Cloning and Expression of Three New Azotobacter vinelandii Genes Closely Related to a Previously Described Gene Family Encoding Mannuronan C-5-Epimerases

The cloning and expression of a family of five modular-type mannuronan C-5-epimerase genes from Azotobacter vinelandii (algE1 to -5) has previously been reported. The corresponding proteins catalyze the Ca²⁺-dependent polymer-level epimerization of β-d-mannuronic acid to α-l-guluronic acid (G) in the commercially important polysaccharide alginate. Here we report the identification of three additional structurally similar genes, designated algE6, algE7, and algY. All three genes were sequenced and expressed in Escherichia coli. AlgE6 introduced contiguous stretches of G residues into its substrate (G blocks), while AlgE7 acted as both an epimerase and a lyase. The epimerase activity of AlgE7 leads to formation of alginates with both single G residues and G blocks. AlgY did not display epimerase activity, but a hybrid gene in which the 5′-terminal part was exchanged with the corresponding region in algE4 expressed an active epimerase. Southern blot analysis of genomic A. vinelandii DNA, using the 5′ part of algE2 as a probe, indicated that all hybridization signals originated from algE1 to -5 or the three new genes reported here.Alginate is a linear copolymer composed of β-d-mannuronic acid (M) and its C-5 epimer, α-l-guluronic acid (G). The M and G residues are organized in blocks of consecutive M residues (M blocks), consecutive G residues (G blocks), or alternating M and G (MG blocks), and the lengths and distributions of the different block types vary among alginates isolated from brown algae or from different bacteria belonging to the genera Azotobacter and Pseudomonas (36, 37). Alginates are the most abundant polysaccharides in brown algae (comprising up to 40% of the dry matter), and their functions are to supply strength and flexibility to the algal tissues (38). The bacterium Azotobacter vinelandii produces alginate both as a vegetative state capsule and as an integrated part of a particular resting stage form (cyst) of this organism (31). The opportunistic pathogen Pseudomonas aeruginosa produces alginate as a capsule-like exopolysaccharide during infection of the lungs of cystic fibrosis patients (12, 23). Alginates from brown algae and A. vinelandii have M, G, and MG blocks (29, 36, 37), while alginates from P. aeruginosa and other Pseudomonas species do not contain G blocks (34, 36). In contrast to the alginates produced by brown algae, bacterial alginates are partially O-acetylated at O-2 and/or O-3 on mannuronic acid residues (36).The relative amount and distribution of G residues determine most of the physicochemical properties of the polymer. Alginates with G blocks can form gels by reversible cross-linking with divalent cations such as Ca²⁺, Ba²⁺, and Sr²⁺ (41), and the gelling and viscosifying properties of alginate are utilized in pharmaceutical, food, textile, and paper industries (26). In addition, alginate has a very interesting potential in a variety of biotechnological applications and in biomedicine. Alginate rich in M blocks stimulates cytokine production (27) and has a much higher antitumor activity than alginates with a high fraction of G blocks (14). G-rich alginates can be used for encapsulation of cells and enzymes (35), and Langerhans islets immobilized in alginates rich in G have been evaluated as a potential treatment for type 1 diabetes (39, 40).Both in brown algae and in alginate-producing bacteria, the polymer is first synthesized as mannuronan, and the enzyme mannuronan C-5-epimerase catalyzes the epimerization of M to G at the polymer level (7, 12, 21, 22). Ertesvåg et al. (7) have previously reported the cloning and expression of five genes encoding a family of Ca²⁺-dependent epimerases in A. vinelandii (algE1 to -5). The deduced AlgE protein sequences consist of two types of structural modules, designated A (385 amino acids each; one or two copies) and R (155 amino acids each; one to seven copies), and each R module contains four to six nine-amino-acid-long repeated sequences corresponding to putative Ca²⁺-binding motifs. The molecular masses of AlgE1 to -5 vary from 57.7 (AlgE4) to 191 kDa (AlgE3), depending on the number of A and R modules in the proteins. Four of the epimerase genes are clustered in the chromosome (algE1 to -4), while algE5 is located in another part of the A. vinelandii genome. Nuclear magnetic resonance (NMR) spectroscopy analyses demonstrate that the reaction products at least of AlgE2 and AlgE4 differ with respect to sequence distributions of M and G residues. AlgE2 leads to formation of mainly G blocks, while AlgE4 forms predominantly alginates with MG blocks.The A. vinelandii chromosome also encodes a Ca²⁺-independent mannuronan C-5-epimerase, designated AlgG (30). Sequence alignments demonstrate that algG does not belong to the algE gene family but shares 66% sequence identity to a mannuronan C-5-epimerase gene (also designated algG) from P. aeruginosa (12). The algG gene in P. aeruginosa is localized in a cluster of alg genes encoding enzymes involved in alginate biosynthesis, and sequence analysis of genomic DNA flanking algG in A. vinelandii suggests that this gene also is part of an alg gene cluster organized as in P. aeruginosa (30).Southern blot analysis of genomic A. vinelandii DNA using the 5′-terminal 800 bp in the A sequence of algE2 as the probe (A probe) demonstrated that the chromosome probably encodes more A-like sequences than are present in algE1 to -5 (7). In this report, we show that the A. vinelandii genome encodes two additional mannuronan C-5-epimerase genes, designated algE6 and algE7, and also a third highly related gene apparently not encoding an active epimerase.