Cloning of erythromycin-resistance determinants and replication origins from indigenous plasmids of Lactobacillus reuteri for potential use in construction of cloning vectors.
Lactobacillus reuteri L1 and N16 strains contain a 7.0-kb plasmid (pTE80) and a 15-kb plasmid (pTE15), respectively, encoding resistance to erythromycin (Em(r)). Physical maps of both plasmids were established. Nucleotide sequences of the genetic determinants encoding Em(r) on pTE80 and pTE15 revealed the existence of a very similar (ca. 99% nucleotide sequence and ca. 98% amino acid sequence identity) open reading frame for an Em(r) transmethylase gene (erm) in both plasmids. These structural erm genes, 753 and 750 bp in length, respectively, were highly related (ca. 98% nucleotide sequence and ca. 97% amino acid sequence identity) to the erm gene of L. fermentum plasmid pLEM3. Sequence analysis showed that these two erm genes from pTE80 and pTE15 could be categorized under the ermB (ermAM) class. These are the first members of the ermB (ermAM) class of Em(r) determinant from L. reuteri to be characterized at the nucleotide sequence level. The Em(r) gene from pTE80 (erm80) was then ligated into pUC18/19 to construct replication origin (RO)-screening vectors pUE80(+) and pUE80(-) (pUE80(+/-)). These plasmids contain the pUC18/19-derived multiple cloning site, ampicillin-resistance trait, and the LacZ' gene, which enable direct screening for recombinants in Escherichia coli. Once the recombinant contains a RO from L. reuteri, the Em(r) trait of erm80 is used as a selection marker for the replication of the chimeric plasmid as it is transformed into L. reuteri using the cloned RO as a replicon. Replication regions from pTE80 and pTE15 were successfully cloned into the constructed vector pUE80(-). The RO cloned from pTE80 was further identified as being highly stable in L. reuteri and also bearing a relatively narrow host range compared with that of pTE15. The Em(r) determinant (erm80) and RO cloned from pTE80 could be used in the future construction of derivatives of cloning vectors for this microbe. Moreover, the pUE80(+/-) and pTE80-RO constructed in this study have the potential to be developed as a suicide vector and an E. coli-L. reuteri shuttle vector, respectively.
Lin
C F
CF
Department of Veterinary Medicine, National Chung-Hsing University, Taichung, Taiwan, ROC.
Chung
T C
TC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D004917:Erythromycin / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D007778:Lactobacillus / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D018741:Replication Origin; D012093:Replicon
D004269:DNA, Bacterial; D004917:Erythromycin