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Alginates are polysaccharides composed of 1-4-linked beta-D-mannuronic acid and alpha-L-guluronic acid. The polymer can be degraded by alginate lyases, which cleave the polysaccharide using a beta-elimination reaction. Two such lyases have previously been identified in the soil bacterium Azotobacter vinelandii, as follows: the periplasmic AlgL and the secreted bifunctional mannuronan C-5 epimerase and alginate lyase AlgE7. In this work, we describe the properties of three new lyases from this bacterium, AlyA1, AlyA2, and AlyA3, all of which belong to the PL7 family of polysaccharide lyases. One of the enzymes, AlyA3, also contains a C-terminal module similar to those of proteins secreted by a type I secretion system, and its activity is stimulated by Ca(2+). All three enzymes preferably cleave the bond between guluronic acid and mannuronic acid, resulting in a guluronic acid residue at the new reducing end, but AlyA3 also degrades the other three possible bonds in alginate. Strains containing interrupted versions of alyA1, alyA3, and algE7 were constructed, and their phenotypes were analyzed. Genetically pure alyA2 mutants were not obtained, suggesting that this gene product may be important for the bacterium during vegetative growth. After centrifugation, cultures from the algE7 mutants form a large pellet containing alginate, indicating that AlgE7 is involved in the release of alginate from the cells. Upon encountering adverse growth conditions, A. vinelandii will form a resting stage called cyst. Alginate is a necessary part of the protective cyst coat, and we show here that strains lacking alyA3 germinate poorly compared to wild-type cells.

Alginate is a linear copolymer of beta-d-mannuronic acid and its C-5-epimer, alpha-l-guluronic acid. During biosynthesis, the polymer is first made as mannuronan, and various fractions of the monomers are then epimerized to guluronic acid by mannuronan C-5-epimerases. The Azotobacter vinelandii genome encodes a family of seven extracellular such epimerases (AlgE1 to AlgE7) which display motifs characteristic for proteins secreted via a type I pathway. Putative ATPase-binding cassette regions from the genome draft sequence of the A. vinelandii OP strain and experimentally verified type I transporters from other species were compared. This analysis led to the identification of one putative A. vinelandii type I system (eexDEF). The corresponding genes were individually disrupted in A. vinelandii strain E, and Western blot analysis using polyclonal antibodies against all AlgE epimerases showed that these proteins were present in wild-type culture supernatants but absent from the eex mutant supernatants. Consistent with this, the wild-type strain and the eex mutants produced alginate with about 20% guluronic acid and almost pure mannuronan (< or =2% guluronic acid), respectively. The A. vinelandii wild type is able to enter a particular desiccation-tolerant resting stage designated cyst. At this stage, the cells are surrounded by a rigid coat in which alginate is a major constituent. Such a coat was formed by wild-type cells in a particular growth medium but was missing in the eex mutants. These mutants were also found to be unable to survive desiccation. The reason for this is probably that continuous stretches of guluronic acid residues are needed for alginate gel formation to take place.

Alginate is an industrially widely used polysaccharide produced by brown seaweeds and as an exopolysaccharide by bacteria belonging to the genera Pseudomonas and Azotobacter. The polymer is composed of the two sugar monomers mannuronic acid and guluronic acid (G), and in all these bacteria the genes encoding 12 of the proteins essential for synthesis of the polymer are clustered in the genome. Interestingly, 1 of the 12 proteins is an alginate lyase (AlgL), which is able to degrade the polymer down to short oligouronides. The reason why this lyase is associated with the biosynthetic complex is not clear, but in this paper we show that the complete lack of AlgL activity in Pseudomonas fluorescens in the presence of high levels of alginate synthesis is toxic to the cells. This toxicity increased with the level of alginate synthesis. Furthermore, alginate synthesis became reduced in the absence of AlgL, and the polymers contained much less G residues than in the wild-type polymer. To explain these results and other data previously reported in the literature, we propose that the main biological function of AlgL is to degrade alginates that fail to become exported out of the cell and thereby become stranded in the periplasmic space. At high levels of alginate synthesis in the absence of AlgL, such stranded polymers may accumulate in the periplasm to such an extent that the integrity of the cell is lost, leading to the observed toxic effects.

Bacterial alginates are produced as 1-4-linked beta-D-mannuronan, followed by epimerization of some of the mannuronic acid residues to alpha-L-guluronic acid. Here we report the isolation of four different epimerization-defective point mutants of the periplasmic Pseudomonas fluorescens mannuronan C-5-epimerase AlgG. All mutations affected amino acids conserved among AlgG-epimerases and were clustered in a part of the enzyme also sharing some sequence similarity to a group of secreted epimerases previously reported in Azotobacter vinelandii. An algG-deletion mutant was constructed and found to produce predominantly a dimer containing a 4-deoxy-L-erythro-hex-4-enepyranosyluronate residue at the nonreducing end and a mannuronic acid residue at the reducing end. The production of this dimer is the result of the activity of an alginate lyase, AlgL, whose in vivo activity is much more limited in the presence of AlgG. A strain expressing both an epimerase-defective (point mutation) and a wild-type epimerase was constructed and shown to produce two types of alginate molecules: one class being pure mannuronan and the other having the wild-type content of guluronic acid residues. This formation of two distinct classes of polymers in a genetically pure cell line can be explained by assuming that AlgG is part of a periplasmic protein complex.

Differentiation in Azotobacter vinelandii involves the encystment of the vegetative cell under adverse environmental circumstances and the germination of the resting cell into the vegetative state when growth conditions are satisfactory again. Morphologically, the encystment process involves the development of a protective coat around the resting cell. This coat partly consists of multiple layers of alginate, which is a copolymer of beta-D-mannuronic acid (M) and alpha-L-guluronic acid (G). Alginate contributes to coat rigidity by virtue of a high content of GG blocks. Such block structures are generated through a family of mannuronan C-5 epimerases that convert M to G after polymerization. Results from immunodetection and light microscopy, using stains that distinguish between different cyst components and types, indicate a correlation between cyst coat organization and the amount and appearance of mannuronan C-5 epimerases in the extracellular medium and attached to the cells. Specific roles of individual members of the epimerase family are indicated. Calcium and magnesium ions appear to have different roles in the structural organization of the cyst coat. Also reported is a new gene sharing strong sequence homology with parts of the epimerase-encoded R-modules. This gene is located within the epimerase gene cluster of Azotobacter vinelandii.