Studies on antibiotic resistance in producing microorganisms

Abstract

The aminoglycoside phosphotransferase (APH) gene of B. circulans has been cloned in E. coli (CC1105) using the vector pBR322 and has been found to confer antibiotic resistance. The 2.7 kb Sal I fragment containing the APH gene was ligated into the plasmid SLP1.2 and transformed into S. lividans 66 (CL1023), where it was also able to confer antibiotic resistance. When the phosphotransferase activity was assayed in crude extracts of B. circulans, E. coli CC1105 and S. lividans CL1023, using a variety of aminoglycosides as phosphate acceptors, a similar spectrum of activity was found in each strain. Using the methods of Sanger and Messing, the sequence of the 2.7 kb Sal I fragment was determined. An examination of the DNA sequence allowed the protein sequence to be deduced. When the deduced protein sequence was compared with those from S. fradiae, TN5 and TN903, significant homology was found, indicating that all the phosphotransferases may have a common origin. 

An examination of the sequence either side of the coding region allowed the identification of possible promotors, a ribosome binding site and a terminator. One of the promotors was similar to the σ³⁷ promotors of B. subtilis, showing that the B. circulans APH gene may be under developmental control.

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