Fusion with ribosomal protein L32 increased the in vivo thermostability of kanamycin nucleotidyltransferase in Thermus thermophilus

Abstract

The kanamycin resistance (Km r) gene widely used as an antibiotic resistance marker in Thermus strains was expressed at temperatures of up to 60°C in Thermus thermophilus HB27. We isolated a clone from HB27 that exhibited the Km r phenotype at 70°C. The clone contained a recombinant plasmid pPP442 in which the promoterless Km r gene was preceded by a 236-bp fragment from HB27 chromosome. The location of the transcriptional start site in the 236-bp fragment was determined. The putative promoter sequence in this fragment exhibited similarity to the consensus promoter sequence of T. thermophilus, but its sequence identities with −35 and −10 consensus sequences were low. The activity of the putative promoter was found not to be very strong based on the results of primer extension. A ribosome binding sequence followed by a truncated open reading frame (ORF) was identified in the 236-bp fragment. The ORF was in frame with the Km r gene. No mutation was found in the Km r structural gene. A pPP442 derivative, pPP442-ΔEcUD, was constructed by introducing a frame shift mutation into the ORF. T. thermophilus carrying pPP442-ΔEcUD could not grow at 70°C in Km-containing medium. The location of the 236-bp fragment on the physical map of T. thermophilus HB27 was determined. The entire ORF was cloned from the genome and the amino acid sequence deduced from the nucleotide sequence showed strong homology to that of ribosomal protein L32 of T. thermophilus HB8. These results suggested that fusion of the ribosomal protein L32 to the N-end of the Km r gene product increased the thermostability of the Km r gene product in T. thermophilus.