Differential CTX-M Expression from a Conserved Promoter: Role of Promoter-Associated Spacer Sequences Downstream of the blaCTX-M Regulon.
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.



The blaCTX-M 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 blaCTX-M in the transconjugants was analyzed using fluorescence microscopy, flow cytometry and qRT-PCR.



The promoters of all the blaCTX-M 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 blaCTX-M was influenced.



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 blaCTX-M gene does not specifically enhance the expression of blaCTX-M, contrary to previous reports. The expression of blaCTX-M may be regulated by changes in promoter activity caused by diverse spacer sequences.
Department of Respiratory and Critical Care Medicine, Guizhou Provincial People's Hospital, Guiyang, PR China.
Lin
L
Liu
Xiangyan
X
Zhang
Siyuan
S
Yang
Yao
Y
Zhai
Weijia
W
Liu
Xiaorong
X
Wang
Zhao
Z
Zhang
Zhancheng
Z
Gao
D005434:Flow Cytometry; D015870:Gene Expression; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D007710:Klebsiella Infections / Q000382:microbiology; D007711:Klebsiella pneumoniae / Q000201:enzymology / Q000302:isolation & purification; D008856:Microscopy, Fluorescence; D011401:Promoter Regions, Genetic; D060888:Real-Time Polymerase Chain Reaction; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000235:genetics*
D049452:Green Fluorescent Proteins; D001618:beta-Lactamases