Alginates from Bacteria

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120

An enzymatic in vitro alginate polymerization assay was developed by using 14 C-labeled GDP-mannuronic acid as a substrate and subcellular fractions of alginate overproducing Pseudomonas aeruginosa FRD1 as a polymerase source. The highest specific alginate polymerase activity was detected in the envelope fraction, suggesting that cytoplasmic and outer membrane proteins constitute the functional alginate polymerase complex. Accordingly, no alginate polymerase activity was detected using cytoplasmic membrane or outer membrane proteins, respectively. To determine the requirement of Alg8, which has been proposed as catalytic subunit of alginate polymerase, nonpolar isogenic alg8 knockout mutants of alginate-overproducing P. aeruginosa FRD1 and P. aeruginosa PDO300 were constructed, respectively. These mutants were deficient in alginate biosynthesis, and alginate production was restored by introducing only the alg8 gene. Surprisingly, this resulted in significant alginate overproduction of the complemented P. aeruginosa ⌬alg8 mutants compared to nonmutated strains, suggesting that Alg8 is the bottleneck in alginate biosynthesis. 1H-NMR analysis of alginate isolated from these complemented mutants showed that the degree of acetylation increased from 4.7 to 9.3% and the guluronic acid content was reduced from 38 to 19%. Protein topology prediction indicated that Alg8 is a membrane protein. Fusion protein analysis provided evidence that Alg8 is located in the cytoplasmic membrane with a periplasmic C terminus. Subcellular fractionation suggested that the highest specific PhoA activity of Alg8-PhoA is present in the cytoplasmic membrane. A structural model of Alg8 based on the structure of SpsA from Bacillus subtilis was developed.

Section: Discussionsupporting

confidence: 68%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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In Vitro Alginate Polymerization and the Functional Role of Alg8 in Alginate Production by Pseudomonas aeruginosa

Remminghorst

Rehm

2006

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120

“…The most characteristic feature of the mucoid phenotype is the secretion of large amounts of highly viscous exopolysaccharides. The copolymer alginate, which is composed of mannuronic acid and guluronic acid, appears to be the major component of the secreted polysaccharide (9,31), and besides nucleic acids and proteins, is the key factor in the development of mucoid biofilms which has been intensively studied (5,16,26,30). Biofilms generated by P. aeruginosa have been shown to enhance the resistant colonization of cystic fibrosis-diseased lungs and to confer several advantages to the respective microorganism, such as a high-level resistance to antibiotics and to the killing effect of leukocytes (1,18).…”

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confidence: 99%

Expression of the psl Operon in Pseudomonas aeruginosa PAO1 Biofilms: PslA Performs an Essential Function in Biofilm Formation

Overhage

Schemionek

Webb

et al. 2005

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The psl gene cluster, comprising 15 cotranscribed genes from Pseudomonas aeruginosa, was recently identified as being involved in exopolysaccharide biosynthesis and biofilm formation. In this study, we investigated the regulation of the psl gene cluster and the function of the first gene in this cluster, the pslA gene. PslA shows strong similarities to UDP-glucose lipid carriers. An isogenic marker-free pslA deletion mutant of P. aeruginosa PAO1 deficient in attachment and biofilm formation was used for complementation studies. The expression of only the pslA gene, comprising a coding region of 1,437 bp, restored the biofilm-forming phenotype of the wild type, indicating that PslA is required for biofilm formation by nonmucoid P. aeruginosa. The promoter region of the psl gene cluster, which encodes PslA-PslO, was identified by rapid amplification of cDNA 5 ends. Promoter assays using transcriptional fusions to lacZ and gfp indicated a constitutive expression of the psl cluster in planktonic cells and a highly regulated and localized expression in biofilms, respectively. Expression of the psl cluster in biofilms was almost exclusively found in the centers of microcolonies, as revealed by confocal laser scanning microscopy. These data suggest that constitutive expression of the psl operon enables efficient attachment to surfaces and that regulated localized psl operon expression is required for biofilm differentiation.

“…An important virulence mechanism is the formation of a mucoid biofilm. Secreted alginate is a crucial constituent of the mucoid biofilm matrix (16,17). However, alginate-negative mutants of P. aeruginosa are also able to form nonmucoid biofilms, showing an architecture different from that of biofilms formed by alginate-overproducing mucoid P. aeruginosa (13,20).…”

mentioning

confidence: 99%

PslD Is a Secreted Protein Required for Biofilm Formation by Pseudomonas aeruginosa

Campisano

Schroeder

Schemionek

et al. 2006

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The function of pslD, which is part of the psl operon from Pseudomonas aeruginosa, was investigated in this study. The psl operon is involved in exopolysaccharide biosynthesis and biofilm formation. An isogenic marker-free pslD deletion mutant of P. aeruginosa PAO1 which was deficient in the formation of differentiated biofilms was generated. Expression of only the pslD gene coding region restored the wild-type phenotype. A C-terminal, hexahistidine tag fusion enabled the identification of PslD. LacZ and PhoA translational fusions with PslD indicated that PslD is a secreted protein required for biofilm formation, presumably via its role in exopolysaccharide export.