System. Appl. Microbiol. 19,501-509 (1996)© Gustav Fischer Verlag

rRNA-Targeted Oligonucleotide Probes for the Identificationof Genuine and Former Pseudomonads

RUDOLF AMANN!, WOLFGANG LUDWIG!, RENATE SCHULZE!, STEFAN SPRING},EDWARD MOORE2, and KARL-HEINZ SCHLEIFER}

Lehrstuhl fur Mikrobiologie, Technische Universitiit Munchen, Arcisstrage 16, D-80290 Munchen, GermanyBereich Mikrobiologie, Gesellschaft fur Biotechnologische Forschung Braunschweig, D-38124 Braunschweig, Germany

Received May 7,1996

Summary

The specificity of previously published oligonucleotide probes war re-evaluated based on the expanded16S and 23S rRNA databases. Moreover, new genus- and (sub)species-specific 16S rRNA-targeted oli­gonucleotide probes were designed in a computer-assisted strategy using the PROBE DESIGN tool of thesoftware package ARB. Probes for Pseudomonas stutzeri and for the genera Acidovorax, Comamonasand Hydrogenophaga were evaluated. Optimal hybridization conditions for the probes have been deter­mined. Using these conditions the probes were tested for binding to 16S rRNA of reference cultures. Fur­thermore, fluorescently labeled derivatives of these probes were tested for their applicability for whole cellhybridization. These rRNA-targeted probes are the basis for high-resolution, rapid, automated microbi­al identification.

Introduction

The genus Pseudomonas formerly represented a phylo­genetically heterogeneous group of bacteria that were of­ten difficult to distinguish by conventional methods. In thecourse of thorough taxonomic studies on this importantgroup of bacteria, numerous pseudomonads have been re­named (Kersters et al., 1996). New genera had to beamended and now harbor, under new names, well knownspecies such as Acidovorax delafieldii (formerly Pseudo­monas delafieldii) (Willems et al., 1990), Burkholderia ce­pacia (formerly Pseudomonas cepacia) (Yabuuchi et al.,1992), Hydrogenophaga (lava (formerly Pseudomonas(lava) (Willems et al., 1989), Comamonas testosteroni(formerly Pseudomonas testosteroni) (De Vos et al., 1985;Tamaoka et al., 1987) and Brevundimonas diminuta (for­merly Pseudomonas diminuta) (Segers et al., 1994) tomention just a few. Today only the rRNA group I, or gen­uine pseudomonads, remain classified in the genus Pseu­domonas sensu stricto.

The heterogeneous group of pseudomonads is a typicalexample wherein the traditional phenotypic approach maylead not only to a wrong classification (Kersters et al.,1996), but also to an unsatisfactory identification of certain

species, as shown for the phenotypically quite diverse P. (lu­orescens, P. putida and P. stutzeri (Bennasar et al., 1996).Other techniques, such as chemotaxonomic (for review seeAuling, 1993) and genotypic methods, have been helpful inimproving the identification of pseudomonads. The advan­tage of genotypic methods is that, in contrast to many phe­notypic, and even chemotaxonomic, properties, they arenot affected by endogenous or exogenous parameters.Among the genotypic methods, nucleic acid hybridizationis a fast and specific method for identifying bacteria at dif­ferent taxonomic levels. A further advantage of this methodis that identification of organisms in mixed cultures is pos­sible, e. g. by in situ colony hybridization (Festl et al.,1986), reverse dot blot hybridization (Ehrmann et al.,1994) or whole cell hybridization (Amann et al., 1995).

Within a large collaborative study funded by the Euro­pean Community full length 165 rRNA sequences havebeen determined for most of the rRNA group I (gamma­group of Proteobacteria) and rRNA group III (beta1­group Proteobacteria) pseudomonads (Moore et al., 1996,Moore, pers. commun.). These sequences were collected inlarge databases (Van de Peer et al., 1996), aligned and

502 R. Amann et al.

used for phylogenetic analysis. The aligned sequenceswere also the basis for the computer-assisted design ofrRNA-targeted oligonucleotide probes using the PROBEDESIGN function of the newly developed ARB softwarepackage (Strunk et aI., in press). Hybridization with suchrRNA-targeted oligonucleotide probes is today a well ac­cepted technique for the identification of microorganisms(Schleifer et aI., 1993; Amann et al. 1995). There are sev­eral reasons for choosing rRNA as target molecules.

(1) All cells contain rRNA. The comparative analysis ofrRNA is today the "gold standard" for phylogenetic stud­ies. Therefore, the database is large with (March 1996)more than 6,000 complete and partial16S rRNA sequenc­es covering the majority of the cultured bacteria.

(2) As a consequence, probes can be designed in a verydirected way. By targeting evolutionary more or less con­served regions probes can be tailored for various phyloge­netic levels to the requirements of the experiments.

(3) rRNA is naturally amplified. In growing bacterialcells usually more than 103 ribosomes, and as many cop­ies of the 5S, 16S, and 235 rRNA molecules, are found.

(4) Due to the natural amplification, whole, fixed indi­vidual cells can be identified in situ with fluorescently la­beled rRNA-targeted probes (Amann et aI., 1995).

Additionally, large amounts of relatively inexpensiveprobes can be produced within few hours with modernsolid-phase oligonucleotide synthesizers. Therefore,rRNA-targeted oligonucleotide probes have become toolsfor a high-resolution, rapid, automated microbial identifi­cation. Numerous probes have already been described andsuccessfully used for the monitoring of bacterial popula­tions in complex environmental samples (for review seeAmann et al. 1995). Among those have been also severalprobes for genuine and former pseudomonads (Schleifer etaI., 1992, Braun-Howland et aI., 1993).

With modern computer programs, such as ARB (Strunket aI., in preparation), the probe design process is indeedstraightforward and, today, achieved within a few minutesto hours. After defining the target organism(s) and probelength, a list of potential probe target sites is generated bycomparative sequence analysis in an ordered form so thatthe number of mismatches, their quality (the estimated de­stabilizing effect) and location is weighted. The length ofthis list reflects, not only the phylogenetic position of theorganism of interest, but also the completeness of therRNA sequence database, especially in the phylogeneticvicinity of the organism of interest. If it is a member of aclosely related cluster of sequenced organisms, only few (ifany) sites are found. If the 165 rRNA sequence of the or­ganism is relatively isolated the researcher can usually se­lect from 5 to 10 different target regions. However, thesubsequent optimization of hybridization conditions ismore laborious. It has to be performed for each new probein a way such that conditions are defined which allow re­liable discrimination of target sequences from closely re­lated non-target sequences. If the probes should also beused for in situ identification of the respective target cellseven more testing is required. After ensuring that the fix­ation has permeabilized the cells by hybridization with ageneral probe serving as a positive control, it has to be

evaluated whether the respective target sites are accessiblein the whole fixed cells (Amann et aI., 1995).

In this study, we evaluated previously published oligo­nucleotide probes and describe the exemplary design, eva­luation and application of several oligonucleotide probestargeted to former and genuine pseudomonads.

Materials and Methods

Probe design, synthesis and labeling. Probe design was per­formed with the PROBE DESIGN tool of the software pack­age ARB (Strunk et al., in preparation). Oligonucleotides werecustom-made by MWG-Biotech (Ebersberg, FRG) with a C6­TFA aminolinker [6-(trifluoracetylamino)-hexyl-(2-cyanoethyl)­(N,N-diisopropyl)phosporamidite] at the 5'-end. Labeling withtetramethylrhodamine-5-isothiocyanate (TRITC; Molecular Pro­bes, Eugene, OR, USA) and 5(6)-carboxyfluorescein-N-hydro­xysuccinimide ester (FLUOS; Boehringer Mannheim, Mannheim,Germany) and purification of the oligonucleotide-dye conjugateswere performed as described by Amann (1995). The bacterialprobe EUB338 (Amann et al., 1990) was used as a positive con­trol. All other sequences are given in Table 1.

Organisms and growth conditions. Various pseudomonadswere obtained from LMG (Laboratory for Microbiology CultureCollection of the University Ghent, Ghent, Belgium), ATCC(American Type Culture Collection, Rockville, MD, USA) orDSM (Deutsche Sammlung von Mikroorganismen und Zellkul­turen GmbH, Braunschweig, Germany) and cultured as describedin the respective catalogues of strains. Strain designations are giv­en in Table 2.

Cell fixation. Cells from exponentially-growing cultures andsamples of activated sludge were fixed with paraformaldehyde asrecently described (Amann, 1995). Cell suspensions were immo­bilized on gelatine-coated microscope slides by air-drying. Cellswere further fixed and dehydrated by immersing the slides in 50,80 and 96% ethanol (Amann, 1995).

Hybridization and microscopy. Whole cell hybridizationswere performed for 1 h at 46°C in 10 ftl of a buffer containing0.9 M sodium chloride, 0.01 % sodium dodecyl sulfate, 10 mMTris/HCI (pH 7.4) and 5 ng/ftl of fluorescently labeled probe. Thesubsequent washing step for 20 min at 48°C was done as de­scribed by Manz et al. (1992). Slides were shortly rinsed with wa­ter to remove salts, airdried and mounted with Citifluor anti­bleaching agent AFI (Citifluor Ltd., Canterbury, UK). A ZeissAxioplan microscope equipped with a 50W mercury high pres­sure bulb and Zeiss filter sets 09 and 15 (lena, Germany) wasused for visualization. Color micrographs were done on KodakEktachrome 1600 at exposure times between 8 and 15s.

Probe evaluation. Stringency of hybridization was graduallyincreased by the addition of deionized formamide in 10% (v/v)increments. The optimal hybridization conditions were deter­mined by monitoring the fluorescence of target and closely relat­ed non-target cells at different stringencies. The formamide con­centrations recommended as optimal in Table 1 are the highestthat still yield good signals with the target cells and also result inlow binding to non-target cells.

Results

Re-evaluation of previously published oligonucleotideprobes

Since the last design of probes for genuine and formerpseudomonads (Schleifer et aI., 1992; Braun-Howland et

rRNA-Targeted Oligonucleotide Probes 503

Table 1. rRNA-targeted oligonucleotide probes for genuine and former pseudomonads, rRNA target sites (E. coli numbering, Brosiuset al. 1981) and optimal hybridization conditions

Probe 5equence (5'-T) rRNA target pos. % formamide ref.

Pst67 AAGCTCTCTTCATCCG 165,67-82 0 this studyPst997 TCTGGCAGGTTCTCAGCA 165,997-1014 0 this studyPae997 TCTGGAAAGTTCTCAGCA 165, 997-1014 0 this studyACI208 CGCGCAAGGCCTTGC 165,208-222 20 this studyCOM1424 ACCTACTTCTGGCGAGA 165,1424-1440 25 this studyHYD208 AATCGCGCGAGGCCTTAC 165,208-225 30 this study

Ps GCTGGCCTAGCCTTC 235, 1432-1446 20 Schleifer et aI., 1992Ppu GCTGGCCTAACCTTC 235, 1432-1446 20 Schleifer et aI., 1992Pce CCCATCGCATCTAACAAT 235, 1406-1423 30 Schleifer et aI., 1992Pdi TTCCACATACCTCTCTCA 165, 659-676 20 Schleifer et aI., 1992Cte TTCCATCCCCCTCTGCCG 165,659-676 20 Schleifer et aI., 1992

PPC CCTCTGTTCCGACCA 165, 1240-1254 N.D." Braun-Howland et aI., 1993P5MG CCTTCCTCCCAACTT 165,440-454 N.D. Braun-Howland et aI., 1993PPT ClTITCGTTCCGTAC 165,441-455 N.D. Braun-Howland et aI., 1993

* N. D. not determined

aI., 1993) (Table 1) the 16S and 23S rRNA databases havebeen considerably enlarged, i. e. instead of little more than1,000 almost full-length 16S rRNA sequences there arenow more than 6,200 16S rRNA sequences available. Asa consequence, it is becoming evident that some probes de­signed to be specific for certain species are also complete­ly complementary to several other species.

Probe Pdi (Schleifer et aI., 1992), for example, once de­signed to detect Brevundimonas diminuta (formerly Pseu­domonas diminuta), is also complementary to three Cau­lobacter sp. and Mycoplana bullata according to the mostrecent database. Even though these species are phyloge­netically closely related, probe Pdi should not be anylonger considered species-specific. The situation is evenworse for the probes Cte (Schleifer et aI., 1992) and PPT(Braun-Howland et aI., 1993), originally designed to thespecific for Comamonas testosteroni. At the time of thedesign of probes, the 16S rRNA database of the beta1­group of Proteobacteria was quite incomplete. Accordingto the current sequence data, these probes are also com­plementary to the respective target sites of the other Co­mamonas species, as well as several Acidovorax and Hy­drogenophaga species and Aquaspirillum metamorphum.Again, both probes remained phylogenetically meaningfulin that they are complementary only to members of a phy­logenetically coherent group, although probes Cte andPPT are neither species- nor genus-specific. The same istrue for probe PPC (Braun-Howland et aI., 1993). Origi­nally targeted to the 16S rRNA of Burkholderia cepacia,it is today evident that this probe is also 100% comple­mentary to Burkholderia caryophylli, B. gladioli, B. mal­lei, B. pseudomallei, B. andropogonis and Sutterella wads­worthia. Also probe Pce (Schleifer et aI., 1992) has beenshown to be not species-specific (Stoffels, unpublished re­sults). It is, e. g., also reacting with other members of thegenus Burkholderia.

In contrast, the specificity of probes targeted to the ge­nus Pseudomonas sensu stricto have not been impairedthrough the increased amount of sequence data and seemto have remained valid. Both, PSMG (Braun-Howland etaI., 1993) and the combination of probes Ps and Ppu(Schleifer et aI., 1992) can be used to differentiate mem­bers of the genuine pseudomonads from other bacteria likethe related genera Acinetobacer and Azotobacter.

Design of new probes

Based on the re-analysis of the previously publishedprobes, we intended to create different new probes. Pseu­domonas stutzeri was selected in order to evaluate the po­tential of 16S rRNA-targeted probes for species identifica­tion within the genus Pseudomonas sensu stricto. Addi­tionally, we attempted to design probes for the identifica­tion and differentiation of the three environmentally im­portant and phylogenetically closely related genera Aci­dovorax, Comamonas and Hydrogenophaga.

Probes for Pseudomonas stutzeri

The PROBE DESIGN tool of the software package ARBidentified three potential probe target sites on the 16SrRNA of P. stutzeri. Two of these probes, Pst67 and Pst997were studied in detail. The sequences are given in Table 1.The third, Pst451 (5'-TATTAACTTACTGCCCTT-3'),was complementary to E. coli positions 451-468 (Brosiuset aI., 1981). However, it has been reported (Amann et aI.,1992) that this region might not be suitable for in situ hy­bridization. Tetramethylrhodamine-Iabeled probe Pst451,indeed, yielded no detectable binding to whole fixed cellsof P. stutzeri and was, therefore, not evaluated further.

Only one of the other two suggested probes, Pst67, wastargeted to a region known to be accessible for in situ hy­bridization. PROBE DESIGN suggested Pst67 to be com-

504 R. Amann et al.

plementary to P. stutzeri DSM 50238 and P. mendocinaLMG 1223T • The probe yielded good signals and the ex­pected specificity when evaluated against reference strainsin dot blot and whole cell hybridization at 46°C withoutaddition of formamide (Table 2). After additional se­quencing (Bennasar et aI., 1996) it is today evident thatnot all strains of P. stutzeri are complementary to probePst67. Whereas P. stutzeri strains DSM 50238, DSM50227, ATCC 17587, ATCC 17591, ATCC 14405,AN10, and AN11 are complementary to Pst67, strainsATCC 17589 and DNSP21 have three central mismatchesand strains ST27MN3 and 19SMN4 have a single centralmismatch (Figure 1; for further information on the strainssee Bennasar et aI., 1996). Additional sequencing (Mooreet aI., 1996) has also revealed that a second strain ofP. mendocina (LMG 5941) has two central mismatches toprobe Pst67.

Probe Pst997 is complementary to a target site identi­fied by PROBE DESIGN to be specific for P. stutzeri basedoriginally on a very limited data set consisting of one rep­resentative of each species of Pseudomonas sensu stricto

including only one sequence of P. stutzeri strain. Evalua­tion of the probe by dot blot hybridization with more than70 reference strains indicated specificity for P. stutzeriDSM 50238 (Table 2). However, after additional sequenc­ing, it is now revealed that 13 other P. stutzeri strains (Ben­nasar et aI., 1996) have three mismatches to Pst997 (Fig­ure 1). Whole cell hybridizations at 46°C without forma­mide yielded detectable but weak signals with cells ofP. stutzeri DSM 50238 and some binding to P. aeruginosa,P. alcaligenes, P. aureofaciens, P. flavescens, P. mendocina,P. oleovorans and P. pseudoalcaligenes. The latter organ­isms have only two mismatches, including one weakly de­stabilizing G-U (Fig. 1). When the stringency of hybridiza­tion was gradually increased by raising the formamideconcentration in 10% steps is became evident that nohigher specificity could be achieved without further de­creasing the already low probe sensitivity to the pointwhere target cells could not be detected reliably. It hasbeen reported previously (Manz et aI., 1992) that compet­itor oligonucleotides offer a possibility to increase thespecificity without a negative influence on the sensitivity.

Table 2. Hybridization of genomic DNA of various bacteria with the specific DNA probes Pst67, Pst997, Pae997, HYD208, COM1424 and ACI208

Strain

Pseudomonas stutzeri DSM50238P. stutzeri DSM50227P. stutzeri DSM2332P. stutzeri sn7MN3P. aeruginosa DSM50071P. alcaligenes LMG2120P. aureofaciens LMG1245P. chlororaphis LMG5004P. cichorii LMG1248p. flavescens NCPPB3063P. fluorescens LMG1794P. mendocina LMG1223P. oleovorans LMG2229p. pseudoalcaligenes LMG1225P. syringae LMG1247P. putida LMG2257Escherichia coliBrevundimonas diminuta DSM1635Burkholderia cepacia DSM50181Acidovorax avenae-avenae LMG1806A. delafieldii LMG5943A. facilis LMG2193A. konjaci LMG5691A. temperans LMG7169Comamonas acidovorans LMG1226C. terrigena LMG1253C. testosteroni LMG1800Hydrogenophaga flava LMG2185H. palleroni LMG2366H. pseudoflava LMG5945H. taeniospiralis LMG7170

rRNAgroup

IVIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

Proteo­bacteriasubclass

GammaGammaGammaGammaGammaGammaGammaGammaGammaGammaGammaGammaGammaGammaGammaGammaGammaAlphaBetaBetaBetaBetaBetaBetaBetaBetaBetaBetaBetaBetaBeta

Hybridization

Pst67 Pst997 Pae997 HYDl08 COM1424 ACI208

+ ++

+++

+

+ +++

Therefore, probe Pae997 was constructed to block theprobe binding site of non-target pseudomonads. ProbePae997 was used either as an unlabeled competitor or la­beled with a different fluorochrome. In the latter case, it isan effective probe for a large group of genuine pseudo-

rRNA-Targeted Oligonucleotide Probes 505

monads, namely P. aeruginosa, P. alcaligenes, P. aureofaci­ens, P. f/avescens, P. mendocina, P. oleovorans and P. pseu­doalcaligenes (Fig. 1). The combination of probes Pst997and Pae997 enables reliable whole cell identification ofP. stutzeri DSM 50238 at 46°C without addition of for-

3'GCCTACTTCTCTCGAA5'5'CGGAUGAAGAGAGCUU3'

Probe Pat67TargetP. stutzeri DSM50238P. stutzeri DSM50227P. stutzeri DSM2332P. stutzeri ST27MN3P. mendocina LMG1223P. mendocina LMG5941P. aeruginosa LMG1242P. aeruginosa DSM50071P. alcaligenes LMG1224P. aureofaciens LMG1245P. flavescens NCPPB3063P. oleovorans DSMI045P. pseudoalcal. LMG1225P. putida DSM291A. temperans LMG7169C. acidovorans LMG1226H. palleroni LMG2366

...... . GUG .

.....•.. •G .

...... . U.G .

.•.•..•.• G..•.••

.UU G .A.CGGAG .. G.U .•..••.••• G••••••... U G.G ........ C.G ..........G ....... . GA .· .. UAAC .. GUCUUCG· .. UAAC .. GUCUUCG· .. UAAC .. CCCG. AA

Probe RYD208 3'CATTCCGGAGCGCGCTAA5'Target 5 'GUAAGGCCUCGCGCGAUU3 ,H. £lava LMG2185 .H. palleroni LMG2366 .H. pseudoflava LMG5945 .H. taeniospir. LMG7170 .R. gela tinosus A3 U .R. gelatinosus DSM1709 .A U .A. avenae-aven. LMG1806.C U ACA. delafieldii LMG5943 .C U UCACA. facilis LMG2193 .C U GACA. konjaci LMG5691 .C U ACA. temperans LMG7169 .C U ACC. acidovorans LMG1226 U.CG .C. terri gena LMG1253 .C A U A.C.C. testoster. LMG1800 U.CG U.U .. U.C .P. aeruginosa DSM50071 UUCG.A A U.. CP. stutzeri DSM50238 UUCG.A A U.. CV. pardoxus IAM12373 .C A U A.

3'ACGACTCTTGGACGGTCT5'5'UGCUGAGAACCUGCCAGA3'

3'AGAGCGGTCTTCATCCA5'5'UCUCGCCAGAAGUAGGU3'

Probe Pat997TargetP. stutzeri DSM50238P. stutzeri DSM50227P. stutzeri DSM2332P. stutzeri ST27MN3P. aeruginosa LMG1242P. alcaligenes LMG1224P. aureofaciens LMG1245P. flavescens NCPPB3063P. mendocina LMG1223P. oleovorans DSMI045P. pseudoalcal. LMG1225P. putida DSM291A. temperans LMG7169C. acidovorans LMG1226H. palleroni LMG2366

..• A•..... U.U .•.. A•..... U.U .... A .....• U.U ...........U.U ........... U.U ........... U.U ...........U.U ........... U.U ........... U.U ...•.••....U.U ..•..... A U.U .· . UACG U. CUUU .· . GCAG GU.U .· .UACG UU .

Probe COM1424TargetC. acidovorans LMG1226C. terri gena LMG1253C. testoster. LMG1800A. avenae-aven. LMG1806A. delafieldii LMG5943A. facilis LMG2193A. konjaci LMGS691A. temperans LMG7169H. flava LMG2185H. palleroni LMG2366H. pseudoflava LMG5945H. taeniospir. LMG7170P. aeruginosa DSM50071P. stutzeri DSM50238V. pardoxus IAM12373

.UCU .

.UCU U.

.UCU .

.UCU .

.UCU .

. U.

. U.. U.. U..UG.U C..UG.U C..UCu u.

Probe Pae997 3'ACGACTCTTGAAAGGTCT5'Target S'UGCUGAGAACUUUCCAGA3'P. aeruginosa LMG1242 ..P. alcaligenes LMG1224 .P. aureofaciens LMG1245 .P. flavescens NCPPB3063 .P. mendocina LMG1223 .P. oleovorans DSMI045 .P. pseudoalcal. LMG1225 .P. putida DSM291 A .P. stutzeri DSM50238 C.G .P. stutzeri DSM2332 A .A. temperans LMG7169 .. UACG UCC.UU .C. acidovorans LMG1226 .. GCAG G .H. palleroni LMG2366 .. UACG U.. G .

Probe ACI208 3'CGTTCCGGAACGCGC5'Target 5'GCAAGGCCUUGCGCG3'A. avenae-aven. LMG1806 .A. facilis LMG2193 .A. konjaci LMG5691 .A. temperans LMG7169 .A. delafieldii LMG5943 UC. acidovorans LMG1226 UUCG C .C. terrigena LMG1253 A AH. flava LMG2185 .U C .H. palleroni LMG2366 .U C .H. pseudoflava LMG5945 .U C .H. taeniospir. LMG7170 .U C .P. stutzeri DSM50238 UUCG.A CA UV. paradoxus IAM12373 A .

Fig. 1. rRNA sequence alignments showing target regions of probes for a selection of reference strains. Nucleotides are only identi­fied for mispairings, pairings are indicated by dots.

506 R. Amann et al.

mamide (Fig. 2, panel A). For the time being, Pst997 is anexample of a strain-specific oligonucleotide probe. How­ever, it should be noted that the efficacy of probes Pst997and Pae997 due to their low sensitivity will be very limit­ed for true in situ hybridization in complex environmentalsamples such as soil or activated sludge.

Probes for the genera Acidovorax, Comamonas,and Hydrogenophaga

The PROBE DESIGN tool indicated that constructionof genus-specific probes for Acidovorax, Comamonas,and Hydrogenophaga was possible. The respective probeswere named ACI208, COM1424 and HYD208. Sequenc­es, target sites and recommended formamide concentra­tions are given in Table 1. According to the most recent se­quence data COM1424 and HYD208 should detect allmembers of the respective genera and no outside organ­isms (Fig. 1). The discrimination of members of the gene­ra Comamonas and Hydrogenophaga by probe COM­1424 (a 17mer) relies on a single strongly destabilizingC-U mismatch at position 1439 (E. coli numberings, Bro­sius et aI., 1981). Specific hybridization is readily achievedat the recommended formamide concentration of 25%(Table 2).

For probe ACI208 the situation is more complicated.Acidovorax delafieldii has one terminal C-U mismatchwith probe ACI208. Although terminal mismatches areless destabilizing than internal ones (Stahl and Amann,1991) A. delafieldii was discriminated at the recommend­ed stringency (20% formamide) (Table 2). At lower strin­gencies (0 or 10% formamide), probe ACI208 also detect­ed A. delafieldii, although discrimination of Variovoraxparadoxus became a problem.

Two of the suggested probes, ACI208 and HYD208,were located in the variable region II, known for good insitu accessibility. This could be confirmed experimentally(Fig. 2, panels B and D). Probe HYD208 should be used at30% formamide. At this stringency the sensitivity in wholecell hybridizations is very good and the specificity was asexpected. The target site of probe COM1424 (E. coli num­berings 1424-1440) was demonstrated to be accessible foroligonucleotide probes in whole fixed cells. Good signalstrength and the expected specificity was achieved at 25%formamide (Figure 2, panel B).

Probing ofactivated sludge with probes AC1208,COM1424 and HYD208

It has been shown previously that beta1-Proteobacteriaare abundant in activated sludge from wastewater treat­ment plants (Wagner et aI., 1993). The three newly de­signed probes ACI208, COM1424 and HYD208 are tar-

rRNA-Targeted Oligonucleotide Probes 507

geted to three important genera of this phylogeneticgroup. When applied to paraformaldehyde-fixed samplesfrom the large municipal wastewater treatment plantMiinchen-GroBlappen, probes ACI208 and HYD208, in­deed, detected cell populations of significant numbersbetween 1 and 7% of all bacteria (Fig. 2, panel C and D).Only very few cells showed signals with probe COM1424(data not shown).

Discussion

In this study we report the design of six new 16S rRNA­targeted oligonucleotide probes was for genuine and for­mer pseudomonads. The design of new probes necessitat­ed by the observation that, in light of the rapidly growing16S rRNA database, certain probes described on a morelimited dataset, no longer show the desired specificity.This was the most obvious for two probes originally de­signed to be specific for C. testosteroni. Comparative dataanalysis today indicates that they are, infact, complemen­tary to 16S rRNA from numerous beta1-Proteobacteria.With the considerably enlarged dataset (Moore, pers. com­mun.) it was possible to design three new probes that iden­tify well defined genera in this environmentally importantgroup of former pseudomonads. These probes are an im­portant contribution to a nested set of oligonucleotideprobes that enables a rapid characterization of complexenvironments following a top-to-bottom approach(Amann et aI., 1995). They should be applied in the courseof characterizing complex environmental samples when­ever previous hybridization with group-specific oligonu­cleotides have indicated significant numbers of beta­subclass Proteobacteria. This group seems to be veryabundant in many freshwater samples.

The situation was different for the probes targeted togenuine pseudomonads. The previously published probesare still applicable for the identification of Pseudomonassensu stricto. This reflects, on the one hand, the fact thatthe rRNA databases at the time of probe design were moreadequate. On the other hand, we frequently find the spec­ificity profiles of group- and genus-specific probes to bemore stable over the years despite the expanded dataset.These probes do not target the most variable regions of16S or 23S rRNA and, therefore, the frequency of muta­tions in the target regions is lower.

Our results on the design of a probe specific for P. stut­zeri indicate that the situation is different for species­specific probes. After the joint effort of the HRAMI pro­ject, Pseudomonas sensu stricto is a very well examinedgenus. The 16S rRNA sequences of many closely relatedspecies have been determined and the sequence similarities

Fig. 2. In situ hybridization with fluorescein- and tetramethylrhodamine-labeled probes. Phase contrast (left) and epifluorescence mi­crographs (right) are shown for identical microscopic fields. Panel A. An artificial mixture of P. stutzeri DSM 50238, P. aeruginosaand B. diminuta was hybridized with fluorescein-labeled probe Pst997 (green) and tetramethylrhodamine-labeled probe Pae997 (red).Panel B. An artifical mixture of Acidovorax temperans and Comamonas testosteroni was hybridized with fluorescein-labeled probeCOM1424 and Cy3-labeled probe ACI208 (yellow). Panel C and D. Activated sludge samples (wastewater treatment plant Miinchen­Groglappen; 8.3.1995) were hybridized with Cy3-labeled probe ACI208 and fluorescein-labeled probe HYD208, respectively.

508 R. Amann et al.

are well above 90%. As a consequence, the PROBE DE­SIGN tool of ARB could only identify one highly specificsite (Pst997) and two more sites that at least allow dis­crimination of P. stutzeri strains from most other genuinepseudomonads. In the course of our studies it became clearthat probe Pst997 would be strain-specific, rather thanspecies-specific. Recently, the 16S rRNA sequences of 14strains of P. stutzeri have been determined (Bennasar et al.,1996) representing all currently described seven genomo­vars (Ursing et al., 1995). Sequence dissimilarities as highas 4.44% were found between different P. stutzeri strainsand it was suggested to rename members of the most dis­tant genomovar 6 as P. balearica (Bennasar et al., 1996).The sequence differences between P. stutzeri strains weredetermined to be located mainly in the most variable re­gions and, not unexpectedly, three differences were alsofound in the target site of probe Pst997. Currently Pst997is complementary only to the 16S rRNA of P. stutzeriDSM 50238, the sole representative of genomovar 7. Asnoted previously (Stahl and Amann, 1991), 16S rRNA­targeted probes, with their potential for discrimination ofsingle mutational events, can be specific below the specieslevel. However, it is also evident from the large number of16S and 23S rRNA sequences of different strains of genu­ine pseudomonads that this is the exemption and not therule.

There are two reasons for the difficulties in designing atruly species-specific probe for P. stutzeri. The first one isspecific for P. stutzeri and a reflection of the fact that itrepresents a very diverse and controversial bacterial spe­cies. Several studies have demonstrated that P. stutzericomprises a heterogeneous set of strains that should be di­vided into several species. However, this was not possibledue to the high phenotypic diversity within strains whichallowed no definitive differentiating phenotypic criteria tobe found (Rossello et al., 1991; Rossello-Mora et al.,1994). Design of a species-specific probe will be muchmore difficult, if not impossible, for such a diverse speciesas compared to a more coherent, genotypically well-de­fined species. The second reason for the difficulty in de­signing species-specific rRNA-targeted probes is a basicone. The probes have to be complementary to the mostvariable regions of the rRNA molecules with the highestmutation frequency. Remembering that evolution is a sta­tistical process, it is therefore quite likely that when manystrains of a valid species are examined one could have a(back)mutation in the probe target site. Therefore, it willbe difficult to find perfect species-specific probes. How­ever, there is a way out of this dilemma. When it is pos­sible to identify two or three good species-specific sites bycomparative analysis of 165 and 23S rRNA sequences spe­cies identification becomes more stable by not relying ononly a single probe.

Throughout this study, we had to consider not only as­pects of probe design by comparative sequence analysis,but also kept in mind that the developed probes should becompatible to a high-resolution, automated, microbialidentification based, e. g., on the flow cytometric analysisof fluorescently labeled cells (Wallner et al., 1996). There­fore, all probes had to be evaluated for in situ accessibil-

ity of target sites in whole fixed cells. As reported beforewe could demonstrate again (Amann et al., 1995) that notall target sites are equally suitable for whole cell hybrid­ization. On a qualitative scale, probes ACI208 andHYD208 were the best, followed by COM1424 and Pst67which were still good, and Pst997 and Pae997 which wereweak but detectable. Another target site in one of the mostvariable regions of 16S rRNA (E. coli positions 451-468)was found to be inaccessible for oligonucleotide probing,confirming earlier findings in which E. coli positions 453­470 were inaccessible in sulfate-reducing bacteria (Amannet al., 1992). It would be very helpful for a more directedprobe design if, in the near future, the whole 16S and 23SrRNA would be quantitatively analyzed in a systematicway for suitable target sites for whole cell hybridization.

In conclusion, we have demonstrated and discussedmodern approaches to the directed design of rRNA­targeted oligonucleotide probes. Considering that com­plete 16S rRNA sequences will be available soon for allvalidly described species and new sequences from hithertouncultured microorganisms will be retrieved from clone li­braries we believe that the golden age of rRNA-basedidentification has just started and that hybridization as­says will likely become routinely used methods in both en­vironmental and medical microbiology.

Acknowledgements. This work was supported by EuropeanUnion HRAMI T-project grant BIOT-CT91-0294. We gratefullyacknowledge the technical assistance of Sibylle Schadhauser.

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Rudolf Amann, Lehrstuhl fiir Mikrobiologie, Technische Universitiit Miinchen, ArcisstraGe 16, D-80290 Miinchen, Germany