Characterization of the Pseudomonas aeruginosa alginate lyase gene (algL): cloning, sequencing, and expression in Escherichia coli

Abstract

Mucoid strains of Pseudomonas aeruginosa produce a viscous exopolysaccharide called alginate and also express alginate lyase activity which can degrade this polymer. By transposon mutagenesis and gene replacement techniques, the algL gene encoding a P. aeruginosa alginate lyase enzyme was found to reside between algG and algA within the alginate biosynthetic gene cluster at 35 min on the P. aeruginosa chromosome. DNA sequencing data for algL predicted a protein product of ca. 41 kDa, including a 27-amino-acid signal sequence, which would be consistent with its possible localization in the periplasmic space. Expression of the algL gene in Escherichia coli cells resulted in the expression of alginate lyase activity and the appearance of a new protein of ca. 39 kDa detected on sodium dodecyl sulfate-polyacrylamide gels. In mucoid P. aeruginosa strains, expression of algL was regulated by AlgB, which also controls expression of other genes within the alginate gene cluster. Since alginate lyase activity is associated with the ability to produce and secrete alginate polymers, alginate lyase may play a role in alginate production.

A novel gene, algK, from the alginate biosynthetic cluster of Pseudomonas aeruginosa

“…The operon encodes proteins such as the dual-function enzyme phosphomannose isomerase-GDP mannose pyrophosphorylase (algA ; Shinabarger et al, 1991) and also GDP-mannose dehydrogenase (algD ; Deretic et al, 1987). Other genes identified in the alginate biosynthetic cluster encode a CS-mannuronate epimerase which is responsible for the incorporation of L-guluronate residues into alginate (algG;Franklin et al, 1994), an 0-acetylase which esterifies mannuronate residues (algF ;Shinabarger et al, 1993), a D-mannuronate-specific alginate lyase (algL; Boyd et al, 1993;Schiller et al, 1993) and an outer-membrane pore which is considered to mediate alginate excretion (algE; Chu et al, 1991;Rehm et al, 1994). Despite this knowledge, however, the mechanism by which alginate polymerization and export occur remains poorly understood.…”

Conversion of Pseudomonas aeruginosa to mucoidy in cystic fibrosis: environmental stress and regulation of bacterial virulence by alternative sigma factors

“…The majority of these and other enzymes modifying alginate are encoded by the large cluster of alginate genes at 34 min (12). This cluster contains in the order of transcription: algD, encoding GDPmannose dehydrogenase, which catalyzes the final step in the precursor biosynthesis (14); algG, encoding the epimerase which converts mannuronic residues into guluronate (8); algL, encoding an enzyme termed alginate lyase, due to its ability to depolymerize alginate (6,67); algF, encoding an acetylase (24,70); and algA, encoding phosphomannose isomerase, which converts fructose 6-phosphate into mannose 6-phosphate (11). The latter enzyme also has a pyrophosphorylase activity implicated in GDPmannose synthesis (69), The alginate biosynthetic cluster is the major point of convergence for various regulatory factors controlling alginate production.…”

Overexpression of algE in Escherichia coli: subcellular localization, purification, and ion channel properties

“…However, there is a lack of information about the final steps of the biosynthesis, i.e., the polymerization and export of alginate. Recently, the genes involved in acetylation (algF) and degradation (algL) of alginate were cloned and mapped in the alginate biosynthesis gene cluster (34 min) (10,30). In addition, the gene product of algG (34 min) was purified and identified as an epimerase located in the periplasm that introduces guluronic acid residues into the alginate (9).…”