use of quantitative real-time rt-pcr to analyse the expression of some quorum-sensing regulated...

ORIGINAL PAPER

Use of quantitative real-time RT-PCR to analysethe expression of some quorum-sensing regulated genesin Pseudomonas aeruginosa

Thomas Schwartz & Sandra Walter &

Silke-Mareike Marten & Frank Kirschhöfer &

Michael Nusser & Ursula Obst

Received: 12 June 2006 /Revised: 4 October 2006 /Accepted: 6 October 2006 / Published online: 28 November 2006# Springer-Verlag 2006

Abstract P. aeruginosa living in biofilm populations sendsout diffusive signalling molecules, called autoinducers, forexample acylated homoserine lactone (AHL) or the P.aeruginosa quinolone signal (PQS). So far, two quorum-sensing systems, LasR and VsmR, have been identified inP. aeruginosa, both of which are required for all virulencedeterminants. The expression of specific genes involved inquorum-sensing regulatory mechanisms has been analysedwith molecular biology methods. Real-time quantitativePCR is a highly sensitive and powerful technique forquantification of nucleic acids. Expression of the genesvsmR, lasI, and PA4296 was studied by use of reversetranscriptase and subsequent quantitative real-time PCR ofthe cDNAs. In parallel, expression of ribosomal 16S rRNA,used as a housekeeping gene that was constitutivelyexpressed in all analyses, was also monitored. Biofilmwas compared with planktonic bacteria, and in contrast tovsmR and Pa4296, the lasI gene was found to be down-regulated in biofilm. Extended experiments were run withsynthetic signal molecules inducing regulated processesin bacterial populations. It was shown that the genesunder investigation were up-regulated in mature biofilm

in the presence of the signal molecule N-(3-oxododeca-noyl)-L-homoserine lactone.

Keywords Gene expression . RT-PCR . Quorum sensing .

Pseudomonas aeruginosa . Biofilm

Introduction

One of the best characteristics of the functional status of acell is its gene expression pattern. Cells belonging todifferent tissues, cells in different development or metabolicstages, and cells affected by specific compounds differ bytheir gene expression patterns and, thus, in their mRNApools. The most important technique for accurate quan-tification of gene expression is quantitative fluorescencereal-time RT-PCR [1, 2]. In the last few years quantitativereal-time RT-PCR has become an important method foranalysis of gene expression in a vast variety of samples [2].

Typically, expression of the target gene is analysed withthat of a reference gene to normalise the amount of the PCRtemplate and, thus, to calculate the relative expression levelof the target gene (i.e. normalised gene expression) [3].Instead of using a standard plot, target gene expressionlevels are calculated relative to the reference. The referencemust therefore be a housekeeping gene, for example therRNA gene, that is not affected by the experimentalsituation [4].

This type of quantitative approach to gene expressionanalysis is the subject of this study, in which selected genesinvolved in bacterial communication, known as quorumsensing, were quantified. The remarkable complexity ofquorum-based systems is exemplified by the variety ofdifferent mechanisms for signal production, signal detec-

Anal Bioanal Chem (2007) 387:513–521DOI 10.1007/s00216-006-0909-0

T. Schwartz : S. Walter : S.-M. Marten : F. Kirschhöfer :M. Nusser :U. ObstDepartment of Environmental Microbiology,Institute for Technical Chemistry – Water Technologyand Geotechnology Division, Forschungszentrum Karlsruhe,P.O. Box 3640,76021 Karlsruhe, Germany

T. Schwartz (*)Department of Environmental Microbiology, ITC-WGT,Forschungszentrum Karlsruhe,76344 Eggenstein-Leopoldshafen, Germanye-mail: [email protected]

tion, signal relay, and signal response [5]. Pseudomonasaeruginosa is a rod-shaped Gram-negative environmentalbacterium known for its antibiotic resistance and forcausing infections, for instance, in the respiratory tract oramong individuals with labile immune systems. P. aerugi-nosa is a typical biofilm bacterium which is very versatile,because it is capable of producing many virulence factors,for example elastase, protease, alkaline protease, andothers. It is, moreover, known that P. aeruginosa living inbiofilm populations sends out signals, for example acylatedhomoserine lactone (AHL) or the P. aeruginosa quinolonesignal (PQS). These signals are diffusive signalling mole-cules, called autoinducers [6]. Two quorum-sensing sys-tems have so far been identified in P. aeruginosa; both arerequired for all virulence determinants. These systems arethought to exist in a hierarchy in which the Las systemtakes over transcriptional control. In P. aeruginosa PAO1LasR and VsmR have been implicated in the regulation ofstructural genes. The las system consists of the transcrip-tional activator LasR and the AHL synthase LasI, whichdirects the biosynthesis of N-3-oxo-dodecanoyl-homoserinelactone. The vsm (rhl) system consists of the transcriptionalactivator VsmR and the enzyme VsmI, which is responsiblefor the biosynthesis of N-butanoyl-homoserine lactone(BHL). Approximately 123 two-component systems(2CSs) are, furthermore, annotated according to the mostrecently updated database of the Pseudomonas aeruginosagenome project [7]. The number of 2CS genes inPseudomonas aeruginosa is relatively high in comparisonwith that in the E. coli and Bacillus genomes. This is likelyto help the bacteria to adapt to different environments,although the function of approximately two thirds of the2CS genes has not yet been characterised.

The quorum-sensing-dependent production of exoprod-ucts by P. aeruginosa is tightly regulated with regard togrowth phase and growth environment. It has been reportedthat provision of exogenous AHLs does not enhance theexpression of several quorum-sensing-dependent genes inwild-type P. aeruginosa PAO1 [8].

The main objective of this study was to detect specificgene expression among Pseudomonas aeruginosa living inbiofilm or in the free water phase. The expression ofregulatory genes involved in quorum sensing and 2CS in P.aeruginosa was investigated in the presence and absence ofthe autoinducer N-(3-oxododecanoyl)-L-homoserine lactone(3O-C12-HSL). The lasI gene is a structural gene codingfor the AHL synthase; the vsmR gene is the transcriptionalactivator of the vsm(rhl) system. The PA4296 gene isdescribed as a probable two-component response regulatorand has been selected from the P. aeruginosa PAO1 genebank entry [7] to study its role in quorum sensing. Differentprimers and probes that amplify genes of regulatorysystems in P. aeruginosa were studied by use of reverse

transcriptase, which converts gene-specific mRNA into acDNA, followed by the real-time polymerase chain reaction(TaqMan-PCR) to quantify the cDNA. To detect such aninduction, P. aeruginosa had to be grown in biofilmcommunities and in planktonic bacterial cultures. Theinduction profiles were also studied after addition of AHLto these cultures.

Materials and methods

Cultivation and quantification

Pseudomonas aeruginosa was isolated from a municipalwastewater sample. Its relationship to those strains forwhich genome sequences have been obtained is, therefore,unknown. The strain was identified using API 20NE(BioMerieux, Nürtingen, Germany). It was cultivated onCetremid agar plates (Oxoid), enriched with brain heartinfusion (BHI) medium (Oxoid), and stored in glycerolsolution at −80 °C.

A 0.5-L plastic cylindrical bioreactor was provided withone vial at each end to connect the bioreactor to anelectrical pump for media supply. When the lid was open abar which contained six steel platelets (15 mm×3.5 mm×1.5 mm) for biofilm growth was inserted into the bioreactor.Plastic tubing of diameter 5 mm was used to circulate BHImedium, which was diluted with sterile drinking water inthe ratio 1:4. The autoclaved bioreactor and tubes wereconnected to the pump. The system was inoculated with40 mL Pseudomonas aeruginosa culture grown overnightin BHI medium (diluted 1:4) at 37 °C. The pump was thenswitched on at a pumping speed of approx. 0.1 L min−1 andthe system was run in circulation for at least 4 h. The outlettube was then inserted into a waste container, whereasdiluted BHI medium passed the bioreactors. The biofilmgrew over a period of 5 days at room temperature.

DAPI staining of bacteria

Biofilm and planktonic bacteria were stained with DAPI toenable monitoring of cell density. DAPI is a bluefluorescent dye used to stain double-stranded DNA. Itbecomes attached to the minor groove of the DNA helixaround A–T clusters. The platelets with biofilm populationswere removed from the bioreactor and bacterial cells werescraped from the platelets into 5 mL phosphate-bufferedsaline solution. DAPI solution (1 mg mL−1, 20 μL) wasalso added to the bacterial mixture and the solution wasincubated for 10 min at room temperature in the dark. Thesolution was then filtered through a polycarbonate filter ofpore size 0.2 μm (Costar). The membrane had to be driedon a glass slide. For planktonic samples, serial dilutions of

514 Anal Bioanal Chem (2007) 387:513–521

the cultures were prepared and known amounts were mixedwith DAPI solution and treated as described for biofilmsamples. One drop of Citifluor (Citifluor, London) wassprinkled over the stained bacterial cells before a glass slipwas used to fully cover the filter. Finally, it was preparedfor microscopic evaluation. For this, a DAPI filter, BP365/FT 395/LP 397, was used to count 10 squares with the helpof 1000× magnification (Zeiss AxioPlan 2, Oberkochen,Germany).

RNA extraction

When incubation had been stopped, the platelets wereremoved from the bioreactor and 1 mL RNAprotect(Qiagen, Hilden, Germany) was added to inhibit RNAdegradation or further gene induction effects. The biofilmswere scraped from the coupons into 1 mL RNAprotect andtransferred to 2-mL reaction tubes. Each planktonic bacteriasolution (2 mL) was also added to 4 mL RNAprotect. Fromthese mixtures, 1 mL was transferred to 2 mL reaction tubesfor further processing. The suspensions were then centri-fuged at 8000 rpm for 5 min at room temperature. Thesupernatant was decanted and the pellets were stored at−80 °C for isolation of RNA.

Extraction of total RNA from the biofilms wasperformed by use of Qiagen (Hilden, Germany) technology,with the Qiagen RNeasy Mini Protocol. DNase digestionwith the RNase-free DNase Set (Qiagen) was, however,advantageous for further application. The samples weretherefore treated with DNase I solution in accordance withthe Qiagen handbook. To determine residual DNA contam-ination of the RNA extracts 10-μL samples of the totalRNA samples were used as templates for eub TaqMan PCRwithout previous reverse transcription. The results verifiedthe purity of the RNA extracts. All devices and solutionswere treated or prepared with diethyl pyrocarbonate(DEPC) to inactivate any RNases. All buffers used wereprovided by Qiagen and are described as RNase-free.

The quantity and purity of the total RNA were measuredby use of a GeneQuant Photometer (Amersham; Freiburg;Germany).

Primer and probe design

The primers were designed for real-time PCR with thePrimer Express software package (PE-ABI, Warrington,UK). All primers and probes of the genes vsmR, lasI,PA4296 are specific for Pseudomonas aeruginosa PAO1(NCBI Genbank accession number AE004091) and theuniversal system eub16S is complementary to sequences ofthe 16S rDNA from Eubacteria. The oligonucleotides usedwere designed in silico using the NCBI database [7] andpurchased from Applied Biosystems (Darmstadt, Germany)(Table 1). The master mixture was prepared in accordancewith the manufacturer’s recommendations. Briefly, itcontained 200 nmol L−1 of each oligonucleotide primer(forward primer, FP; reverse primer, RP) and 100 nmol L−1

fluorescent labelled probe; 6-FAM is 6-carboxyfluoresceinand TAMRA is 6-carboxytetramethylrhodamine.

Controls

Negative control templates (NTC) consisted of the mastermixture with sterile water as a template. NA (2 μL, i.e.50 ng) extracted from Pseudomonas aeruginosa in sterilewater was added to the master mixture in the same way, asthe positive control.

Reverse transcriptase and quantitative TaqMan PCR

The reverse transcriptase (RT) reaction combined with real-time PCR enables more sensitive quantification of geneexpression. Reverse transcription (RT) was performed ac-cording to the manufacturer’s instructions (Applied Bio-systems) with 5 μL tenfold concentrated RT buffer, 11 μL25 mmol L−1 MgCl2, 10 μL 200 μmol L−1 dNTP mixture,

Table 1 List of sequences ofgene-specific primers (FP andRP) and probes (P) used forreal-time TaqMan PCR. Re-verse primers (RP) were usedfor the reverse transcriptasereaction. All primers andprobes were designed and test-ed in this study

Primers and probes Sequences Target

eub16 FP 5′-GATCAGCCACACTGGGACTGA-3′ Eubacteriaeub16 RP 5′-TCAGGCTTGCGCCCATT-3′ Eubacteriaeub16 P FAM-5′-TCCTACGGGAGGCAGCAGTGGG-3′-TAMRA EubacterialasI FP1 5′-GCCCCTACATGCTGAAGAACA-3′ P. aeruginosalasI RP1 5′-CGAGCAAGGCGCTTCCT-3′ P. aeruginosalasI P FAM-5′-CTTCCCGGAGCTTCTGCACGGC-3′-TAMRA P. aeruginosaPA4296 FP 5′-CGGCAACGGCAGGTTCT-3′ P. aeruginosaPA4296 RP 5′-CATGGCCTCGATCACTTCCT-3′ P. aeruginosaPA4296 P FAM-5′-AATCGATCATCCTCACCGGTCACGA-3′-TAMRA P. aeruginosavsmR FP 5′-TGTTCGCCGTCCTGGAA-3′ P. aeruginosavsmR RP 5′-CGCCATAGGCGTAGTAATCGA-3′ P. aeruginosavsmR P FAM-5′-AGGAAGTGCGGCGCCTGGG-3′-TAMRA P. aeruginosa

Anal Bioanal Chem (2007) 387:513–521 515

2.5 μL complementary reverse primer (2.5 μmol L−1), 1 μLRNase inhibitor, 1.25 μL MultiScribe polymerase, 100 ngtotal RNA, and water to give a final reaction volume of50 μL. The mixture was incubated for 30 min at 48 °C forreverse transcription and then for 5 min at 95 °C toinactivate the polymerase.

Subsequent TaqMan PCR is a sensitive method forquantification of the cDNA yield of the previous RTreaction. Amplification of the desired DNA sequence isperformed in a Thermocyler combined with a fluorescencespectrometer supplied by Applied Biosystems (ABI 7700Sequence detection system). This measuring device reportsevery increase in concentration of the amplified DNAsequence of each PCR cycle. The principle is addition of afluorogenic gene probe which carries a reporter dye at the5′-end (FAM) and a quencher at the 3′-end (TAMRA)(Fig. 1). For each reaction, 25 μL of the twofold UniversalMaster Mix buffer (Applied Biosystems) was mixed with5 μL forward primer (5 μmol L−1), 5 μL reverse primer(5 μmol L−1), 5 μL fluorescent labelled probe (5 μmolL−1), and water to give a final volume of 50 μL. The cDNAyield of each reverse transcriptase reaction was first dilutedwith sterile PCR water at a ratio of 1:10 and a second timeat a ratio of 1:4. Of this dilution, 23 μL was used astemplate for TaqMan PCR.

Applied Biosystems supply the user with an optimisedUniversal Master Mix (uMM) for quantitative PCR assays,including dNTPs, AmpliTaq Gold DNA Polymerase,AmpErase UNG (uracil-N-glycosidase), MgCl2, buffercomponents, and the fluorogenic dye ROX as passivereference.

The AmpliTaq Gold polymerase used for this TaqMansystem is a recombinant form of the AmpliTaq DNAPolymerase, which was reversibly activated after incubationfor 9 to 12 min at 95 °C. To optimise probe hybridisation,so-called two-step PCR was performed under standardconditions. This was possible because of the substantialactivity of the AmpliTaq Gold at temperatures >55 °C.Choice of primers with a Tm of approximately 60 °Cfacilitated the two-step PCR procedure. To protect thereaction against carry-over contamination, the AmpEraseUNG had to be incubated for 2 min at 50 °C.

The ABI7700 was run for 2 min at 50 °C, 10 min at95 °C, then 40 cycles of 15 s at 95 °C and 1 min at 60 °C.

Synthesis of 3-oxo-12C-homoserine lactone (3-O-12C-HL)

L-Homoserine, decanoylic acid, Meldrum’s acid, 4-(dimeth-ylamino)pyridine, N,N-dicyclohexylcarbodiimide, and otherstarting chemicals were purchased from Sigma–Aldrich

Fig. 1 Principle of TaqManPCR based on the 5′-3′-exonu-clease activity of Taq polymer-ase, in accordance with http://edoc.hu-berlin.de/dissertationen/kuner-ruprecht-2002-07-02/HTML/objct8.png


http://edoc.hu-berlin.de/dissertationen/kuner-ruprecht-2002-07-02/HTML/objct8.png




(Munich, Germany). N-(3-Oxododecanoyl)-L-homoserinelactone (3-O-C12-HSL, 4) was synthesised in accordancewith Chhabra [9]. In brief, one equivalent of Meldrum’sacid, 1.1 equivalents of 4-(dimethylamino)-pyridine(DMAP), and 1.1 equivalents of N,N-dicyclohexylcarbodi-imide (DCC) were added to a solution of decanoylic acid indichloromethane. This furnished acylated Meldrum’s acidwhich was used in the next step without further purifica-tion. Homoserine lactone and triethylamine were stirredunder reflux in acetonitrile. The desired 3O-C12-HSL wasisolated and purified by liquid chromatography on silicagel. Structure and identity were verified by electrosprayionisation mass spectrometry (ESI–TOFMS) and 1H NMRspectroscopy.

In ESI–TOFMS (Applied Biosystems Mariner API-TOFWorkstation), calculation for C16H28NO4 (MH+) yieldedm/z 298.19 and the measured values correlated with m/z298.19 (Fig. 2). In 1H NMR (Bruker AMX 500) withCDCl3 as solvent, the following chemical shifts weredetected for 3O-12C-HSL, as expected: 0.81 (3H, t, CH3),1.19 (12H, m, CH3(CH2)6), 1.57 (2H, m, CH2CH2CO),2.16 (1H, m, 4R-H), 2.46 (2H, t, CH2CO), 2.69 (1H, m,4-H), 3.40 (2H, s, COCH2CO), 4.21 (1H, m, 5R-H), 4.42(1H, td, 5-H), 4.53 (1H, m, 3-H), 7.62 (1H, d, NH).

Statistics

Two independent experiments (A, B) were performed todetect differences between gene expression in planktonic

and biofilm populations of P. aeruginosa. In biofilmanalysis at least three of the six platelets were removedfrom bioreactor for RNA extraction in each experiment.The RNA extracts were pooled for photometrical quantifi-cation and molecular biology testing. Three replicates wereused for cDNA quantification of each target gene inexperiments A and B. Similar to these experiments twoindependent experiments were performed to determine theimpact of N-(3-oxododecanoyl)-L-homoserine lactone ongene expression in biofilms. Biofilm from three plateletswas used for RNA extraction and pooled RNA were usedfor molecular analysis. Again, several replicate analyseswere performed for cDNA quantification of each targetgene.

Results

Cell counts and RNA content

Cell densities of Pseudomoas aeruginosa biofilms werecalculated to range from 104 to 105 cells cm−2 per plateletafter incubation for 5 days. In contrast with biofilms, theconcentration of planktonic bacteria reached mean values of108 cells mL−1, which may be used for RNA extraction. Asa consequence, the total RNA isolated from P. aeruginosacultures ranged from 7.2 to 10.4 μg mL−1 for P. aeruginosagrown in a biofilm compared with 354 to 524 μg mL−1 forP. aeruginosa grown in planktonic communities. As

Fig. 2 Spectrum obtained fromN-(3-oxododecanoyl)-L-homo-serine lactone (3O-C12-AHL)by ESI-TOFMS, with its chem-ical structure and molecularweight


expected, the biomass yield of planktonic bacteria wasmuch greater than for biofilm populations.

Gene expression in biofilm and planktonic populations

The combination of a reverse transcriptase and TaqManPCR enabled sensitive quantification of gene expression.The intersection of the amplification curve and thethreshold value yielded the Ct–value (threshold cycle). AΔCt-value of 3.3 corresponds to a difference of targetsequence concentration of a factor of 10. Universaloligonucleotide primers specific to the conserved regionof the eubacterial 16S rRNA gene were designed for use inthe real-time PCR (TaqMan) system. Analysis of RNAexpression using techniques like real-time PCR traditional-ly uses reference or housekeeping genes to control errorsamong the samples. In this work we used the eubacterialTaqMan system to determine expression in Pseudomonasaeruginosa. To verify the systems, several dilutions of totalRNA from 100 to 10−3 were analysed in a ReverseTranscriptase-TaqMan PCR (Fig. 3). The Ct-values of theundiluted RNA extract corresponded to cycle 19, the 10−1

dilution to cycle 22.4, the 10−2 dilution to 25.7, and the10−3 dilution to cycle 28.9. Average ΔCt-values forconsecutive dilution steps were 3.2 and met the analyticalrequirement for gene expression analysis. The experimentsalso revealed that the amounts of total RNA used coveredthe range for optimum fluorescence detection during Taq-Man PCR amplification. TaqMan PCR cycles from 1 to 15were required for baseline equilibration, and Ct valueshigher than 38 are believed to be close to the detectionlimit.

The Ct-value for eub16 TaqMan PCR was 21 for bothbiofilms and planktonic cultures. Thus, the concentration of16S rRNA was found to be constant in relation to the totalamount of RNA. On addition, the outcome of thisexperiment also proved that photometric quantification ofthis method was correct. The no-template controls (NTC)for eub16 occasionally had Ct-values close to the detectionlimits, indicating possible weak contamination of reagentssuch as the polymerase with ribosomal nucleic acids.

Comparison of Ct-values from different experimentsrevealed expression of the ribosomal 16S gene was highlyexpressed in all experiments with constant Ct values(Tables 2, 3 and 4). Because of the constancy of the Ctvalues of this reference gene normalization of the Ct valuesof the compared target genes was not required. Bycomparing different expression experiments a ΔCt valuewas calculated and from the exponential fluorescenceincrease of the signal the gene induction factor wasestimated to be 2ΔCt . Comparison of biofilms, for whichthe Ct-values were approximately 32, with planktonicbacteria yielded an average ΔCt of 5.5, corresponding toexpression in biofilms that is smaller by a factor of 47.Compared with the lasI gene, the amount of vsmR geneinduced in biofilms was greater by a factor of 6. For the2CS gene PA4296 increased induction of gene expressionwas also found in biofilms, but the induction factorsdiffered in both independent experiments by 4.5 and 1.8,respectively. The gene-expression data, in particular forlasI, were, nevertheless, indicative of specific regulation ofthe quorum-sensing-relevant genes in populations of P.aeruginosa, depending on their lifestyle.

Fig. 3 Graphical display of aTaqMan reaction with dilutions(100 to 103) of the total RNAextracted from a Pseudomonasaeruginosa biofilm. Eubacterialprimers and probe were used forreal-time PCR after the reversetranscriptase reaction with thereverse primer of the eub16oligonucleotide set. The ampli-fication plot shows three repli-cates for the undiluted sampleand for the three consecutivediluted RNA samples


Expression in the presence of N-(3-oxododecanoyl)-L-homoserine lactone

The impact of signal molecules such as N-(3-oxododeca-noyl)-L-homoserine lactone (3O-C12-AHL) on gene ex-pression was analysed by use of another approach. It wasimpossible to grow biofilms in a reactor-like systembecause of the large amounts of synthetic AHL necessary.BHI medium (20 mL) diluted at a ratio of 1:4 was thereforesupplemented with N-(3-oxododecanoyl)-L-homoserine lac-tone in a final concentration 1 μmol L−1. After inoculationwith P. aeruginosa the biofilms grew on the steel plateletswithin 5 days of incubation at room temperature. The

results in Table 3 show induction of gene expression inbiofilms with N-(3-oxododecanoyl)-L-homoserine lactone.

3-Oxo-C12-homoserine lactone was responsible for up-regulation of the three genes PA 4296, lasI, and vsmR in P.aeruginosa biofilm populations (Table 3). In planktonicpopulations only weak induction of gene expression byAHL (a factor of 1–1.5) were observed (Table 4). Incontrast with the biofilm population, application of exog-enous N-(3-oxododecanoyl)-L-homoserine lactone to bacte-rial suspensions did not increase target gene expressionsignificantly. Occasionally the Ct value for negativecontrols (NTC) targeting the 16S reference gene wasslightly below Ct 40. But, these Ct values were within the

Table 3 Gene expression measured by TaqMan PCR after the reverse transcription of specific mRNA from P. aeruginosa biofilms

Target Experiment A Experiment B

Ct ΔCt Factor Ct ΔCt Factor

with AHL no AHL with AHL no AHL

vsmR 32.85±0.07 36.54±0.12 3.69 +12.9 32.59±0.19 36.52±0.2 3.93 +15.2NTC >40 >40 – >40 >40 –lasI 30.09±0.09 33.62±0.22 3.53 +11.5 30.07±0.34 33.59±0.25 3.53 +11.5NTC >40 >40 – – >40 >40 – –PA4296 32.87±0.11 37.47±0.32 4.6 +24.3 32.54±0.33 37.98±0.35 5.4 +42.2NTC >40 >40 – >40 >40 –eub16 20.12±0.12 20.52±0.32 n.d. n.d. 20.65±0.29 20.43±0.26 n.d. n.d.NTC 39.57 >40 >40 39.58

Ct is the cycle threshold value±standard error of three replicates in experiments A and BNTC: no template control; n.c.: not calculated; n.d.: not determinedThe Ct is the mean of three replicates and ΔCt is the difference between AHL-incubated and untreated biofilms. The induction factor wasobtained from the formula 2ΔCt (+, up-regulation; −, down-regulation)Biofilms grown in the presence of N-(3-oxododecanoyl)-L-homoserine lactone were compared with untreated biofilms

Table 2 Gene expression measured by TaqMan PCR after the reverse transcription of specific mRNA from P. aeruginosa



Biofilm Planktonic Biofilm Planktonic

vsmR 32.77±0.2 35.45±0.34 2.68 +6.4 33.09±0.2 35.53±0.2 2.44 +5.4NTC >40 >40 – >40 >40 –lasI 31.72±0.22 26.24±0.18 5.48 −44.6 33.64±0.2 28.01±0.2 5.63 −49.5NTC >40 >40 – – >40 >40 – –PA4296 31.66±0.32 33.84±0.27 2.18 +4.5 33.38±0.2 34.22±0.2 0.84 +1.8NTC >40 >40 – >40 >40 –eub16 20.82±0.11 20.12±0.18 0.7 n.c. 20.25±0.23 20.93±0.36 0.68 n.c.NTC >40 39.68 n.d. n.d. 39.54 39.12 n.d. n.dPositive 16.22±0.2 16.32±0.18 n.c. n.c. 16.19±0.12 16.25±0.13 n.d. n.d.

Ct is the cycle threshold value±standard error of three replicates in experiments A and BNTC: no template control; n.c.: not calculated; n.d.: not determinedThe Ct is the mean of three replicates and ΔCt is the difference between biofilm and planktonic samples. The induction factor was obtained fromthe formula 2ΔCt (+, up-regulation; −, down-regulation)Biofilms were compared with planktonic cultures


detection limit range of the system and were negligible. Init is also known that polymerase enzymes are contaminatedwith ribosomal DNA, which could be targeted by the eubTaqMan system used.

Discussion

Bacterial-adhesion and biofilm-formation processes onnatural and abiotic surfaces are discussed in differentmodels. The most advanced model includes five stages ofbiofilm development:

1. reversible attachment;2. irreversible attachment;3. maturation-1 with cell clusters embedded in the EPS

matrix;4. maturation-2 with cell clusters reaching their maximum

thickness; and5. dispersion with motile cells swimming away from their

cell clusters [10].

In this study selected gene expression systems wereestablished with the objective of quantifying the effects ofenvironmental stress factors (pharmaceutical products,temperature, osmotic changes, etc.) on biofilm formationin other experimental approaches. Because of the biologicalsignificance as a model system some genes of the quorum-sensing regulon were selected for study of differential geneexpression.

It is known that P. aeruginosa has at least two AHL-dependent quorum-sensing systems which are composed ofLasRI and VsmRI (RhlI) [11]. LasI directs the synthesis of

3O-C12-HSL and is under the regulatory control of LasRwhereas VsmRI directs the synthesis of (C4-HSL) [12, 13].Each system modulates a regulon comprising an over-lapping set of genes, however [8]. The authors havedemonstrated that quorum-sensing-dependent genes cannotbe enhanced by addition of either 3O-C12-HSL or C4-HSLin P. aeruginosa. In contrast with this, addition of therespective cognate AHL signal modules induced quorum-sensing-dependent production of antibiotics in Erwiniacarotovora [14] and bioluminescence in Vibrio fischeri[15].

In this study the methods of molecular biology wereused to quantify the expression of quorum sensing-regulated genes and a two-component regulatory gene inPseudomonas aeruginosa biofilms and planktonic bacteria.From the total RNA, specific cDNA was amplified by areverse transcriptase reaction and subsequent real-timePCR. In planktonic P. aeruginosa the expression of thegenes under investigation was quantified in the same range,irrespective of the presence or absence of 3O-C12-HSL. Inbiofilm bacteria the presence of 3O-C12-HSL induced theexpression of quorum-sensing-related genes (lasI, vsmR),but also a gene of the two-component system (2CS). AHLwas used in fivefold higher concentration than in thestudies of Diggle [8], which might have an inductive impacton genes in P. aeruginosa biofilm cells. In addition,quantification of gene expression by reverse transcriptaseand subsequent real-time PCR did not use any reporter geneapproach, but is reported to be very sensitive to and specificfor selected genes. Housekeeping genes such as theribosomal 16S gene served as standards to compare thesimilarities of different experiments with regard to calcu-lated RNA concentrations and their effective on reverse

Table 4 Gene expression measured by TaqMan PCR after the reverse transcription of specific mRNA from P. aeruginosa planktonic cultures



with AHL no AHL with AHL no AHL

vsmR 32.71±0.31 32.91±0.36 0.2 +1.5 32.97±0.29 33.12±0.32 0.15 +1.1NTC >40 >40 – >40 >40 –lasI 27.27±0.19 27.62±0.28 0.35 +1.27 28.22±0.27 28.59±0.23 0.37 +1.29NTC >40 >40 – – >40 >40 – –PA4296 34.47±0.4 34.11±0.35 0.36 +1.28 34.01±0.23 34.04±0.17 0.03 +1.0NTC >40 >40 – >40 >40 –eub16 21.12±0.24 21.02±0.18 n.d. n.d. 20.77±0.24 20.89±0.33 n.d. n.d.NTC 39.57 39.65 >40 >40

Ct is the cycle threshold value±standard error of three replicatesNTC: no template control; n.d.: not determinedThe Ct is the mean of three replicates and ΔCt is the difference between AHL-incubated and untreated cultures. The induction factor was obtainedfrom the formula 2ΔCt (+, up-regulation; −, down-regulation)Cultures grown in the presence of N-(3-oxododecanoyl)-L-homoserine lactone were compared with untreated cultures


transcription in cDNA. Some workers have quantified theamount of rRNA per cell, by determining the cDNA/genomic DNA ratio, and have demonstrated the regulationof structural genes during biofilm formation [16, 17]. By useof this method rRNA expression in Staphylococcus epi-dermidis was compared in in-vivo and in-vitro experiments.A decrease of 16S rRNA content was observed in a late in-vivo infection approach [18]. Kinetic studies to evaluatemetabolic changes in ribosome content and bacterial celldensities were not performed in this work. Instead, totalRNA from different Pseudomonas aeruginosa populationswere isolated and the 16S ribosomal cDNAwas quantified ina defined amount of RNA to correct for potential variationsin general metabolic activity between different experiments.In all experiments the ribosomal 16S cDNA Ct valuesremained constant during analysis and no correction of theCt values of the other regulated genes was necessary.

It is commonly accepted that quorum-sensing-regulatedgenes depend on environmental conditions and growthphysiology [19, 20]. In contrast with the more comprehen-sive transcriptome analysis [6, 19, 21], in which cDNAmicroarray technology was used to identify Pseudomonasaeruginosa genes differentially expressed in growing anddeveloping biofilms and planktonic cultures, this approachwas directed toward analysis of specific genes in P.aeruginosa populations.

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use of quantitative real-time rt-pcr to analyse the expression of some quorum-sensing regulated...

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