Cloning, sequence analysis and expression of Pseudoalteromonas elyakovii IAM 14594 gene (alyPEEC) encoding the extracellular alginate lyase.

A gene (alyPEEC) encoding an alginate lyase of Pseudoalteromonas elyakovii IAM 14594 was cloned using the plasmid vector pUC118 and expressed in Escherichia coli. Sequencing of a 3.0kb fragment revealed a 1,197bp open reading frame encoding 398 amino acid residues. The calculated molecular mass and isoelectric point of the alyPEEC gene product are 43.2 kDa and pI 5.29. A region G(165) to V(194) in the AlyPEEC internal sequence is identical to the N-terminal amino acid sequence of the previously purified extracellular alginate lyase of P. elyakovii, and the calculated molecular mass (25.4 kDa) and isoelectric point (pI 4.78) of the region resembled those of the purified enzyme. Expression of enzymically-active alginate lyase from alyPEEC required growth of recombinant E. coli in LB broth containing 50% (v/v) artificial seawater (ASW). Alginate lyase activity with broad substrate specificity was detected in both 42 and 30 kDa products. Subcloning of the region G(165) to N(398) of AlyPEEC corresponding to the 30 kDa protein confirmed that this region of the alyPEEC gene encoded the active site of the enzyme. A region A(32) to G(164) corresponding to about 13 kDa of the N-terminal region of AlyPEEC showed about 30% identity to a putative chitin binding domain of Streptomyces chitinases, but did not exhibit any catalytic activity.

Enzyme kinetics and chemical modification of alpha-1,4-glucan lyase from Gracilariopsis sp.

The kinetic properties and active site amino acids of alpha-1,4-glucan lyase from the marine red macroalga Gracilariopsis sp. were examined. Using 1H NMR spectroscopy the alpha-1,4-glucan lyase was found to degrade alpha- and beta-maltose at different rates. The effect of pH on the kinetic constants suggested the presence of two catalytically important amino acids in the active site with pKa values of 3.5 and 6.2. The former indicated the presence of an ionised aspartate or glutamate residue in the active site. This was tested using the carboxyl specific reagent EDAC, which inhibited enzyme activity in a time dependent manner when an external nucleophile was added. No protection against the inactivation was obtained by addition of amylopectin, maltitol or 1-deoxinojirimycin. Inactivation decreased Vmax over 2.5-fold with little effect on Km which supports the direct involvement of a carboxyl group in catalysis.

Isolation, characterisation and expression of a cDNA for pea cholinephosphate cytidylyltransferase.

In plants, phosphatidylcholine is the major phospholipid in extra-plastid membranes and is synthesised mainly by the CDP-choline pathway. Evidence from studies in animals, as well as in plants, suggests that the intermediate step catalysed by cholinephosphate cytidylyltransferase (CPCT) has a major control in carbon flux to this lipid. We have isolated a full-length CPCT cDNA (designated PCT2) from Pisum sativum cv. Feltham First using an Arabidopsis probe and the polymerase chain reaction (PCR). The deduced amino acid of PCT2 is 48%, 43% and 76% identical to the rat, yeast and Brassica napus amino acid sequences, respectively. Expression of the CPCT protein in Escherichia coli confirmed the activity of the enzyme. Expression of the PCT2 mRNA in pea roots and stems was increased by treatment with 0.1 microM indole-3-acetic acid.

Heterologous expression of full-length and truncated forms of the recombinant guluronate-specific alginate lyase of Klebsiella pneumoniae.

The alyA gene, which encodes a guluronate-specific alginate lyase from Klebsiella pneumoniae, was cloned into the plasmid pCT54 to facilitate heterologous expression of the enzyme in Escherichia coli. The greatest enzyme specific activity was observed when the complete gene and flanking regions of DNA (contained within a 1.95 kb HindIII fragment) were cloned in the correct orientation relative to the vector-encoded trp promoter. Heterologous expression of the intact gene complete with flanking regions resulted in a 20-fold increase in enzyme yield compared to the original construct, pRC5. PCR mutagenesis and/or restriction endonuclease digestion was used to generate a series of fragments containing either the whole or truncated versions of the gene. Active enzyme was produced from constructs in which the region encoding the signal peptide had been deleted, although the recombinant protein was retained within the bacterial cells. An internal methionine (position 74) could be used as a start site for translation but resulted in the accumulation of inactive protein within inclusion bodies.

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.

Pseudomonas aeruginosa AlgG is a polymer level alginate C5-mannuronan epimerase.

Alginate is a viscous extracellular polymer produced by mucoid strains of Pseudomonas aeruginosa that cause chronic pulmonary infections in patients with cystic fibrosis. Alginate is polymerized from GDP-mannuronate to a linear polymer of beta-1-4-linked residues of D-mannuronate and its C5-epimer, L-guluronate. We previously identified a gene called algG in the alginate biosynthetic operon that is required for incorporation of L-guluronate residues into alginate. In this study, we tested the hypothesis that the product of algG is a C5-epimerase that directly converts D-mannuronate to L-guluronate. The DNA sequence of algG was determined, and an open reading frame encoding a protein (AlgG) of approximately 60 kDa was identified. The inferred amino terminus of AlgG protein contained a putative signal sequence of 35 amino acids. Expression of algG in Escherichia coli demonstrated both 60-kDa pre-AlgG and 55-kDa mature AlgG proteins, the latter of which was localized to the periplasm. An N-terminal analysis of AlgG showed that the signal sequence was removed in the mature form. Pulse-chase experiments in both E. coli and P. aeruginosa provided evidence for conversion of the 60- to the 55-kDa size in vivo. Expression of algG from a plasmid inan algG (i.e., polymannuronate-producing) mutant of P. aeruginosa restored production of an alginate containing L-guluronate residues. The observation that AlgG is apparently processed and exported from the cytoplasm suggested that it may act as a polymer-level mannuronan C5-epimerase. An in vitro assay for mannuronan C5 epimerization was developed wherein extracts of E. coli expressing high levels of AlgG were incubated with polymannuronate. Epimerization of D-mannuronate to L-guluronate residues in the polymer was detected enzymatically, using a L-guluronate-specific alginate lyase of Klebsiella aerogenes. Epimerization was also detected in the in vitro reaction between recombinant AlgG and poly-D-mannuronate, using high-performance anion-exchange chromatography. The epimerization reaction was detected only when acetyl groups were removed from the poly-D-mannuronate substrate, suggesting that AlgG epimerization activity in vivo may be sensitive to acetylation of the D-mannuronan residues. These results demonstrate that AlgG has polymer-level mannuronan C5-epimerase activity.

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.

Heterologous expression of an alginate lyase gene in mucoid and non-mucoid strains of Pseudomonas aeruginosa.

A 1.95 kb DNA fragment containing the aly gene from Klebsiella pneumoniae, which encodes an alginate lyase, has been ligated into the broad-host-range vector pLAFR3. Transfer of the resultant recombinant plasmid, pALY8, into mucoid and non-mucoid strains of Pseudomonas aeruginosa resulted in expression of the alginate lyase. The heterologously expressed alginate lyase, which had the same isoelectric point and substrate specificity as the native enzyme, altered the morphology of mucoid strains. Analysis of the extracellular material from mucoid strains revealed that lyase expression reduced the M(r) and overall yield of alginate produced. The mature form of the recombinant enzyme was the same as that produced extracellularly by Klebsiella pneumoniae; however, most of the alginate lyase was retained intracellularly by P. aeruginosa.

Isolation of a mucoid alginate-producing Pseudomonas aeruginosa strain from the equine guttural pouch.

The isolation and characterization of a mucoid, alginate-producing strain of Pseudomonas aeruginosa from a nonhuman host, namely, in chondroids from an equine guttural pouch, is reported for the first time. Pure cultures of P. aeruginosa 12534 were isolated from a 17-month-old pony mare with a history of chronic bilateral mucopurulent nasal discharge from the right guttural pouch. Transmission electron microscopy of chondroids showed mucoid P. aeruginosa growing as microcolonies within a matrix of extracellular material. On the basis of expression of the mucoid phenotype under different growth conditions, P. aeruginosa 12534 belongs to group 1 and resembles other isolates carrying the muc-23 mutation. The bulk of the extracellular material was characterized as being alginate by chemical and 1H nuclear magnetic resonance analyses, which showed that it had a composition similar to that produced by isolates of P. aeruginosa from human patients with cystic fibrosis.