Sequence of the algL gene of Pseudomonas aeruginosa and purification of its alginate lyase product.
The alginate lyase-encoding gene (algL) of Pseudomonas aeruginosa was localized to a 1.7-kb EcoRI-XbaI fragment within the alginate biosynthetic gene cluster at 34 minutes on the chromosome. The nucleotide sequence of this DNA fragment revealed an ORF encoding a protein of M(r) 40,885 which is transcribed in the same orientation as the other alg genes within the biosynthetic gene cluster. The predicted protein has a potential N-terminal signal peptide which is consistent with its proposed periplasmic location. The AlgL protein was overproduced in Escherichia coli and purified. The purified protein was shown to have alginate lyase activity. In addition, an algL insertion mutant of the mucoid P. aeruginosa 8830 was constructed. This mutant (alm1) had a nonmucoid phenotype due to a polar effect on the transcription of an essential alg gene, algA. Thus, the algL gene is located within a region of the alginate biosynthetic gene cluster that appears to be non-essential for alginate production.
Boyd
A
A
Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago 60612.
Ghosh
M
M
May
T B
TB
Shinabarger
D
D
Keogh
R
R
Chakrabarty
A M
AM
D000464:Alginates / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis; D004926:Escherichia coli; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D005810:Multigene Family; D016254:Mutagenesis, Insertional; D010957:Plasmids; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011135:Polysaccharides, Bacterial / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA
D000464:Alginates; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011135:Polysaccharides, Bacterial; D011993:Recombinant Fusion Proteins; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase