Bacterial Alginate Research Articles

Bacterial alginate biosynthesis--recent progress and future prospects.

The extracellular polysaccharide alginate has been widely associated with chronic Pseudomonas aeruginosa infections in the cystic fibrosis lung. However, it is clear that alginate biosynthesis is a more widespread phenomenon. Alginate plays a key role as a virulence factor of plant-pathogenic pseudomonads, in the formation of biofilms and with the encystment process of Azotobacter spp.

Rheology of alginate from Azotobacter vinelandii in aqueous dispersions

The rheological behaviour of a range of alginate-like polymers produced by Azotobacter vinelandii DSM 576 was determined at different alginate concentrations ( y), temperatures ( T) and shear rates (γ) in the ranges 0.3–1.5% w/v, 278.16–308.16 K, and 1.1–1400s −1, respectively. All aqueous dispersions exhibited the typical pseudoplastic behaviour, that was described by the well known Ostwäld-de Waele model. The consistency coefficient ( K ow ) was found to be a complex function of T, y, and number-average molecular mass ( M n ) of the biopolymer used. The flow behaviour index ( n) showed a hyperbolic reduction with y, thus tending to a limiting value progressively deviating from unity as M n increased from 40 to 193 kDalton. By maximising the least squares regression coefficient ( r 2) up to 0.99, it was possible to reconstruct the experimental apparent viscosities ( μ a ) of aqueous dispersions containing either bacterial or algal alginates with a mean standard error of ca. 15%, thus resulting in straightforward estimation of the thickening capability of any alginate, whatever is its algal or bacterial origin based only upon its number-average molecular mass.

The separation of alginate biosynthesis and acetylation in Pseudomonas syringae

Seaweed alginate was acetylated by resting cells of Pseudomonas syringae subsp. phaseolicola ATCC 19304. Physiological studies on this strain and its UV-induced mutants showed no correlation between bacterial alginate biosynthesis and acetylation. Specific yields of alginate and degree of acetylation in these polymers varied with strain and culture medium. This was indirect evidence that alginate biosynthesis is separate from polysaccharide acetylation. It indicated that the enzyme system involved in alginate biosynthesis was not directly linked to alginate acetylation and explained why microbial acetylation of seaweed alginates was possible.

Bacterial alginates: biosynthesis and applications.

Alginate is a copolymer of beta-D-mannuronic acid and alpha-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.

Role of acetylation on metal induced precipitation of alginates

Acetylation dramatically effects both the solution properties and the metal induced precipitation of alginates. The presence of acetyl groups on both bacterial and seaweed alginate polymers marginally increased the weight average molecular weight ( M w ) of each polymer by 7% and 11%, respectively. Acetylated bacterial alginate showed a significant increase in solution viscosity compared to its deacetylated counterpart. However microbial acetylation of seaweed alginate did not change its solution viscosity. Acetylation altered the calcium induced precipitation of both alginates. The presence of acetyl groups decreased the ability of each polymer to bind with calcium but increased their ability to bind with ferric Ion (Fe 3+). By controlling the degree of acetylation on the alginate chains, it was possible to modify solution viscosity and cation induced precipitation of these polymers.

Distribution of DNA and alginate in purulent cystic fibrosis sputum: implications to pulmonary targeting strategies.

Cystic fibrosis (CF) patients frequently experience recurring airway infections characterized by thick, viscous sputum. The consistency and nature of these purulent secretions may produce a significant barrier to the successful delivery of drugs and gene therapy vectors designed to treat CF. We have carried out a series of in vitro studies to determine the distribution of two macromolecular components typically present in purulent sputum, bacterial alginate and neutrophil-derived DNA. Sputum samples were obtained from hospitalized CF patients. DNA and alginate were disrupted, respectively, by the in vitro additions of human recombinant deoxyribonuclease I (rhDNase) or alginate lyase prepared from a mucoid strain of Pseudomonas aeruginosa. N-acetyl-L-cysteine (acetylcysteine) was similarly used to collapse the mucin matrix of these samples for comparison. Using a centrifugation-based rheological method known as the compaction assay, a greater maximal response was observed for rhDNase compared to alginate lyase treatment. A simultaneous addition of these enzymes to purulent sputum produced an additive compaction response. Electron microscopy was used to identify alginate and DNA components within the mucin matrix of sputa and to evaluate changes following treatment with high concentrations of alginate lyase or rhDNase. DNA was more widely distributed throughout purulent samples than alginate. Differences in the distribution of DNA and alginate may explain, at least in part, the larger compaction response to rhDNase versus alginate lyase treatment. An improved understanding of DNA and alginate distribution within purulent CF sputum may lead to improvements in drug and vector delivery to airway epithelial cells.

Yields of alginates produced by fluorescent pseudomonads in batch culture

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.

Addition of a Bacterial Alginate Lyase to Purulent CF Sputum In Vitro Can Result in the Disruption of Alginate and Modification of Sputum Viscoelasticity

Summary: Alginate is a large molecular weight exopolysaccharide present in the purulent airway secretions of cystic fibrosis (CF) patients. This polymer, produced by some of the opportunistic pathogens associated with the recurrent lung infections characteristic of CF, has been suggested to effect an increase in the viscoelastic properties of purulent CF airway secretions. We have investigated the use of an enzyme targeted at this exopolysaccharide, an alginate lyase obtained from a bacterial source, to disrupt its polymeric nature and effect a change in the rheological properties of CF sputum in vitro. Expectorated sputum samples obtained from hospitalized CF patients were found to contain 80-200 μg alginate per ml sputum with no measurable endogenous alginate lyase activity. Treatment with exogenous alginate lyase prepared from a mucoid strain of Pseudomonas aeruginosa resulted in the disruption of alginate and a decrease in sputum viscoelasticity in a small percentage of the samples tested. Similar treatment of these samples with recombinant human deoxyribonuclease I to cleave DNA present in purulent sputum and the use of alginate extracted from sputum as an alginate lyase assay substrate suggested that the inability of the exogenous alginate lyase to disrupt sputum alginate was not due to substrate inaccessibility or an unresponsive substrate. Concentrations of Ca 2+ and Zn 2+ in alginate lyase-resistant sputum samples, determined by metal ion analysis, were found to inhibit enzyme activity in studies using seaweed alginate as a substrate. High concentrations of Ca 2+ and Zn 2+ in sputum samples initially resistant to lyase activity could be reduced significantly in some samples by dialysis and these same samples acquired sensitivity to the lyase. Other sputum samples did not show reduced concentrations of Ca 2+ and Zn 2+ following dialysis and these samples remained lyase-insensitive. Together, these results suggest that bacterial alginate present within purulent CF sputum may be quite stable, that endogenous alginate lyase activities appear to be limited and that the in vitro addition of exogenous alginate lyase can lead to the disruption of alginate and a change in the viscoelastic properties of some purulent CF sputum samples.

Chemical analysis of alginates from mucoid strains of Pseudomonas aeruginosa.

Mucoid or alginate-producing strains of Pseudomonas aeruginosa are the major pathogens in chronic pulmonary infections in patients with cystic fibrosis (CF), in which alginate is considered a major virulence factor. Bacterial alginates are linear exopolysaccharides comprising (1 +4)-linked p-D-mannuronate (M) and its C5-epimer a-L-guluronate (G), in which mannuronate residues may be either monoor di-0-acetylated [l] . Alginate biosynthetic regulation is complex and involves environmentally responsive signal transduction systems, histone-like elements [2] and the possible metabolic regulation of key enzymic activities [3]. Expression of the mucoid phenotype involves three chromosomal loci: biosynthetic genes are located at 341nin on the P. aeruginosu genome, regulatory genes at 91Nn. and the controlling rnuc or master switch region at 70min [4]. Laboratory-derived strains containing rnuc mutations allowed the isolation of stable mucoid variants [5] that were designated PA0568 (rnuc2). PA0578 (muc22) and PA0579 (muc23) respectively. These exhibit differing medium-dependent expression of the mucoid phenotype when grown in virro on various agar-based media [5,6]. This medium-dependent expression of mucoidy is also exhibited by a variety of wildtype CF isolates and therefore facilitates their classification into muc-like groups. In this study 'H-nmr has been used to compare the structural composition of alginates produced both by rnuc strains and CF isolates. Experimental procedures have been described previously

Detection of Alginate Lyase by Activity Staining after Sodium Dodecil Sulfate-Polyacrylamide Gel Electrophoresis and Subsequent Renaturation

A slab gel electrophoretic method for the study of bacterial alginate lyase has been developed. By incorporating alginate in acrylamide gels, the method is based on renaturation of the enzyme after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, subsequent staining with cetylpyridinium chloride, and quantification of the spot by densitometric scanning. The molecular mass of alginate lyase can be determined from its position in the gel.

Structure-function relationships in microbial exopolysaccharides

Sufficient well-characterized microbial exopolysaccharides are now available to permit extensive studies on the relationship between their chemical structure and their physical attributes. This is seen even in homopolysaccharides with relatively simple structures but is more marked when greater differences in structure exist, as are found in several heteropolysaccharides. The specific and sometimes unique properties have, in the case of several of these polymers, provided a range of commercial applications. The existence of “families” of structurally related polysaccharides also indicates the specific role played by certain structures and substituents; the characteristics of several of these microbial polysaccharide families will be discussed here. Thus, microbial exopolysaccharides frequently carry acyl groups which may profoundly affect their interactive properties although these groups often have relatively little effect on solution viscosity. Xanthan with or without acylation shows marked differences in synergistic gelling with plant gluco- and galacto-mannans, although the polysaccharides with different acylation patterns show similar viscosity. Similarly “gelrite” from the bacterium originally designated as Auromonas ( Pseudomonas) elodea is of greater potential value after deacetylation, when it provides a valuable gelling agent, than it is as a viscosifier in the natural acylated form. The Klebsiella type 54 polysaccharide only forms gels when it, too, has been chemically deacetylated to give a structure equivalent to the Enterobacter XM6 polymer. Both these polysaccharides form gels due to the enhanced interaction with cations following deacylation and to the conformation adopted after removal of the acyl groups. Recent work in our laboratory suggests that deacetylation of certain bacterial alginates also significantly increases ion binding by these polysaccharides, making them more similar in their properties to algal alginates even although the alginates from some Pseudomonas species lack poly- L-guluronic acid sequences. The existence within families of polysaccharides of types in which monosaccharides are altered within a specific structure, or with varying side-chains, also gives an indication of the way in which such substituents affect the physical properties of the polymers in aqueous solution.

Crystallization and preliminary X-ray diffraction studies of bacterial alginate lyase

Alginate lyase (EC 4.2.2.3) from a soil bacterium ( Flavobacterium sp.), which was overexpressed in Bacillus subtilis, has been crystallized by the vapor-diffusion method. Tetragonal crystals were obtained using ammonium sulfate as a precipitant. The crystal was resistant to X-ray radiation damage and diffracted to about 2.2 Å resolution. The diffraction pattern indicated that the crystal belongs to the orthorhombic system, space group P2 12 12 1 with unit-cell dimensions of a=82.7, b=142.1 and c=80.9 A ̊ . It is supposed that the asymmetric unit consists of two alginate lyase molecules.

On the self-processing of bacterial alginate lyase

All of the alginate lyase species (A1-I, A1-II and A1-III) in a bacterium (strain A1) isolated from a ditch are generated from a common precursor protein (A1-0) having a molecular size of 69 kDa (Murata et al., J. Ferment. Bioeng., 76, 427–437, 1993). A1-I (63 kDa) generated after removal of an N-terminal peptide (6 kDa) from A1-0 was found to be converted into A1-II (23 kDa) and A1-III (40 kDa) by a self-processing reaction of the A1-I molecule itself.

Inhibition of adherence of mucoid Pseudomonas aeruginosa by alginase, specific monoclonal antibodies, and antibiotics.

The adherence of pseudomonal species was investigated by using a newly developed radiometric dacron fiber microcolumn assay. Pseudomonas aeruginosa, P. stutzeri, and Xanthomonas maltophilia were more adherent (approximately 20%) than P. pseudomallei, P. fluorescens, and P. cepacia (approximately 10%). Mucoid strains of P. aeruginosa were consistently more adherent than nonmucoid strains (30% versus 20%). Alginase was shown to inhibit the adherence of mucoid but not nonmucoid P. aeruginosa. Monoclonal antibodies to alginate were also shown to inhibit the adherence of mucoid but not nonmucoid P. aeruginosa. In addition, antibiotics active against P. aeruginosa were shown to inhibit the adherence of both mucoid and nonmucoid strains. Furthermore, synergism between dyadic combinations of monoclonal antibodies and antibiotic (ciprofloxacin), as well as alginase and antibiotic, was also observed. These results indicate that bacterial alginate has an intrinsic role in the adherence of mucoid P. aeruginosa and may have evolved not only for protection against dehydration in the water and soil ecosystem of this bacterium, but also as a means of attaching to soil substrates in the same ecosystem to enhance survival. They also suggest that synergistic combinations of antibiotics with alginase or monoclonal antibodies to alginate may be of value in the therapy of some pseudomonal infections.

Bacterial alginate lyase highly active on acetylated alginates

A bacterium (strain A1) isolated from a ditch synthesized three kinds of intracellular alginate lyases: A1-I (molecular weight [M.W.] 60,000), A1-II-1 (M.W. 60,000) and A1-II-2 (M.W. 25,000) in laboratory-scale cultures. However, when cells of strain A1 were grown on an industrial scale, another lyase (A1-III) was produced other than A1-I, A1-II-1 and A1-II-2. The A1-III lyase was a monomer with a M.W. of about 38,000, and its activity toward bacterial (acetylated) alginates was much higher (2-fold) than that toward seaweed (non-acetylated) alginates. The N-terminal amino acid sequence of A1-III lyase was consistent with that of A1-I lyase.

Bacterial alginate lyase inactive on alginate biosynthesized by Pseudomonas aeruginosa

A bacterium (strain Al) isolated from a ditch produces three kinds of intracellular alginate lyases [Al-I (molecular weight: M.W. 60,000), Al-II-1 (M.W. 60,000) and Al-II-2]; the former two lyases have been purified and characterized (Yonemoto et al., J. Ferment. Bioeng., 72, 152–157, 1991). As part of a series of studies, Al-II-2 lyase was purified from cell-free extract of the bacterium. The lyase, with a M.W. of 25,000, depolymerized sodium-, potassium- and propyleneglycol alginates most efficiently at pH 8.0, 70°C, but it was inactive toward bacterial alginates with O-acetyl groups.

アルギン酸から酵素分解によって調製したオリゴ糖の静菌作用

Oligosaccharides were prepared from alginate, guluronate-rich blocks, and mannuronate-rich blocks of alginate by bacterial alginate lyases, and were examined for antimicrobial action against four strains of aerobic and two strains of anaerobic bacteria, as well as six strains of fungi. O1-igosaccharides from guluronate-rich and mannuronate-rich blocks showed significant activity against the aerobic and anaerobic bacteria, but none of the oligosaccharides were antifungal. The bacteriostatic action of the oligosaccharides differed depending on the degree of polymerization and hepta- and hexa-saccharides were highly bacteriostatic. None of the saccharides tested were utilized by any of the seven strains of intestinal bacteria.

Bacterial alginate lyase gene: Nucleotide sequence and molecular route for generation of alginate lyase species

A bacterium (strain A1) isolated from a ditch synthesized three types of intracellular alginate lyases: A1-I (molecular weight [M.W.] 60,000), A1-II-2 (M.W. 25,000) and A1-III (M.W. 38,000). The nucleotide sequence of the gene for A1-I lyase, which has been cloned in Escherichia coli DH1 was determined. The open reading frame of the gene encoded 622 amino acids with a calculated M.W. of 69,153. The N-terminal amino acid sequence of A1-I lyase purified from strain A1 or E. coli DH1 cells transformed with the A1-I lyase gene was consistent with the deduced sequence from 55His to 74Ala, indicating that the A1-I lyase was synthesized as a precursor with a M.W. of 69,153 and then processed to a mature form with a M.W. of 63,681. The N-terminal sequence of the first twenty amino acids of A1-III lyase was found to match that of A1-I lyase. The N-terminal sequence of the first twenty amino acids of A1-II-2 lyase was consistent with the deduced amino acid sequence from 414Ala to 433Val in the nucleotide sequence of the A1-I lyase gene. These results indicated that the A1-I lyase was further processed to generate A1-II-2 and A1-III lyase species.

Promotion of germination and shoot elongation of some plants by alginate oligomers prepared with bacterial alginate lyase

Depolymerization of sodium alginate (average molecular weight: 25,700) from an edible seaweed Eisenia bicyclis with bacterial alginate lyase yielded oligosaccharide(s) with an average molecular weight of 1,800 together with other components. The proliferation and/or differentiation of plants was markedly enhanced in the presence of the oligosaccharide(s), although that of mammalian (HeLa) cells and an algae ( Chlamidomonas sp.) was not. The results indicate that the depolymerization products of alginate containing oligosaccharine-like compounds specifically affect the proliferation and/or differentiation of higher plants.

Alginases from Azotobacter species

Alginate lyases (alginases) have been prepared from strains of Azotobacter vinelandii and Azotobacter chroococcum in which they were located in the periplasm. The enzymes are present in wild-type strains of each species and in mutants failing to encyst or produce bacterial alginate. The lyases have been partially purified by ion exchange chromatography and by affinity chromatography on a matrix prepared from poly-D-mannuronic acid. Although several bacterial and algal alginate preparations were degraded by the enzymes, highest activity was found on poly-D-mannuronic acid or on algal alginates with high mannuronic acid content. The major product from enzymes of either bacterium was an unsaturated uronic acid, when either alginates or poly-D-mannuronic acid were used as substrates. When tested against a series of algal alginates of increasing D-mannuronic acid content, the enzyme activity was highest against alginates of high D-mannuronic acid content, indicating that the enzymes are endo-D-mannurono-lyases. The alginases from the two bacterial species are not identical in their substrate specificity although both show the same generalized type of action.