Dissection of the promoter/operator region and evaluation of N-acylhomoserine lactone mediated transcriptional regulation of elastase expression in Pseudomonas aeruginosa.
In Pseudomonas aeruginosa, expression of the lasB gene which codes for the metalloprotease, elastase, depends on small diffusible N-acylhomoserine lactones. lasB expression is regulated through the interactions of N-3-oxododecanoyl-L-homoserine lactone and N-butanoyl-L-homoserine lactone with the transcriptional activators LasR and VsmR(RhlR), respectively. To investigate lasB expression further, we first located the transcriptional start site to a position 141 bp upstream from the translational start site. Using this information, we constructed a series of plasmids containing consecutive 5' deletions of the upstream region of lasB fused to a promoterless chloramphenicol acetyltransferase reporter gene. The results obtained indicate that three regions are required for efficient transcription of lasB; a 35 bp palindromic sequence located at +26 to +60 bp upstream from the translation start site, and two regions located upstream of the transcription start site, at -135 to -85 bp and -63 to -26 bp, respectively. Deletion of the latter region results in the loss of both N-butanoyl-L-homoserine lactone- and N-3-oxododecanoyl-L-homoserine lactone-mediated stimulation of lasB expression and provides further support for the role of this operator site as a target for either or both LasR and VsmR.
Fukushima
J
J
Department of Bacteriology, Yokohama City University School of Medicine, Japan. jfukusim@med.yokohama-cu.ac.jp
Ishiwata
T
T
You
Z
Z
Ishii
T
T
Shigematsu
T
T
Kurata
M
M
Chikumaru-Fujita
S
S
Bycroft
B W
BW
Stewart
G S
GS
Kawamoto
S
S
Morihara
K
K
Williams
P
P
Okuda
K
K
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D008666:Metalloendopeptidases / Q000235:genetics; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D014158:Transcription, Genetic / Q000502:physiology
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C095458:EsaI protein, Erwinia stewartii; D011994:Recombinant Proteins; D015500:Chloramphenicol O-Acetyltransferase; D010196:Pancreatic Elastase; D008666:Metalloendopeptidases; C043859:pseudolysin, Pseudomonas aeruginosa