Abstract
Aims: The aim of this project was to explore the different CTX-M expression levels occurring from a single conserved promoter with different spacer sequences, the variation of which is hypothesized to be a key factor in fluctuating levels of CTX-M. Methods: The bla<sub>CTX-M</sub> promoter fragments with five different spacer sequences were amplified, sequenced and cloned into the pUA66 expression vector carrying the green fluorescent protein (GFP) gene. The expression of bla<sub>CTX-M</sub> in the transconjugants was analyzed using fluorescence microscopy, flow cytometry and qRT-PCR. Results: The promoters of all the bla<sub>CTX-M</sub> genes were provided by ISEcp1 and were extremely conserved. The promoter-associated spacer sequences varied from 42 to 127 bp and variations in GFP expression in the five transconjugants were observed. A nucleic acid deletion and point mutation were detected in the spacer sequences by variations in which the expression of bla<sub>CTX-M</sub> was influenced. Conclusion: The different spacer sequences have a significant impact on the activity of the conserved promoter. The shorter spacer sequence between the conserved promoter and the bla<sub>CTX-M</sub> gene does not specifically enhance the expression of bla<sub>CTX-M</sub>, contrary to previous reports. The expression of bla<sub>CTX-M</sub> may be regulated by changes in promoter activity caused by diverse spacer sequences.
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