Dataset Information

Characterization of alginate lyase from Pseudomonas syringae pv. syringae.

ABSTRACT: The gene encoding alginate lyase (algL) in Pseudomonas syringae pv. syringae was cloned, sequenced, and overexpressed in Escherichia coli. Alginate lyase activity was optimal when the pH was 7.0 and when assays were conducted at 42 degrees C in the presence of 0.2 M NaCl. In substrate specificity studies, AlgL from P. syringae showed a preference for deacetylated polymannuronic acid. Sequence alignment with other alginate lyases revealed conserved regions within AlgL likely to be important for the structure and/or function of the enzyme. Site-directed mutagenesis of histidine and tryptophan residues at positions 204 and 207, respectively, indicated that these amino acids are critical for lyase activity.

SUBMITTER: Preston LA

PROVIDER: S-EPMC94769 | biostudies-literature | 2000 Nov

REPOSITORIES: biostudies-literature

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Characterization of alginate lyase from Pseudomonas syringae pv. syringae.

Preston L A LA Wong T Y TY Bender C L CL Schiller N L NL

Journal of bacteriology 20001101 21

The gene encoding alginate lyase (algL) in Pseudomonas syringae pv. syringae was cloned, sequenced, and overexpressed in Escherichia coli. Alginate lyase activity was optimal when the pH was 7.0 and when assays were conducted at 42 degrees C in the presence of 0.2 M NaCl. In substrate specificity studies, AlgL from P. syringae showed a preference for deacetylated polymannuronic acid. Sequence alignment with other alginate lyases revealed conserved regions within AlgL likely to be important for t ...[more]

PMID: 11029455

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Project description:BackgroundPseudomonas syringae pv. phaseolicola 1448a (P. syringae 1448a), the causative agent of bean halo blight, is a bacterium capable of occupying diverse biological niches. Under conditions of iron starvation P. syringae 1448a secretes siderophores for active uptake of iron. The primary siderophore of P. syringae 1448a is pyoverdine, a fluorescent molecule that is assembled from amino acid precursors by non-ribosomal peptide synthetase (NRPS) enzymes. Whereas other species of Pseudomonas often exhibit structural variations in the pyoverdine produced by different strains, all P. syringae pathovars previously tested have been found to make an identical pyoverdine molecule. P. syringae 1448a also appears to have the genetic potential to make two secondary siderophores, achromobactin and yersiniabactin, each of which has previously been detected in different P. syringae pathovars.ResultsFive putative pyoverdine NRPS genes in P. syringae 1448a were characterized in-silico and their role in pyoverdine biosynthesis was confirmed by gene knockout. Pyoverdine was purified from P. syringae 1448a and analyzed by MALDI-TOF and MS/MS spectroscopy. Peaks were detected corresponding to the expected sizes for the pyoverdine structure previously found in other P. syringae pathovars, but surprisingly P. syringae 1448a appears to also produce a variant pyoverdine species that has an additional 71 Da monomer incorporated into the peptide side chain. Creation of pyoverdine null mutants of P. syringae 1448a revealed that this strain also produces achromobactin as a temperature-regulated secondary siderophore, but does not appear to make yersiniabactin. Pyoverdine and achromobactin null mutants were characterized in regard to siderophore production, iron uptake, virulence and growth in iron limited conditions.ConclusionsThis study provides the first evidence of a P. syringae pathovar producing a side chain variant form of pyoverdine. We also describe novel IC₅₀ and liquid CAS assays to quantify the contribution of different siderophores across a range of iron starvation conditions, and show that although achromobactin has potential to contribute to fitness its contribution is masked by the presence of pyoverdine, which is a significantly more effective siderophore. Neither pyoverdine nor achromobactin appear to be required for P. syringae 1448a to cause bean halo blight, indicating that these siderophores are not promising targets for crop protection strategies.

Dataset Information

Characterization of alginate lyase from Pseudomonas syringae pv. syringae.

Publications

Characterization of alginate lyase from Pseudomonas syringae pv. syringae.

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