isogenic mucR knockout mutant. (C) Swarming phenotype of a mutant derived from the mucoid PDO300 strain incapable of producing alginate due to the loss of the alg8 gene, which is essential for alginate biosynthesis.

FIG. 5.

Outer membrane protein profiles of various P. aeruginosa strains. (Left) Outer membrane protein profiles of the ΔmucR mutant derived from PDO300; (right) outer membrane protein profiles of the ΔmucR mutant derived from PAO1. The arrows indicate the identity of proteins whose levels differed between mutants. Plasmids harbored by certain strains are indicated in parentheses.

FIG. 6.

The alginate stimulating activity of MucR is dependent on the GGDEF and EAL domains containing the C terminus. (Left) Schematic representations of the various MucR fusion proteins. Plasmids harbored by the PDO300ΔmucR strain are indicated in parentheses. (Right) Alginate production levels of the PDO300ΔmucR strain harboring the genes encoding the various MucR fusion proteins. Right, CDM, cell dry mass. Alginate quantification data represent the results of three independent experiments.

FIG. 7.

Subcellular localization and purification of MucR. (A) Immunoblot analysis of whole-cell lysate (T), soluble cytosol (S), and envelope (M) fractions. (B) SDS-PAGE and immunoblot analysis of hexahistidine-tagged MucR (MucRhis) purified from the solubilized membrane fraction of the PDO300ΔmucR(pBBR1MCS-5:mucRhis) strain using Ni-NTA affinity purification. MucR was identified by tryptic peptide fingerprinting and MALDI-TOF mass spectrometry. WT, wild type.

FIG. 8.

Proposed model for the MucR-mediated regulation of alginate biosynthesis in P. aeruginosa. The membrane topology and domain structure of MucR, as predicted by SMART and Pfam, are shown. The circles represent the conserved MHYT residues suggested to be involved in binding a copper atom and sensing oxygen, NO, or CO. The hexagon represents the putative signal. The stars represent c-di-GMP. Various domains are shown. MFP, membrane fusion protein domain, similar to multidrug efflux systems.