Isolation of a neuraminidase gene from Actinomyces viscosus T14V.
A genomic library of Actinomyces viscosus T14V DNA in lambda gt11 was screened for expression of neuraminidase activities. Four recombinant clones were detected that gave blue fluorescence upon incubation with a fluorogenic substrate, 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid. Of these, two were identical, and all of the neuraminidase-positive clones shared a common 3.4-kbp DNA region. Expression of the enzyme activities in Escherichia coli carrying the cloned DNA was independent of the lacZ promoter of the vector. Maxicell analysis revealed that the 3.4-kbp DNA insert directed synthesis of a protein with an apparent molecular mass of 100,000 Da. The protein from cell extracts of E. coli clones migrated as a single band that stained for enzyme activity after electrophoresis in a nondissociating polyacrylamide gel. Moreover, human erythrocytes incubated previously with cell lysates from neuraminidase-positive E. coli were hemagglutinated by Actinomyces spp. The enzyme expressed by E. coli was active on substrates containing alpha-2,3 and alpha-2,6 ketosidic linked sialyl residues. Similar substrate specificities were obtained for both the extracellular and cell-associated neuraminidases from A. viscosus T14V. The 3.4-kbp insert hybridized to DNA fragments in a Southern blot containing A. viscosus T14V chromosomal DNA that had been digested with various restriction endonucleases. Data from hybridization studies show that A. viscosus T14V contains a single copy of the neuraminidase gene.
Yeung
M K
MK
Department of Pediatric Dentistry, University of Texas Health Science Center, San Antonio 78284.
Fernandez
S R
SR
D016924:Actinomyces viscosus / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005784:Gene Amplification; D005798:Genes, Bacterial; D006386:Hemagglutination Tests; D009439:Neuraminidase / Q000737:chemistry / Q000235:genetics*; D015183:Restriction Mapping; D013379:Substrate Specificity
D004269:DNA, Bacterial; D009439:Neuraminidase