Use of quantitative real-time RT-PCR to analyse the expression of some quorum-sensing regulated genes in Pseudomonas aeruginosa.
P. aeruginosa living in biofilm populations sends out diffusive signalling molecules, called autoinducers, for example acylated homoserine lactone (AHL) or the P. aeruginosa quinolone signal (PQS). So far, two quorum-sensing systems, LasR and VsmR, have been identified in P. aeruginosa, both of which are required for all virulence determinants. The expression of specific genes involved in quorum-sensing regulatory mechanisms has been analysed with molecular biology methods. Real-time quantitative PCR is a highly sensitive and powerful technique for quantification of nucleic acids. Expression of the genes vsmR, lasI, and PA4296 was studied by use of reverse transcriptase and subsequent quantitative real-time PCR of the cDNAs. In parallel, expression of ribosomal 16S rRNA, used as a housekeeping gene that was constitutively expressed in all analyses, was also monitored. Biofilm was compared with planktonic bacteria, and in contrast to vsmR and Pa4296, the lasI gene was found to be down-regulated in biofilm. Extended experiments were run with synthetic signal molecules inducing regulated processes in bacterial populations. It was shown that the genes under investigation were up-regulated in mature biofilm in the presence of the signal molecule N-(3-oxododecanoyl)-L-homoserine lactone.
Schwartz
Thomas
T
Department of Environmental Microbiology, Institute for Technical Chemistry - Water Technology and Geotechnology Division, Forschungszentrum Karlsruhe, P.O. Box 3640, 76021, Karlsruhe, Germany. thomas.schwartz@itc-wgt.fzk.de
Walter
Sandra
S
Marten
Silke-Mareike
SM
Kirschhöfer
Frank
F
Nusser
Michael
M
Obst
Ursula
U
D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000494:pharmacology; D018441:Biofilms; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects / Q000502:physiology; D006714:Homoserine / Q000031:analogs & derivatives / Q000494:pharmacology; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000502:physiology; D053038:Quorum Sensing / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
C109860:N-(3-oxododecanoyl)homoserine lactone; D012333:RNA, Messenger; D006714:Homoserine; D015107:4-Butyrolactone