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.
Caswell
R C
RC
Department of Biochemistry, University of Wales College of Cardiff, U.K.
Gacesa
P
P
Lutrell
K E
KE
Weightman
A J
AJ
D003001:Cloning, Molecular; D003470:Culture Media; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D005822:Genetic Vectors; D007711:Klebsiella pneumoniae / Q000235:genetics*; D009154:Mutation; D010957:Plasmids; D011133:Polysaccharide-Lyases / Q000235:genetics* / Q000378:metabolism
D003470:Culture Media; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase