APPLICATION OF THE RESIDENT PLASMID INTEGRATION TECHNIQUE TO CONSTRUCT A STRAIN OF STREPTOCOCCUS-GODRONII ABLE TO EXPRESS THE BACILLUS-CIRCULANS CYCLOISOMALTOOLIGOSACCHARIDE GLUCANOTRANSFERASE GENE, AND SECRETE ITS ACTIVE GENE-PRODUCT

Citation
T. Shiroza et al., APPLICATION OF THE RESIDENT PLASMID INTEGRATION TECHNIQUE TO CONSTRUCT A STRAIN OF STREPTOCOCCUS-GODRONII ABLE TO EXPRESS THE BACILLUS-CIRCULANS CYCLOISOMALTOOLIGOSACCHARIDE GLUCANOTRANSFERASE GENE, AND SECRETE ITS ACTIVE GENE-PRODUCT, Gene, 207(2), 1998, pp. 119-126
Citations number
16
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Genetics & Heredity
Journal title
GeneACNP
ISSN journal
0378-1119
Volume
207
Issue
2
Year of publication
1998
Pages
119 - 126
Database
ISI
SICI code
0378-1119(1998)207:2<119:AOTRPI>2.0.ZU;2-V
Abstract
A novel transformation technique, resident plasmid integration, for th e cloning of foreign DNA in oral streptococci was described recently ( T. Shiroza and H.K. Kuramitsu, Plasmid 34 (1995) 85-95. This technique is based on the integration of linearized foreign genes by recombinat ion-proficient bacteria onto a resident plasmid, if an appropriate sel ection marker is flanked by the same anchor sites present in the resid ent plasmid. Since the transforming vehicles for this system included a pUC-derived replication origin, the high level expression in Escheri chia coli cells hindered the cloning of certain genes. In the present study, new plasmids were constructed, two resident plasmids, four inte gration plasmids, and four cloning plasmids, all of which possess the medium-copy number replication origin, p15A ori, isolated from pACYC17 7. The resident plasmids consisted of the following three components: the p15A ori (0.65-kb Bg/II fragment), the pVA380-1 basic replicon fun ctional in mutans streptococci (2.5-kb BamHI fragment), and either an erythromycin resistance or a spectinomycin resistance gene (0.9- or 1. 1-kb BamHI fragment, respectively). Most of the basic replicon of pVA3 80-1, except for the 3'-portion of the 0.2-kb region, in the resident plasmid was replaced with a kanamycin resistance gene to construct the four integration plasmids. Therefore, the upstream and downstream anc hor sites for the double cross-over event in this new system were 0.65 -kb p15A ori and the 0.2-kb portion of the 3'-end of pVA380-1 replicon , respectively. This system was used to clone the gene coding for cycl oisomaltooligosaccharide glucanotransferase which produces cycloisomal tooligosaccharide, a potent inhibitor of oral streptococcal glucosyltr ansferase, isolated from Bacillus circulans chromosome, into Streptoco ccus gordonii, and its gene product was successfully secreted into the culture media. Plasmids described here should be useful tools for int roducing heterologous DNA into resident plasmids following integration in oral streptococci. (C) 1998 Elsevier Science B.V.