aeruginosa suicide

vector, AmpR [26] pUCGmlox AmpR, GmR, pUC18-based vector containing the lox see more flanked aacC1 [26] pCM157 cre expression vector, TcR [33] pGAB10 Deleted rhlG cloned in pEX100Tlink, AmpR This study pFAB1 Deleted PA3388 cloned in pEX100Tlink, AmpR This study pJBB1 Deleted rhlG-PA3388 operon cloned in pEX100Tlink, AmpR This study pGAB10.14 lox flanked aacC1 from pUCGmlox cloned in pGAB10, AmpR GmR This study PFAB1.13 lox flanked aacC1 from pUCGmlox cloned in pFAB1, AmpR GmR This study pJBB11 lox flanked aacC1 from pUCGmlox cloned in pJBB, AmpR GmR This study pGAB Complementation, rhlG cloned in pBBR1MCS-5, GmR This study Rhamnolipid and PQS analyses PQS and selleckchem the p38 MAPK apoptosis major rhamnolipid species (di-rhamnolipid Rha–Rha–C10–C10) were identified and quantified from culture supernatants and cellular

pellet using LC-MS as previously reported [17, 18]. Biofilm formation Biofilms were grown for 24 h in flow cell chambers under dynamic conditions (2.5 ml.h−1 of LB medium) at 37°C as previously described [21], stained with 5 μM SYTO 9 green (Molecular Probes, Invitrogen), observed and quantified by Confocal Laser Scanning Microscopy (CLSM) with a TCS-SP2 microscope (Leica Microsystems, Heidelberg, Germany) using a 63x oil immersion objective. Bioluminescence assays Induction of bioluminescence in bacteria carrying luxCDABE reporter plasmids was detected in optiplateTM 96 wells using the Lumicount apparatus (PerkinElmer, Boston,

Ma.), with a gain set at 1 or 6 and with photomultiplier tubes (PMT) set at 1100. 100 μl of bacterial suspensions were adjusted to the lowest optical density of the different samples, and bioluminescence values of a negative control strain (containing pAB133) were subtracted from values resulting from pAB134-containing strain(s) [34]. Bioluminescence was expressed in RLU/0.5 s. mRNA quantification by quantitative reverse transcription-PCR (qRT-PCR) RNAs SB-3CT were extracted using RNA protect bacteria reagent, RNeasy Midi Kit, and RNase-Free DNase Set (Qiagen, Hilden, Germany). RNAs were converted to cDNAs using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, Ca.). rhlG mRNAs were quantified by real-time PCR amplification of their cDNAs with the 7300 Real Time PCR System apparatus and SYBR Green PCR Master Mix (Applied Biosystems), using procedures previously described [21] and the primers shown in Table 2.