Peter Gacesa

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.

The purification and chemical characterisation of the alginate present in extracellular material produced by mucoid strains of Pseudomonas aeruginosa

A rapid ion-exchange method has been used to purify the alginate from the extracellular material of mucoid strains of Pseudomonas aeruginosa isolated from the lungs of cystic fibrosis patients. The structure has been investigated by chemical analysis, infrared spectroscopy, paper chromatography, and gas-liquid chromatography. The alginates contain mainly random or poly(D-mannuronic acid) block structures, and are highly acetylated. The relative viscosity is not correlated with the ratio of D-mannuronic acid to L-guluronic acid residues, or the degree of acetylation. The chemical/physical properties of the alginate from P. aeruginosa are considered in the context of the growth of the organism in the lung.

The determination of the uronic acid composition of alginates by anion-exchange liquid chromatography

Abstract An isocratic, anion-exchange, liquid chromatography system has been developed to separate and quantify the uronic acids ( d -mannuronic and l -guluronic acids) that are present in acid hydrolysates of alginates. The sensitivity of the technique will allow 10 μg of uronic acid to be detected. The method has the advantages of speed (typical analysis time, 20 min) and a small-sample requirement (routine analysis, 140 μg of hydrolysate), whilst retaining the accuracy of the traditional method.

A recommended procedure for the estimation of bovine testicular hyaluronidase in the presence of human serum

A procedure is described for the assay of bovine testicular hyaluronidase in human blood following intravenous administration of the enzyme. Inhibition of hyaluronidase by the reported nonspecific serum inhibitor is minimal. However, the presence of human serum does alter the pH profile of hyaluronidase and enhances the activity of the enzyme at low pH values. Preliminary data indicates that the effects caused by serum on the pH optimum and activity of the enzyme are largely associated with the albumin fraction and are not due to the presence of endogenous serum hyaluronidase. The activation effect is not specific for any particular blood type and is independent of whether serum or citrated plasma is used. A similar effect to that of serum on hyaluronidase activity is produced by different buffer mixtures or increased NaCl concentration. It is recommended that bovine testicular hyaluronidase be measured at pH 4.0 in 0.1 m sodium citrate buffer containing 0.15 m NaCl as under these conditions the addition of human serum or citrated plasma does not alter the pH optimum of the enzyme. These recommendations necessitate certain modifications of the reducing N-acetylhexosamine assay method of Reissig et al. (J. L. Reissig, J. L. Strominger, and L. F. Leloir, 1955, J. Biol. Chem.217, 959–966).

Molecular cloning and heterologous expression of a Klebsiella pneumoniae gene encoding alginate lyase

The alginate lyase (Aly; guluronate specific)-coding gene of Klebsiella pneumoniae was cloned using the cosmid vector pMMB33, transduced into Escherichia coli and expressed in this host. Four Aly-positive clones with unstable phenotypes were identified out of 700 kanamycin-resistant transductants. A stable derivative of one of the clones was studied further and contained 12.1-kb of insert DNA. The Aly-coding gene (aly), still partially under the control of its native promoter, was localised within a 1.95-kb HindIII fragment by transposon gamma delta mutagenesis and sub-cloning. Most of the Aly produced was secreted into the medium by both the original K. pneumoniae strain (71.7%) and the E. coli recombinant clones (85.1%). The enzyme from both K. pneumoniae and the E. coli clones had a pI of 8.9 and comprised a single 28-kDa polypeptide chain. Other minor bands were also observed on isoelectric focusing and these were attributed to processing intermediates of a single gene product. It is concluded that E. coli can recognise and process the signal peptide of Aly to produce a mature polypeptide that is identical to that synthesised by K. pneumoniae.

GDP-mannose dehydrogenase is the key regulatory enzyme in alginate biosynthesis in Pseudomonas aeruginosa : evidence from metabolite studies

The Pseudomonas aeruginosa enzyme GDP-mannose dehydrogenase (GMD) is encoded by the algD gene, and previous genetic studies have indicated that it is a key regulatory and committal step in the biosynthesis of the polysaccharide alginate. In the present study the algD gene has been cloned into the broad-host-range expression vector pMMB66EH and GMD overexpressed in mucoid and genetically-related non-mucoid strains of P. aeruginosa. The metabolic approach of P. J. Tatnell, N. J. Russell & P. Gacesa (1993), J Gen Microbiol 139, 119-127, has been used to investigate the subsequent effect of GMD overexpression on the intracellular concentrations of the key metabolites GDP-mannose and GDP-mannuronate, which have been related to GMD activity and total alginate production. The overexpression of algD in mucoid and non-mucoid strains resulted in elevated GMD activities compared to wild-type strains; there was a concomitant reduction in GDP-mannose concentrations and greatly increased GDP-mannuronate concentrations. However, significantly, alginate biosynthesis was detected only in mucoid strains and GMD overexpression resulted in only a marginal increase in exopolysaccharide production. The GDP-mannuronate concentrations in mucoid strains which overexpressed GMD were always significantly greater than those of GDP-mannose, indicating that GMD was no longer the major kinetic control point in the biosynthesis of alginate by these genetically-manipulated strains. The small but significant increase in alginate production by such strains together with the increased GDP-mannuronate concentrations is interpreted as meaning that a later enzyme of the alginate pathway has become the major kinetic control point and now determines the extent of alginate production. This study has provided direct metabolic evidence that GMD is the key regulatory enzyme in alginate biosynthesis in P. aeruginosa.

Alginate lyase from Klebsiella pneumoniae, subsp. aerogenes : gene cloning, sequence analysis and high-level production in Escherichia coli

The alyA gene, encoding a secreted guluronate-specific alginate lyase (Aly) from Klebsiella pneumoniae subsp. aerogenes type 25, has been cloned. DNA sequence analysis reveals two possible translation start sites for the precursor form of Aly and a long open reading frame (ORF) predicted to encode a 287-amino-acid (aa) mature form of Aly, in agreement with N-terminal aa sequence analysis of the protein. Aly has a calculated molecular mass of 31.4 kDa, in good agreement with SDS-PAGE analysis, and a calculated pI of 9.39. Comparison of the deduced aa sequence with a mannuronate-specific lyase from a marine bacterium reveals 19.3% identity and 28.8% similarity with a 9-aa conserved region close to the C terminus, probably of functional or structural significance. There is no obvious sequence similarity with pectate lyases which also catalyse a beta-elimination reaction. Heterologous expression of K. pneumoniae alyA in Escherichia coli yields 10 mg of Aly per litre of culture supernatant, apparently due to non-specific release from the periplasm.

A metabolic study of the activity of GDP-mannose dehydrogenase and concentrations of activated intermediates of alginate biosynthesis in Pseudomonas aeruginosa

GDP-mannose dehydrogenase (GMD) is a key regulatory enzyme and the committal step in alginate biosynthesis. In this study, a metabolic approach has been used to investigate GMD activity in non-mucoid and isogenically related mucoid strains of Pseudomonas aeruginosa. Intracellular concentrations of GDP-mannose and GDP-mannuronate have been quantified using HPLC separation methods, and their concentrations have been related to GMD activity and total alginate production. In all strains of P. aeruginosa tested, GDP-mannose accumulated particularly during the exponential phase of growth in batch culture; the GDP-mannose concentrations in mucoid strains were significantly lower compared with isogenic non-mucoid strains. The product of GMD activity, GDP-mannuronate, was detectable only in mucoid strains, albeit at low but relatively constant levels irrespective of growth phase. The GDP-mannose concentrations in mucoid strains were always significantly greater than those of GDP-mannuronate, indicating that GMD is a rate-limiting enzyme in the biosynthesis of alginate. Significant GMD activity and extracellular alginate production were detected only in mucoid strains. The metabolic data reported here, together with previous genetic studies, provide strong evidence that GMD is the key regulatory enzyme controlling alginate biosynthesis in mucoid strains of P. aeruginosa.

Detection of alginate lyases by isoelectric focusing and activity staining

Abstract A sensitive method has been developed for the rapid analysis of mutliple forms of alginate lyases in crude bacterial extracts. The technique is based on isoelectric focusing with a substrate-overlay technique for the direct measurement of enzyme activity. Isoelectric point values have been determined for the alginate lyases present in five strains of bacteria using, typically, 5.7 × 10 − units of activity. Multiple forms of these enzymes have been observed in three of the five bacterial strains studied. The method has also been used to compare the pI value of the l -guluronate lyase from Klebsiella pneumoniae with those for the cloned gene products in strains of Escherichia coli .

Modification of functional arginine residues in purified bovine testicular hyaluronidase with butane-2, 3-dione.

Purified bovine testicular hyaluronidase (hyaluronate 4-glycanohydrolase, EC 3.2.1.35) was inactivated by butane-2,3-dione in either borate or Hepes buffer, pH 8.3. The presence of borate enhanced the inactivation process which followed pseudo-first-order kinetics with a calculated second-order rate constant of 13.54M-1 min-1. Using kinetic data it was estimated that the modification of 1 mol arginine per mol enzyme was sufficient for inactivation to occur, whereas amino acid analysis indicated that 4 mol arginine had been modified. The inactivation process was partially prevented by using either competitive inhibitors or substrates of the enzyme, thus indicating that the essential arginine residue is close to the active site of hyaluronidase. A full kinetic analysis of the enzyme with either hyaluronic acid or chondroitin 6-sulphate as substrate showed that the activity of hyaluronidase was uncompetitively activated by either protons or NaCl. The product obtained by reduction of the corboxyl groups of hyaluronic acid to the corresponding alcohol groups was a competitive inhibitor. The possibility that the microenvironment of hyaluronic acid was responsible for the observed kinetic effects of pH and ionic strength was dispelled. It is concluded that these data are compatible with a mechanism that involves anionic interaction between a carboxyl group on the substrate and an arginine residue on the enzyme.