Bacterial alginates: biosynthesis and applications

Alginate is a copolymer of β-d-mannuronic acid and α-l-guluronic acid (GulA), linked together by 1–4 linkages. The polymer is a well-established industrial product obtained commercially by harvesting brown seaweeds. Some bacteria, mostly derived from the genus Pseudomonas and belonging to the RNA superfamily I, are also capable of producing copious amounts of this polymer as an exopolysaccharide. The molecular genetics, regulation and biochemistry of alginate biosynthesis have been particularly well characterized in the opportunistic human pathogen Pseudomonas aeruginosa, although the biochemistry of the polymerization process is still poorly understood. In the last 3 years major aspects of the molecular genetics of alginate biosynthesis in Azotobacter vinelandii have also been reported. In both organisms the immediate precursor of polymerization is GDP-mannuronic acid, and the sugar residues in this compound are polymerized into mannuronan. This uniform polymer is then further modified by acetylation at positions O-2 and/or O-3 and by epimerization of some of the residues, leading to a variable content of acetyl groups and GulA residues. In contrast, seaweed alginates are not acetylated. The nature of the epimerization steps are more complex in A. vinelandii than in P. aeruginosa, while other aspects of the biochemistry and genetics of alginate biosynthesis appear to be similar. The GulA residue content and distribution strongly affect the physicochemical properties of alginates, and the epimerization process is therefore of great interest from an applied point of view. This article presents a survey of our current knowledge of the molecular genetics and biochemistry of bacterial alginate biosynthesis, as well as of the biotechnological potential of such polymers. Received: 14 March 1997 / Received revision: 7 May 1997 / Accepted: 11 May 1997     

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.     

Bacterial alginate production: an overview of its biosynthesis and potential industrial production

Alginate is a linear polysaccharide that can be used for different applications in the food and pharmaceutical industries. These polysaccharides have a chemical structure composed of subunits of (1–4)-β-d-mannuronic acid (M) and its C-5 epimer α-l-guluronic acid (G). The monomer composition and molecular weight of alginates are known to have effects on their properties. Currently, these polysaccharides are commercially extracted from seaweed but can also be produced by Azotobacter vinelandii and Pseudomonas spp. as an extracellular polymer. One strategy to produce alginates with different molecular weights and with reproducible physicochemical characteristics is through the manipulation of the culture conditions during fermentation. This mini-review provides a comparative analysis of the metabolic pathways and molecular mechanisms involved in alginate polymerization from A. vinelandii and Pseudomonas spp. Different fermentation strategies used to produce alginates at a bioreactor laboratory scale are described.     

The acetylation degree of alginates in Azotobacter vinelandii ATCC9046 is determined by dissolved oxygen and specific growth rate: studies in glucose-limited chemostat cultivations

Alginates are polysaccharides that may be used as viscosifiers and gel or film-forming agents with a great diversity of applications. The alginates produced by bacteria such as Azotobacter vinelandii are acetylated. The presence of acetyl groups in this type of alginate increases its solubility, viscosity, and swelling capability. The aim of this study was to evaluate, in glucose-limited chemostat cultivations of A. vinelandii ATCC9046, the influence of dissolved oxygen tension (DO) and specific growth rate (μ) on the degree of acetylation of alginates produced by this bacterium. In glucose-limited chemostat cultivations, the degree of alginate acetylation was evaluated under two conditions of DO (1 and 9 %) and for a range of specific growth rates (0.02–0.15 h^?1). In addition, the alginate yields and PHB production were evaluated. High DO in the culture resulted in a high degree of alginate acetylation, reaching a maximum acetylation degree of 6.88 % at 9 % DO. In contrast, the increment of μ had a negative effect on the production and acetylation of the polymer. It was found that at high DO (9 %) and low μ, there was a reduction of the respiration rate, and the PHB accumulation was negligible, suggesting that the flux of acetyl-CoA (the acetyl donor) was diverted to alginate acetylation.     

Structural investigation of the capsular polysaccharide produced by a novel Klebsiella serotype (SK1). Location of O-acetyl substituents using NMR and MS techniques

The capsular polysaccharide of Klebsiella SK1 was investigated by methylation analysis, Smith degradation, and ¹H NMR spectroscopy. The oligosaccharides (P1 and P2) obtained by bacteriophage ΦSK1 degradation of the polymer were studied by methylation analysis, and 1D- and 2D-NMR spectroscopy. The resulting data showed that the patent repeating unit is a branched pentasaccharide having a structure identical to the revised structure recently proposed for Klebsiella serotype K8 capsular polysaccharide.

The 2D-NMR data showed that one third of the glucuronic acid residues in the SK1 polymer are acetylated at O-2, O-3, or O-4. FABMS studies confirmed the presence of monoacetylated glucuronic acid residues. Thus, the relationship between the Klebsiella K8 and SK1 polymers is akin to that found for Klebsiella polysaccharides K30 and K33, which have been typed as serologically distinct yet their structures differ only in the degree of acetylation.     

Structure of a new galactomannan from the ascocarps of Cordyceps cicadae shing

A water-soluble galactomannan (C-3), [α]_D²⁰ +30°, isolated from the rod-like ascocarps of Cordyceps cicadae, was determined to be homogeneous, and the molecular weight was estimated by gel filtration to be 27,000. The polysaccharide is composed of d-mannose and d-galactose in the molar ratio of 4:3. The results of methylation analysis, Smith degradation, stepwise hydrolysis with acid, and ¹³C-n.m.r. spectroscopy indicated that the polysaccharide is of highly branched structure, and composed of α-d-(1→2)-linked and α-d-(1→6)-linked mannopyranosyl residues in the core; some of these residues are substituted at O-6 and O-2 with terminal β-d-galactofuranosyl and α-d-mannopyranosyl groups, and with short chains of β-d-(1→2)-linked d-galactofuranosyl units.     

Biochemical Properties and Substrate Specificities of a Recombinantly Produced Azotobacter vinelandii Alginate Lyase

Alginate is a polysaccharide composed of β-d-mannuronic acid (M) and α-l-guluronic acid (G). An Azotobacter vinelandii alginate lyase gene, algL, was cloned, sequenced, and expressed in Escherichia coli. The deduced molecular mass of the corresponding protein is 41.4 kDa, but a signal peptide is cleaved off, leaving a mature protein of 39 kDa. Sixty-three percent of the amino acids in this mature protein are identical to those in AlgL from Pseudomonas aeruginosa. AlgL was partially purified, and the activity was found to be optimal at a pH of 8.1 to 8.4 and at 0.35 M NaCl. Divalent cations are not necessary for activity. The pI of the enzyme is 5.1. When an alginate rich in mannuronic acid was used as the substrate, the K_m was found to be 4.6 × 10⁻⁴ M (sugar residues). AlgL was found to cleave M-M and M-G bonds but not G-M or G-G bonds. Bonds involving acetylated residues were also cleaved, but this activity may be sensitive to the extent of acetylation.

Alginate is a family of 1-4-linked copolymers of β-d-mannuronic acid (M) and α-l-guluronic acid (G). It is produced by brown algae and by some bacteria belonging to the genera Azotobacter and Pseudomonas (8, 17, 18, 31). The polymer is widely used in industry and biotechnology (36, 44), and the genetics of its biosynthesis in Pseudomonas aeruginosa has been extensively studied due to its role in the disease cystic fibrosis (33). In bacterial alginates, some of the M residues may be O-2- and/or O-3-acetylated (42). The polymer is initially synthesized as mannuronan, and the G residues are introduced at the polymer level by mannuronan C-5-epimerases (13, 22, 23). The epimerized alginates contain a mixture of blocks of consecutive G residues (G blocks), consecutive M residues (M blocks), and alternating M and G residues (MG blocks). Alginates from Pseudomonas sp. do not contain G blocks (42).Alginate lyases catalyze the depolymerization of alginates by β-elimination, generating a molecule containing 4-deoxy-l-erythro-hex-4-enepyranosyluronate at the nonreducing end. Such lyases have been found in organisms using alginate as a carbon source, in bacteriophages specific for alginate-producing organisms, and in alginate-producing bacteria (45). An alginate molecule may contain four different glycosidic bonds, M-M, G-M, M-G, or G-G, and the relative rates at which each of these bonds are cleaved vary among different lyases (36a). The lyases also differ in the extent to which they are affected by acetylation (35, 43, 46).Davidson et al. (10) described an Azotobacter vinelandii lyase which preferred M blocks as a substrate. Kennedy et al. (28) later reported the purification of periplasmic alginate lyases from A. vinelandii and from Azotobacter chroococcum which also seemed to prefer deacetylated, M-rich alginate. The activities of these enzymes were found to be optimal at pH 6.8 and 7.2, respectively, while the enzyme reported by Davidson et al. (10) was found to display optimal activity at pH 7.8.A gene, algL, encoding an alginate lyase has been cloned from P. aeruginosa (2, 41). The gene was found to be located in a cluster containing most of the genes necessary for the biosynthesis of alginate. A homologous gene cluster has recently been identified in A. vinelandii (38) and shown to encode an alginate lyase (32). In our previous report, we showed that plasmid pHE102, which contains a part of this gene cluster, contains a DNA sequence sharing homology with algL from P. aeruginosa (38). We have now subcloned, sequenced, and expressed this gene in Escherichia coli. The lyase was shown to preferentially cleave deacetylated M-M and M-G bonds, but acetylated substrates were also cleaved.     

Glycosylation by d-fructofuranose thio-orthoesters

The capsular polysaccharide from Klebsiella type K54, containing both O-formyl and O-acetyl groups, has been investigated by using the techniques of methylation analysis (by gas-liquid chromatography), periodate oxidation-Smith degradation, and both ¹H- and ¹³C-n.m.r. spectroscopy. Degradation of the native polysaccharide with a bacteriophage-induced glucosidase generated a formylated, as well as a formylated and acetylated, tetrasaccharide, whereas similar depolymerization of the deacetylated polysaccharide yielded a single tetrasaccharide; the corresponding, O-acylated octasaccharides were also isolated and characterized. These oligosaccharides, utilized in chemical and spectroscopic studies in order to determine the location of the O-acyl substituents in the repeating sequence, indicated formylation at O-4 of each lateral d-glucosyl group and acetylation at O-2 of alternate l-fucosyl residues. A new structure for the repeating unit in the polysaccharide is proposed.     

The complete structure of the trifoliin a lectin-binding capsular polysaccharide of Rhizobium trifolii 843

The complete structure of the acidic, extracellular, capsular polysaccharide of Rhizobium trifolii 843 has been elucidated by a combination of chemical, enzymic, and spectroscopic methods, confirming an earlier proposed sugar sequence and assigning the locations of the acyl substituents. The polysaccharide was depolymerized by a lyase into octasaccharide units which were uniform in carbohydrate composition and linkage. These units also contained a uniform distribution of acetyl and pyruvic acetal [O-(1-carboxyethylidene)] groups, and half of them were further acylated with d-3-hydroxybutanoyl groups. A much smaller proportion (<5%) of the oligomers was further acylated by a second d-3-hydroxy-butanoyl group. The locations of the substituents were determined chemically and by J-correlated, ¹H-n.m.r. spectroscopy, proton nuclear Overhauser effect (n.O.e.)_ measurements, doubie-resonance ¹H-n.m.r. spectroscopy, and ¹³C-n.m.r. spectroscopy. The composition and structure of the carbohydrate chain were determined by methylation analysis using g.l.c.-m.s. fast-atom-bombardment mass spectrometry, and n.m.r. studies on the reduced, deacylated oligomer. Structural studies were supplemented by n.m.r. analyses on the original polymer. The oligosaccharides were found to be branched octasaccharides with four sugar residues in each branch, and the carbohydrate sequence agreed well with that expected from earlier work. In the abbreviated sequence and structure (1a), the sugar residues are labelled “a” through “h”. The main chain (a–d) is composed of a 4-deoxy-α-l-threo-hex-4-enopyranosyluronic acid group (a) that is linked to O-4 of a 3-O-acetyl-d-glucosyluronic acid residue (b) which is β-linked to O-4 of a d-glucosyl residue (c). Residue c is β-linked to O-4 of the branching d-linked to O-4 of a d-glucosyl residue (d). The side chain consists of a substituted d-galactosyl group (h) which is β-linked to O-3 of residue 9 of a β-(1→4)-linked d-glucose trisaccharide (fragment e–f–g). The reducing end of the resulting tetrasaccharide (e–f–g–h) is β-linked to O-6 of the branching d-glucose residue (d). In the native polymer, this branching residue is α-linked to O-4 of the modified d-glucuronic acid residue (a) which is the unsaturated sugar in the oligomer. A small proportion of the O-2 atoms of the acetylated d-glucosyluronic acid residues is acetylated because of ester migration. The two terminal sugars (g and h) of the branch chain bear 4,6-O-(1-carboxyethylidene) groups. The d-galactosyl groups of half of the oligomers are acylated by d-3-hydroxybutanoyl groups at O-3. About 5% of the oligomers bear a second d-3-hydroxybutanoyl group at O-2 of the d-galactosyl group (h).     

Configuration of the acetal carbon atom of pyruvic acid acetals in some bacterial polysaccharides

The configuration at the acetal carbon atom of pyruvic acid acetals present in some extracellular bacterial polysaccharides has been investigated. Assignment of the absolute configuration was made by comparing signals in the ¹³C- and ¹H-n.m.r. spectra of the polysaccharides with those of model substances. The S-configuration was demonstrated in eight polysaccharides in which pyruvic acid is linked to O-4 and O-6 of D-glucopyranosyl or D-mannopyranosyl residues. The R-configuration was demonstrated in four polysaccharides in which pyruvic acid is linked to O-4 and O-6 of D-galactopyranosyl residues. Consequently, in each of these acetals, which form 1,3-dioxane rings, the methyl group is equatorial and the carboxyl group axial. The S-form was further demonstrated in four polysaccharides in which the pyruvic acid is linked to O-3 and O-4 of D-galactopyranosyl groups.     

Yields of alginates produced by fluorescent pseudomonads in batch culture

Summary Saprophytic and plant pathogenic fluorescent pseudomonads are possible sources of bacterial alginates to be used as substitutes for algal alginates for certain commercial applications. In this study, a total of 115 strains of fluorescentPseudomonas species (P. cichorii, P. fiuorescens, P. syringae andP. viridiflava) were tested for yields of alginates when grown in batch culture in a proprietary liquid medium (PLM). The PLM contained either fructose or glucose (both at 5%, w/v) as the primary carbon and energy source. For comparison, selected strains were also grown in a modified Vogel and Bonner medium (MVBM) containing gluconate (5%, w/v) and formulated to support maximal alginate production by the human pathogenP. aeruginosa. After five days of incubation at 24°C with shaking (250–300 r.p.m.), alginates were harvested from the culture fluids by precipitation with three volumes of isopropanol. Maximum yields of alginates, based on assays for uronic acid content of precipitable material, were 5 g L^–1 for PLM with fructose, 3 g L^–1 for PLM with glucose and 9 g L^–1 for MVBM.Reference to a brand or firm name does not constitute an endorsement by the US Department of Agriculture over others of a similar nature not mentioned.     

13C n.m.r. studies of the carbohydrate portion of glucoamylase from Aspergillus oryzae

Proton-decoupled, natural abundance ¹³C n.m.r. spectroscopy was used to investigate the carbohydrate structure and content of glucoamylase from Aspergillus oryzae. We found α-d-mannopyranose was the dominant sugar present (⋍91 residues). The Elson-Morgan assay showed that hexosamine was also present as a minor component (2.6% of the total carbohydrate). The intermannose linkages appear to be random. Integration data suggest that 41 α-d-mannopyranose residues are O-2 and O-3 glycosylated and 17 α-d-mannopyranose residues are involved in O-4 glycosylation. Treatment of glucoamylase with α-mannosidase appeared to remove all the carbohydrate residues present.     

Characterization of Three New Azotobacter vinelandii Alginate Lyases,One of Which Is Involved in Cyst Germination

Alginates are polysaccharides composed of 1-4-linked β-d-mannuronic acid and α-l-guluronic acid. The polymer can be degraded by alginate lyases, which cleave the polysaccharide using a β-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 Ca²⁺. 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.Azotobacter vinelandii is a nitrogen-fixing bacterium found in soil. A. vinelandii and several species belonging to the related genus Pseudomonas have been found to produce the polymer alginate. This linear, extracellular polysaccharide is composed of 1-4-linked β-d-mannuronic acid (M) and its C-5 epimer α-l-guluronic acid (G) (35), and the relative amount and distribution of these two residues vary according to the species and growth conditions. Some of the M residues in bacterial alginates may be O acetylated at C-2, C-3, or both C-2 and C-3 (34).Alginate is first synthesized as mannuronan, and the G residues are introduced by mannuronan C-5 epimerases. All genome-sequenced alginate-producing bacteria have been found to encode a periplasmic epimerase, AlgG, that epimerizes some of the M residues in the polymer into G residues (40). AlgG seems to be unable to epimerize an M residue next to a preexisting G residue in vivo. A. vinelandii also encodes a family of secreted mannuronan C-5 epimerases (AlgE1-7) (40), some of which are able to form stretches of consecutive G residues (G blocks). Alginates containing G blocks can be cross-linked by divalent cations and thereby form gels (35).Polysaccharide lyases (EC 4.2.2.-) are a group of enzymes which cleave the polymer chains via a β-elimination mechanism, resulting in the formation of a double bond at the newly formed nonreducing end. For alginate lyases, 4-deoxy-l-erythro-hex-4-enepyranosyluronate (denoted as Δ) is formed at the nonreducing end. Several such lyases have been purified from both alginate-producing and alginate-degrading organisms, as reviewed by Wong et al. (42). When they are classified according to primary structure, the alginate lyases belong to the polysaccharide-degrading enzyme families PL5, PL6, PL7, PL14, PL17, and PL18 (http://www.cazy.org). Alginate molecules may contain four different bonds (M-M, M-G, G-M, and G-G), and alginate lyases may therefore be classified according to their preferred substrate specificities. It is now possible to obtain pure mannuronan and nearly pure (MG)_n and G blocks (17, 19, 20), and this allows for an improved assessment of the substrate specificities of the alginate lyases.The following two alginate lyases have been characterized in A. vinelandii: the periplasmic AlgL that belongs to the PL5 family (15) and the extracellular bifunctional mannuronan C-5 epimerase and alginate lyase AlgE7 (36, 37). AlgL is encoded by the alginate biosynthesis operon, similar to what has been found in all characterized alginate-producing bacteria. This enzyme cleaves M-M and M-G bonds (15), while AlgE7 preferably degrades G-MM and G-GM bonds (37). The latter enzyme is also able to introduce G residues in the alginate, thus creating the preferred substrate for the lyase.When A. vinelandii experiences a lack of nutrients, it will develop into a dormant cell designated cyst (30). The cell is then protected against desiccation by a multilayered coat, of which gel-forming alginate is a necessary part. Resuspension of cysts in a medium containing glucose leads to a germination process in which vegetative cells eventually escape from the cyst coat. It has been proposed that an alginate lyase may be involved in the rupture of the coat (43). AlgL is dispensable for germination (38), while the biological function of AlgE7 is unknown. In this report, we use the available draft genome sequence of A. vinelandii to identify three additional putative lyases and evaluate their and AlgE7''s role in growth, encystment, and germination of the bacterium.     

Defining the <Emphasis Type="Italic">Pseudomonas</Emphasis> Genus: Where Do We Draw the Line with <Emphasis Type="Italic">Azotobacter</Emphasis>?

The genus Pseudomonas has gone through many taxonomic revisions over the past 100 years, going from a very large and diverse group of bacteria to a smaller, more refined and ordered list having specific properties. The relationship of the Pseudomonas genus to Azotobacter vinelandii is examined using three genomic sequence-based methods. First, using 16S rRNA trees, it is shown that A. vinelandii groups within the Pseudomonas close to Pseudomonas aeruginosa. Genomes from other related organisms (Acinetobacter, Psychrobacter, and Cellvibrio) are outside the Pseudomonas cluster. Second, pan genome family trees based on conserved gene families also show A. vinelandii to be more closely related to Pseudomonas than other related organisms. Third, exhaustive BLAST comparisons demonstrate that the fraction of shared genes between A. vinelandii and Pseudomonas genomes is similar to that of Pseudomonas species with each other. The results of these different methods point to a high similarity between A. vinelandii and the Pseudomonas genus, suggesting that Azotobacter might actually be a Pseudomonas.     

Methyl derivatives of 2-acetamido-2-deoxy-3-O-(β-d-glucopyranosyluronic acid)-d-glucose (hyalobiouronic acid) from methylated hyaluronic acid

Methanolysis of methylated hyaluronic acid, followed by acetylation, gave, in 70% yield, crystalline methyl 2-acetamido-2-deoxy-4,6-di-O-methyl-3-O-(methyl 4-O-acetyl-2,3-di-O-methyl-β-d-glucopyranosyluronate)-α-d-glucopyranoside. Removal of the O-acetyl and methyl ester groups gave compounds that are useful in the investigation, by ¹H-n.m.r. spectroscopy, of interaction within chains of hyaluronic acid in solution.     

Pseudomonas gallaeciensis sp. nov., isolated from crude-oil-contaminated intertidal sand samples after the Prestige oil spill

Strains V113^T, V92 and V120 have been isolated from sand samples taken at the Atlantic intertidal shore in Galicia, Spain, after the Prestige oil spill. A preliminary analysis of the 16S rRNA and the partial rpoD gene sequences indicated that these strains belonged to the Pseudomonas genus, but they were distinct from any known Pseudomonas species. They were extensively characterized by a polyphasic taxonomic approach and phylogenetic data that confirmed that these strains belonged to the Pseudomonas pertucinogena group. Phylogenetic analysis of 16S rRNA, gyrB and rpoD gene sequences showed that the three strains were 99% similar and were closely related to members of the P. pertucinogena group, with less than 94% similarity to strains of established species; Pseudomonas pachastrellae was the closest relative. The Average Nucleotide Index based on blast values was 89.0% between V113^T and the P. pachastrellae type strain, below the accepted species level (95%). The predominant cellular fatty acid contents and whole cell protein profiles determined by MALDI-TOF mass spectrometry also differentiated the studied strains from known Pseudomonas species. We therefore conclude that strains V113^T, V92 and V120 represent a novel species of Pseudomonas, for which the name Pseudomonas gallaeciensis is proposed; the type strain is V113^T (= CCUG 67583^T = LMG 29038^T).     

Pseudomonas protegens sp. nov., widespread plant-protecting bacteria producing the biocontrol compounds 2,4-diacetylphloroglucinol and pyoluteorin

Fluorescent Pseudomonas strains producing the antimicrobial secondary metabolite 2,4-diacetylphloroglucinol (Phl) play a prominent role in the biocontrol of plant diseases. A subset of Phl-producing fluorescent Pseudomonas strains, which can additionally synthesize the antimicrobial compound pyoluteorin (Plt), appears to cluster separately from other fluorescent Pseudomonas spp. based on 16S rRNA gene analysis and shares at most 98.4% 16S rRNA gene sequence identity with any other Pseudomonas species. In this study, a polyphasic approach based on molecular and phenotypic methods was used to clarify the taxonomy of representative Phl⁺ Plt⁺ strains isolated from tobacco, cotton or wheat on different continents. Phl⁺ Plt⁺ strains clustered separately from their nearest phylogenetic neighbors (i.e. species from the ‘P. syringae’, ‘P. fluorescens’ and ‘P. chlororaphis’ species complexes) based on rpoB, rpoD or gyrB phylogenies. DNA-DNA hybridization experiments clarified that Phl⁺ Plt⁺ strains formed a tight genomospecies that was distinct from P. syringae, P. fluorescens, or P. chlororaphis type strains. Within Phl⁺ strains, the Phl⁺ Plt⁺ strains were differentiated from other biocontrol fluorescent Pseudomonas strains that produced Phl but not Plt, based on phenotypic and molecular data. Discriminative phenotypic characters were also identified by numerical taxonomic analysis and siderotyping. Altogether, this polyphasic approach supported the conclusion that Phl⁺ Plt⁺ fluorescent Pseudomonas strains belonged to a novel species for which the name Pseudomonas protegens is proposed, with CHA0^T (=CFBP 6595^T, =DSM 19095^T) as the type strain.     

Structural studies of an extracellular polysaccharide (S-198) elaborated by Alcaligenes ATCC 31853

The structure of an extracellular polysaccharide, S-198, elaborated by Alcaligenes ATCC 31853 has been investigated; methylation analysis, specific degradations, and ¹H-n.m.r. spectroscopy were the main methods used. It is suggested that the polysaccharide is composed of “repeating units” with the structure

A sugar residue in the chain may be either L-rhamnose or L-mannose and only ≈50% of the residues contain the branching α-L-rhamnopyranosyl group. The polysaccharide further contains O-acryl groups. It belongs to a group of polysaccharides, elaborated by Alcaligenes and Pseudomonas species, which all have the same linear backbone (except that some of them do not contain L-mannose) without branching or with branches that differ in their chemical structures and/or positions.     

Effect of nutrient amendments and sterilization on mineralization and/or biodegradation of 14C-labeled MCPP by soil bacteria under aerobic conditions

Mineralization and/or degradation of the phenoxy herbicide mecoprop (MCPP) by a group of soil bacteria under the effects of nutrient amendments and sterilization were investigated. Five different species of Pseudomonas (P. paucimobilis, P. aeruginosa, P. mallei, P. pseudomallei, and P. pickettii) were isolated from sediments of Lake Mariut, a freshwater lake in south Alexandria, Egypt. MCPP mineralization and/or removal were tested by the selected Pseudomonas species as active and dead masses in minimal and nutrient-rich media supplemented with ¹⁴C-MCPP at a final concentration of 10 μg l⁻¹ for 6 successive weeks. Results revealed significant variations in the removal percentages of MCPP by either mineralization or biodegradation. Pseudomonas spp. exhibited high selectivity toward MCPP. Considering the short duration of the experiment (45 days) Pseudomonas spp. investigated in this study provide an effective and selective potential for MCPP decontamination. As a general trend, all of the investigated species exhibited higher biodegradation and removal efficiency of MCPP (1.3–89.5%) compared to their mineralization abilities (0.10–9.28%) under the experimental conditions. Also the highest MCPP mineralization and degradation by the selected Pseudomonas spp. were achieved by their inactive (dead) followed by active-rich cultures (both were inoculated in nutrient-rich medium), confirming the positive effects of nutrient amendments and sterilization on MCPP decontamination. Efficiency of Pseudomonas spp. was positively correlated with time up to the 3rd week for biodegradation and up to the 6th week for mineralization, indicating high mineralization efficiency provided enough time. Finally, Pseudomonas spp. showed selective preferences among them toward MCPP with the highest mineralization efficiency achieved by P. aeruginosa (1SB) and P. mallei (2SA), while the highest biodegradation efficiency was achieved by P. pickettii (5SB) and P. pseudomallei (3S). They seemed very promising but require longer exposure and higher MCPP concentration to stimulate and enhance their metabolic and mineralization capabilities. Results of this study can be manipulated efficiently to select the most promising Pseudomonas species for decontaminating polluted systems providing the optimum degradation conditions.     

Phenotypic and molecular characterisation of efficient nitrogen-fixing Azotobacter strains from rice fields for crop improvement

Biological nitrogen fixation (BNF) is highly effective in the field and potentially useful to reduce adverse effects chemical fertilisers. Here, Azotobacter species were selected via phenotypic, biochemical and molecular characterisations from different rice fields. Acetylene reduction assay of Azotobacter spp. showed that Azotobacter vinelandii (Az3) fixed higher amount of nitrogen (121.09 nmol C₂H₄?mg^-1 bacteria h^-1). Likewise, its plant growth functions, viz. siderophore, hydrogen cyanide, salicylic acid, IAA, GA₃, zeatin, NH₃, phosphorus solubilisation, ACC deaminase and iron tolerance, were also higher. The profile of gDNA, plasmid DNA and cellular protein profile depicted inter-generic and inter-specific diversity among the isolates of A. vinelandii. The PCR-amplified genes nifH, nifD and nifK of 0.87, 1.4 and 1.5 kb , respectively, were ascertained by Southern blot hybridisation in isolates of A. vinelandii. The 16S rRNA sequence from A. vinelandii (Az3) was novel, and its accession number (JQ796077) was received from NCBI data base. Biofertiliser formulation of novel A. vinelandii isolates along with commercial one was evaluated in rice (Oriza sativa L. var. Khandagiri) fields. The present finding revealed that treatment T4 (Az3) (A. vinelandii) are highly efficient to improved growth and yield of rice crop.