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Applied and Environmental Microbiology, December 2003, p. 7173-7180, Vol. 69, No. 12
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.12.7173-7180.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Heat Shock Treatment Increases the Frequency of Loss of an Erythromycin Resistance-Encoding Transposable Element from the Chromosome of Lactobacillus crispatus CHCC3692

Department of Genomics and Strain Development, Chr. Hansen A/S, DK-2970 Hørsholm, Denmark

Received 31 March 2003/ Accepted 12 September 2003

A 3,165-bp chromosomally integrated transposon, designatedTn3692, of the gram-positive strain Lactobacillus crispatus CHCC3692 contains an erm(B) gene conferring resistance to erythromycin at concentrations of up to 250 µg/ml. Loss of this resistance can occur spontaneously, but the rate is substantially increased by heat shock treatment. Heat shock treatment at 60°C resulted in an almost 40-fold increase in the frequency of erythromycin-sensitive cells (erythromycin MIC, 0.047 µg/ml). The phenotypic change was followed by a dramatic increase in transcription of the transposase gene and the concomitant loss of an approximately 2-kb DNA fragment carrying the erm(B) gene from the 3,165-bp erm transposon. In cells that were not subjected to heat shock, transcription of the transposase gene was not detectable. The upstream sequence of the transposase gene did not show any homology to known heat shock promoters in the gene data bank. Significant homology (>99%) was observed between the erythromycin resistance-encoding gene from L. crispatus CHCC3692 and the erm(B) genes from other gram-positive bacteria, such as Streptococcus agalactiae, Streptococcus pyogenes, Enterococcus faecium, and Lactobacillus reuteri, which strongly indicates a common origin of the erm(B) gene for these species. The transposed DNA element was not translocated to other parts of the genome of CHCC3692, as determining by Southern blotting, PCR analysis, and DNA sequencing. No other major aberrations were observed, as judged by colony morphology, growth performance of the strain, and pulsed-field gel electrophoresis. These observations suggest that heat shock treatment could be used as a tool for the removal of unwanted antibiotic resistance genes harbored in transposons flanked by insertion sequence elements or transposases in lactic acid bacteria used for animal and human food production.

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