rRNA based identification and detection systems for rhizobia and otherbacteria
Wolfgang Ludwig1,∗, Rudolf Amann1, Esperanza Martinez-Romero2, Wilhelm Schönhuber1,Stephan Bauer1, Alexander Neef1 and Karl-Heinz Schleifer1
1Lehrstuhl für Mikrobiologie, Technische Universität München, D-80290 München, Germany and2Centro deInvestigacion sobre Fijacion de Nitrogeno, 565-A, Cuernavaca, Mor., M´exico
Ribosomal ribonucleic acids are excellent marker molecules for the elucidation of bacterial phylogeny; they alsoprovide useful target sites for identification and detection with nucleic acid probes. Based on the currently avail-able 16S rRNA sequence data, bacteria of the rhizobial phenotype (plant nodulation, nitrogen fixation) are membersof three moderately related phylogenetic sub-groups of theα-subclass of theProteobacteria: i.e. the rhizobia group,the bradyrhizobia group, and the azorhizobia group. All rhizobia, azo-, brady-, meso- and sinorhizobia are closelyrelated to and in some cases phylogenetically intermixed with, non-symbiotic and/or non-nitrogen-fixing bacteria.Especially in the case ofBradyrhizobium japonicumstrains, the 16S rRNA sequence data indicate substantialheterogeneity. Specific probe design and evaluation are discussed. A multiprobe concept for resolving specificityproblems with group specific probes is presented.In situ identification with group specific probes of rhizobia incultures as well as rhizobia and cyanobacteria within plant material is shown.
Introduction
The capability of rhizobia and some other plant-symbiotic bacteria to convert atmospheric nitrogen toammonia and to turn it over to the host is of greateconomic importance in agriculture. Cultivation ofleguminous crops helps to improve soil by natural ni-trogen fixation and protects ground water from nitratecontamination resulting from excessive application ofN-fertilizer. Legumes are routinely inoculated withrhizobia in many countries. Major efforts are beingmade to improve rhizobia used for plant inoculation.The major targets of such efforts are the ability of thebacteria to fix nitrogen, nodulate the host plant, andcompete with the indigenous bacteria in soil for col-onization of rhizosphere and root. To screen naturalresources for better strains, reliable identification anddetection methods are needed.
∗ FAX No: 498928922360.E-mail: [email protected] and Soilis the original source of publication of this article.It is recommended that this article is cited as:Plant and Soil204:1–19, 1998.
During the past decade, ribosomal RNA basedidentification and detection methods have becometechniques routinely used in all disciplines of micro-biology. As molecular chronometers (Woese, 1987)these molecules have preserved their evolutionary his-tory. Highly conserved portions carry the informationon early evolutionary events and more recent changesare documented within less conserved positions orstretches. The degree of divergence of present dayrRNA sequences gives an estimate of their phyloge-netic distances. Phylogenetic trees based on rRNAsequence data roughly reflect the evolutionary historyof the organisms as shown by comparative sequenceanalyses of alternative non-rRNA phylogenetic markermolecules (Ludwig and Schleifer, 1994; Ludwig et al.,1993). Comparative analysis of comprehensive data-bases of bacterial 16S rRNA sequences (De Rijk etal., 1996; Ludwig, 1995; Maidak et al., 1996; Vande Peer et al., 1996) with appropriate software allowsrapid identification of unknown bacteria based on theirrRNA sequence data.
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Besides the phylogenetic information rRNAs alsoprovide targets for specific hybridization probes(Amann and Ludwig, 1994; Amann et al., 1995).These targets are diagnostic sequence stretches whichare unique to defined phylogenetic groups. Specificnucleic acid probes can be designed complementary tosequence stretches containing the diagnostic residues.Mainly, synthetic oligonucleotides are used for targetmonitoring. The presence of the target is indicatedby stable hybrid formation which is detected by mea-suring probe or target associated labels. The classicalradioactive labels have been more and more replacedby non-radioactive labelling and detection systems(Schleifer et al., 1993). The rRNA based specificprobe technology permits the detection of microbialnucleic acids or organisms even within complex sam-ples. Specificin situ identification of individual cellscan be achieved by whole cell hybridization (Amann etal., 1995). The so called rRNA cycle was the first tech-nique to combine the phylogenetic analysis andin situvisualisation of so far uncultured bacteria (Amann andLudwig, 1994; Amann et al., 1995). Ribosomal rRNAgenes can bein vitro amplified from environmentalsamples and subsequently cloned and sequenced. Thesequence data allow the phylogenetic positioning aswell as specific probe design.In situ cell hybridiza-tion closes the cycle by assigning the sequence to amorphotype within the original sample.
With rhizobia, most of the validly describedspecies have been phylogenetically characterized bycomparative analyses of almost complete 16S rRNAsequences (Ludwig et al., 1995; Nour et al., 1994,1995; Sawada et al., 1993; Willems and Collins, 1992;Willems et al., 1993; Yanagi et al., 1993). Manystrains and isolates can be (roughly) assigned to phylo-genetic groups by full (Dupuy et al., 1994; Wong et al.,1994) and partial 16S rRNA sequence analyses (Chenet al., 1995; Eardly et al., 1992; So et al., 1994; VanBerkum et al., 1994; Van Rossum et al., 1995; Xu etal., 1995; Young et al., 1991). Not many reports on theuse of specific probe technology for the identificationof rhizobia exist however (Springer et al., 1993).
The phylogeny of rhizobia
Currently, 10 species of the genusRhizobiumas wellas 5 of Mesorhizobium, 6 of Sinorhizobium, 3 ofBradyrhizobium, and 1 ofAzorhizobiumhave beenvalidly described. Full and/or partial 16S rRNA se-quence data are available in public databases (De Rijk
Figure 1. Major groups of theα-subclass of theProteobacteria.The consensus tree is based on distance matrix, maximum parsi-mony and maximum likelihood analyses of all available (>90%complete) 16S rRNA sequences. Only alignment positions whichshare common residues in at least 50% of all available sequencesfromα-subclass proteobacteria were included for calculations. Mul-tifurcations indicate branches for which a relative order cannotunambiguously be determined or a common order is not supportedapplying different treeing methods. The bar indicates estimated basechanges (10%). The quadrilaterals indicate phylogenetic groups.The lengths of the horizontal sides indicate the overall shortest andlongest branches within the cluster. The vertical extensions roughlyindicate the number of sequences used. The brevundimonads groupincludesMycoplana bullata, M. segnis, Caulobacter crescentusandC. bacteroides. Rhodobacters includeParacoccus, Rhodovulumand(less significantly)Hirschia and Hyphomonas. Rickettsia includeEhrlichia, Cowdria, Neorickettsiaand (less significantly)Caedibac-ter andHolospora. Magnetospirilli includeRhodospirillum fulvumandR. molischianum. Azospirilli includeRhodocista. Acetobactersinclude Gluconobacter, Acidiphilium andRoseococcus. The mag-netic bacteria group comprises magnetic non-cultured bacteria notrelated to the magnetospirilli. The phylogentic substructure of therhizobia, bradyrhizobia and azorhizobia groups is shown in Figures2–5.
et al., 1994; Ludwig, 1995; Maidak et al., 1994; Vande Peer et al., 1994) as specified in Table 1. Accordingto 16S rRNA sequence data, all genera are mem-bers of theα-subclass of the proteobacteria. The classProteobacteriarepresents one of the major lines ofdescent within the domainBacteria. The separation ofthe initially described genusRhizobiuminto the generaRhizobiumandBradyrhizobiumbased on phenotypic
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Table 1. Validly described rhizobia species. Accession numbers and references for 16S rRNA sequence data
Genus Species Accession Reference
Azorhizobium caulinodans D13948, D11342, M55491, Rainey and Wiegel, 1996
U12913, X67221, X77126, Sawada et al., 1993;
X94200 Van Rossum et al., 1995;
Willems and Collins, 1993;
Bradyhizobium elkanii M55490, U35000 So et al., 1994
Bradyhizobium liaoningensis X86065 Xu et al., 1995
Bradyhizobium japonicum D11345, D12781, D13429, Kuendig et al., 1995
D13430, L23330, L23331, Ludwig et al., 1995;
M55485–M55490, Sawada et al., 1993;
S46916, U12911, U12912, Springer et al. 1993;
U50164, U50165, U69638, Van Rossum et al., 1995;
X66024, X71840, X87272, Willems and Collins, 1992;
Z35330 Wong et al., 1994;
Yanagi and Yamasato, 1993;
Young et al., 1991;
Mesorhizobium ciceri U07934 Nour et al., 1994
Mesorhizobium huakuii D12797, D13431, S74905 Chen et al., 1995;
Sawada et al., 1993;
Yanagi and Yamasato, 1993;
Mesorhizobium loti D01270, D12791, D14514, Sawada et al., 1993;
U50166, X63825, X67229, Willems and Collins, 1993;
X67230 Yanagi and Yamasato, 1993
Young et al., 1991;
Mesorhizobium mediterraneum L38825 Nour et al., 1994
Mesorhizobium tianshanense S74904 Chen et al., 1995
Rhizobium etli L20762–L20766, U28916, Hernandez-Lucas et al., 1995;
U28939, U47303, Van Berkum et al., 1996
Rhizobium galegae D11343, D12793, X63823, Huber and Selenska-Pobell, 1994
X67226, Z79620 Sawada et al., 1993;
Willems and Collins, 1993;
Yanagi and Yamasato, 1993;
Rhizobium gallicum U86343, AF008127– Anarger et al., 1997;
AF008130 Sessitsch et al., 1997
Rhizobium giardinii U86344 Anarger et al., 1997
Rhizobium hainanensis U71078 Chen et al., 1997
Rhizobium leguminosarum D01269, D14513, D12782, Breil et al., 1996;
M55233, M55235– Eardley et al. 1992;
M55240, M55494, Sawada et al., 1993;
M63183, U09271, U29386, Segovia et al., 1991;
U29388, U31074, X67227, Van Berkum et al. 1996;
X67233, X77122, X91211 Willems and Collins, 1993;
Yanagi and Yamasato, 1993:
Young et al., 1991;
Rhizobium lupini Ludwig et al., 1995
Rhizobium tropici D11344, D12798, L20697, Hernandez-Lucas et al., 1995;
L20765, L21837, U38469, Sawada et al., 1993;
X67234, X77125, X77127 Van Berkum et al., 1994;
Yanagi and Yamasato, 1993;
Sinorhizobium fredii D01272, D12792, X77123, Sawada et al., 1993;
X67231 Willems and Collins, 1993;
Yanagi and Yamasato, 1993
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Table 1. Continued
Sinorhizobium meliloti D01265, D12783, D14509, Eardley et al., 1992;
D14516, M55242, Sawada et al., 1993;
M55243, M55495, Willems and Collins, 1993;
X67222, X67231, X77121, Young et al., 1991;
X67231
Sinorhizobium medicae L39882 Rome et al., 1996
Sinorhizobium saheli X68390 Lajudie et al., 1994
Sinorhizobium terangae X68387, X68388 Lajudie et al., 1994
Sinorhizobium xinjiangensis D12796 Yanagi and Yamasato, 1993
Figure 2. The rhizobia group. The tree is based on the results ofa maximum likelihood analysis of all (>90% complete) 16S rRNAsequences from the members of the rhizobia, azorhizobia, bradyrhi-zobia groups and from selected representatives of other majorgroups of theα-subclass. The tree topology was evaluated and cor-rected according to the results of distance and maximum parsimonyanalyses of the complete sequence data set fromα-subclass pro-teobacteria. Only sequence positions which share common residuesin at least 50% of the members of the rhizobia group were includedfor calculations. The triangles indicate the phylogenetic subgroups.Multifurcations indicate branches for which a relative order cannotbe unambigousely determined or a common order is not supportedby different treeing methods. The bar indicates estimated basechanges (%).
characters and nucleic acid hybridization data (Jor-dan, 1982) reflects the phylogenetic situation as waslater corroborated by comparative 16S rRNA sequenceanalysis (Figure 1). Later, the genusRhizobiumwasfurther divided intoMesorhizobum, RhizobiumandSinorhizobium(De Lajudie et al., 1994; Jarwis et al.,
Figure 3. Phylogenetic relationships ofMesorhizobium(M.), Rhi-zobium(R.), andSinorhizobium(S.) species. Only type strains aredepicted. Tree reconstruction was as decribed for Figure 2.
1997; Figure 2). With the exception ofRhizobiumlupini all rhizobial, meso- and sinorhizobial speciesare members of one phylogenetic subgroup of theα-subclass of the proteobacteria (rhizobia group inFigure 1). This subgroup also comprises non-nitrogenfixing species of other genera associated (Agrobac-terium and Phyllobacterium) or not associated withplants (Blastobacter, Chelatobacter, MycoplanaandZoogloea) or even (facultative) pathogens such asBartonella, BrucellaandOchrobactrum(Figure 2).
Species of the genusSinorhizobiumrepresent acoherent phylogenetic group (Figure 2). The genushas been amended based on the results of polyphasictaxonomic investigations (De Lajudie et al., 1994) tocontain the renamed formerRhizobium frediiandR.
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melilotias well as several newly isolated and describedspecies (Sinorhizobium medicae, S. saheli, S. terangaeandS. xinijangensis) (Figure 3).
Another monophyletic group moderately related toPhyllobacteriumcomprises the species of the genusMesorhizobium(Figure 2).This genus was recentlycreated to harbour the formerRhizobium ciceri, R.huakuii, R. loti, R. mediterraneumandR. tianshanense(Jarvis et al., 1997; Figure 3).
The current genusRhizobium(Figure 2) comprisesRhizobium etli, R. galegae, R. gallicum, R. giardinii,R. hainanense, R. leguminosarum, R. lupini, R. phase-oli, R. trifolii and R. tropici (Figure 3). From thephylogenetic point of view, however, the genusRhi-zobium as currently defined is not a monophyleticgroup. According to 16S rRNA sequence data, therecently describedR. giardinii (Amarger et al., 1997)does not cluster with the otherRhizobiumspecies butrather represents a new line of descent within therhizobia group (Figures 2 and 3). Furthermore, thegenuineRhizobiumcluster (Rhizobium etli, R. gale-gae, R. gallicum, R. hainanense, R. leguminosarum, R.lupini, R. tropici) also contains probably misclassifiedAgrobacterium tumefaciensas well asA. rhizogenesstrains.
There is a number of phylogenetic groups com-prising non-rhizobial bacteria which are related to oneanother and to the mesorhizobia, sinorhizobia as wellas the rhizobia groups at comparable levels. The typestrains ofAgrobacterium rubiandA. tumefaciensarerelated to one another and separated from rhizobialspecies. However, only part of otherAgrobacteriumrubi and A. tumefaciensstrains group with their re-spective type strains. Some of the strains designatedas Agrobacterium tumefaciensare only moderatelyrelated to the type strain but phylogenetically inter-mixed with R. etli, R. leguminosarum, R. tropici andAgrobacterium rhizogenesstrains (Eardly et al., 1992;Sawada et al., 1993; Segovia et al., 1991; Willemsand Collins, 1993; Yanagi and Yamasoto, 1993). Inthe case ofAgrobacterium rhizogenes, the type strainis not related to the species of the present group butwas reclassified asSphingomonas rosa(Takeuchi etal., 1995). TheSphingomonasspecies are also mem-bers of theα-subclass of the proteobacteria, however,phylogenetically separated from the rhizobia and rel-atives (Figure 1). Species of the generaBrucella andBartonellarepresent two entities which, with respectto the level of phylogenetic relatedness, are equiva-lent to the agrobacteria, rhizobia, sino- and mesorhi-zobia groups (Figure 2).Ochrobactrumapparently
Figure 4. Bradyrhizobia group. Tree reconstruction and presenta-tion is as described for Figure 2 except that only alignment positionswhich share common residues in at least 50% of the members ofthe brady- and azorhizobia group were included for calculations.Abbreviations:Af – Afipia; Ag – Agromonas; Az – Azorhizobium;Bl – Blastomonas; Br – Bradyrhizobium; N – Nitrobacter; Rhi –Rhizobium; Rho– Rhodopseudomonas.
shares a common origin withBrucella. A moderaterelationship to this group is indicated for twoMy-coplanaspecies (M. dimorphaandM. ramosa; Yanagiand Yamasoto, 1993) applying different treeing meth-ods. (The statistical significance is low, however.)Phylogenetically, otherMycoplana species are notmembers of this group but are related toCaulobac-ter and Brevundimonas, other proteobacteria of theα-subclass. Partial 16S rRNA sequence data indi-cate thatChelatobacter heintziiand Chelatococcusasaccharovoransalso belong to the major rhizobiacluster (Auling et al., 1993). A remarkable exampleof mis-classification based on conventional taxonomicmethods is the speciesZoogloea ramigera. While thetype strain (ATCC 19544) and another strain (ATCC25935) cluster in two different groups of proteobac-teria of theβ-subclass, a third strain (ATCC 19623)clearly is a member of the rhizobia group (Rosselló-Mora et al., 1993; Shin et al., 1993).BlastobacteraggregatusandB. capsulatusemerge from a commonbranch rooting at the basal level of the rhizobia group.Rhizobium lupiniis a mis-classified species and sharesidentical 16S as well as 23S rRNA sequences with thetype strain ofBradyrhizobium japonicum(Ludwig etal., 1995).
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Table 2. Ranges of overall 16S rRNA sequence similarities (% identicalhomologous bases) of non-Bradyrhizobium japonicumandBradyrhizo-bium japonicumstrains within theBradyrhizobiumgroup
Species Similarity (%) with
Bradyrhizobium japonicumstrains
Bradyrhizobium elkanii 98.8–96.6
Blastobacter denitrificans 98.8–96.3
Rhodopseudomonas palustris98.2–95.6
Nitrobacter hamburgensis 97.5–95.3
Nitrobacter vulgaris 97.5–95.2
Nitrobacter winogradskyi 98.5–95.5
Afipia clevelandis 98.5–95.5
Afipia felis 98.0–95.0
Figure 5. Azorhizobia group. Tree reconstruction and presentationis as described for Figure 2 except that only alignment positionswhich share common residues in at least 50% of the members ofthe brady- and azorhizobia group were included for calculations.Abbreviations:An – Ancylobacter; Aq – Aquabacter; Az– Azorhi-zobium; B – Bradyrhizobium; R– Rhizobium; T – Thiobacillus; X –Xanthobacter.
The bradyrhizobia together withRhodopseudomo-nas palustris, Blastomonas denitrificansas well asAfipia, andNitrobacterspecies represent a subgroupof closely related proteobacteria of theα-subclasswhich is rather distant from the rhizobia group (Fig-ure 1). The current genusRhodopseudomonasisphylogenetically diverse. All species analyzed sofar are members of theα-subclass, however, onlyRhodopseudomonas palustrisis among the organsims
of the bradyrhizobia group. TheNitrobacter species(N. hamburgensis, N. vulgarisand N. winogradskyi)share a common root in phylogenetic trees. How-ever, a relative branching order of theBradyrhizo-bium species, the Agromonas / Blastomonasline,Rhodopseudomonas palustrisandNitrobactercannotunambiguously be determined (Figure 4). This is alsoreflected by overall similarity values of more than 95%for all available 16S rRNA sequences from membersof the group includingAfipia. The tree of Figure 4is based on sequences from type strains (if avail-able). A number of complete and partial sequencesfrom Bradyrhizobium japonicumstrains and relatedphotosynthetic strains have been published (Ludwiget al., 1995; So et al., 1994; Swada et al., 1993;Van Rossum et al., 1995; Willems and Collins, 1992;Wong et al., 1994; Xu et al., 1995; Yanagi andYamasoto, 1993; Young et al., 1991). No clearcutbranching pattern can be reconstructed by applyingdifferent treeing methods. Furthermore, a cluster ofBradyrhizobiumstrains separated from the other gen-era is not significantly supported. The heterogeneityof these strains is also evident from a comparison ofoverall sequence similarity values. The lowest simi-larity values for the differentBradyrhizobium japon-icum strains are 96.6%. As shown in Table 2, thecorresponding overall 16S rRNA sequence similarityvalues resulting from comparison of theB. japonicumstrains with non-Bradyrhizobiumstrains of the groupare in the range of 98.6%–95.0% and thereby at leastpartly within the range of intra-Bradyrhizobium japon-icum relationships. As discussed earlier,Bradyrhi-zobium japonicumcould probably be reclassified asRhodopseudomonas palustris(Young et al., 1991).
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Later it was argued that this should not be donegiven the phylogenetic heterogeneity of the genusRhodopseudomonas(Wong et al., 1994). It is evidentfrom the sequence data, that major taxonomic revi-sion of the whole group is needed. At this level ofclose relationships the resolution power of rRNA datais insufficient as a sole basis for reclassification how-ever (Stackebrandt and Goebel, 1994). DNA–DNAhybridization studies and evaluation of chemotaxo-nomic and phenotypic data should be combined withthe rRNA data in a polyphasic approach to resolve thetaxonomic status of this group.
The closest relative of the single species genusAzorhizobiumis currentlyXanthobacter flavus. Thegenus Xanthobacter lacks phylogenetic coherence(Rainey and Wiegel, 1996).Aquabacter spiritensisand Azorhizobium caulinodansare intermixed withthe Xanthobacter species (Figure 5). These organismscluster phylogenetically withAncylobacter aquaticusandThiobacillus novellus(Figure 5). The azorhizobiagroup is only moderately related to the rhizobia andthe bradyrhizobia groups, but is somewhat closer tothe latter (Figure 1).
Nucleic acid probe technology
Molecular background
Specific probe hybridization techniques are based onthe capacity of complementary nucleic acid stretchesto anneal by specific base pairing. Formation and sta-bility of the formed hybrids depends on a number ofphysical and chemical parameters, e.g. temperature,ionic strength, denaturant concentration and pH. Thecrucial factor for specific probe hybridization is thedegree of complementarity of a probe to target andnon-target molecules as defined by the number of mis-matches within the corresponding hybrids (Amannand Ludwig, 1994; Schleifer et al., 1993). Given acareful design of probe sequences and properly ad-justed experimental conditions, single mis-matches inprobe target hybrids of 15–30 base pairs can be suffi-cient to allow the differentiation of imperfect hybridsfrom perfectly matched ones. A variety of techniquescan be used for specific probe hybridization (Schleiferet al., 1993). Three basic steps are typical for mostof these methods: hybrid formation of probe andtarget, removal of non-bound probe or target mole-cules (washing), and detection of hybrids via probeor target associated labels. The classical radioactive
Figure 6. Artificial mixture of Rhizobium etliandEscherichia colihybridized with probe GAM42a (green, specific for theγ -subclassof the Proteobacteria) and the rhizobia probe RHI1247 (red).Phase-contrast (upper panel) and epifluorescence (double exposureusing filter sets for red and green fluorescence; lower panel) micro-graphs are shown for identical microscopic fields. Bar represents 5µm.
labelling procedures have been replaced by a variety ofnon-radioactive labelling and detection methods basedon coloured products of enzymic reactions, chemi-luminescence, or fluorescence (Amann et al., 1995;Schleifer et al., 1993).
Design of specific rRNA targeted probes
As mentioned above, rRNA sequences contain se-quence motifs which are diagnostic for phylogeneticentities and may be used as targets for taxon-specificprobes. Successful design of reliable probes dependson the availability of comprehensive sequence infor-mation (De Rijk et al., 1994; Ludwig et al., 1995;Maidak et al., 1994; Van De Peer at al., 1994) andsuitable software for database handling, target searchand probe evaluation (Strunk et al., 1998). At first,
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Table 3. Sequence and target positions of specific hybridization probes. The homologous regionsare missing within the partial 16S rRNA sequences ofMesorhizobium ciceriandM. tianshanense
Probe Specificity Sequence Target1
RHI1247 Rhizobia2 5′-TCGCTGCCCACTGTC-3′ 1246
RHI820 Rhizobia3 5′-CCGACGGCTAACATTC-3′ 820
BLA999 Blastobacter 5′-CTCCACTGTCCGCGACC-3′ 999
RLM820 Mesorhizobium loti4, M. huakuii, 5′-CCAACGGCTAGCTTCC-3′ 820
15′-position,Escherichia colinumbering (Brosius et al., 1981).2Agrobacterium, Bartonella, Blastobacter aggregatus, B. capsulatus, Brucella, Chelatobacter,Mycoplana dimorpha, M. ramosa, Ochrobactrum, Phyllobacterium, Rhizobium, Mesorhizobium,SinorhizobiumandZoogloea ramigerastrain ATCC 19623.3Agrobacterium, Bartonella, Brucella, Chelatobacter, Mycoplana dimorpha, M. ramosa, Rhizo-bium, SinorhizobiumandZoogloea ramigerastrain ATCC 19623.4Mesorhizobium lotiIAM 13588.5Mesorhizobium lotistrains ATCC 33669, LMG 6125T and LMG 4248.
all database sequences (currently>10.000) have to bechecked for unique motifs for the respective phyloge-netic group. These potential target sites then have tobe evaluated with respect to base composition, length,intraprobe self complementarity, and optimal posi-tion and quality of hybrid destabilizing mis-matchesin potential non-target hybrids. A major criterion forcomputer aided probe optimization is the experimentalease to clearly differentiate probe target hybrids frompotential non-target hybrids. Central mis-matches inpotential non-target hybrids are more destabilizingthan lateral ones. A-A, A-C, C-C, T-T and C-T mi-spairings are more destabilizing than A-G, G-T andG-G. Types of base pairs next to mis-matches havealso to be taken into account. The destabilizing ef-fect of mis-matches can be partly compensated by thenext neighbouring base pairs in correlation with thestrengths of the latter. Probe length is usually chosenin a way that the difference of the predicted disso-ciation temperature of the (perfectly matched) targethybrid and that of the most stable potential non-targethybrids is sufficient (at least 2◦C). Mostly, probesare designed as oligonucleotides comprising 15–20nucleotides. Self complementarities within probe andtarget sequences may result in competition of the for-mation of intra-molecular secondary structures withprobe-target hybridization. A selection of target andthe most similar potential non-target sites of a broadrange probe (RHI1247, Table 3) specific for the rhizo-bia group as defined above is shown in Table 4.
In case the establishment of experimental condi-tions ensuring probe specificity is difficult, the si-
multanous use of competitor probes during the hy-bridization procedure often helps to keep the probespecific (Manz et al., 1992). Competitor probes aredesigned complementary to non target sites. The unla-belled competitor probes displace the labelled specificprobes from the non-target sites and thereby improvethe signal to noise ratio significantly.
In principle, the rRNAs or the corresponding genesare excellent targets for phylogenetic probes. In theorythe alternating sequence of highly conserved to highlyvariable positions should provide potential probe tar-get sites for many different phylogenetic levels orgroups. Indeed, in many cases there are diagnos-tic sequence signatures (single nucleotides, sequencestretches, base pairs, deletions, insertions) common toall members of a given phylogenetic group which mayprovide unique group specific target regions. How-ever, in praxis it is often difficult or even impossible todesign specific probes exclusively targeting all organ-isms of a phylogenetic group. The reconstruction andcurrent definition of phylogenetic groups is based onthe comparative analysis of full 16S rRNA sequenceswhereas probe target sites comprise only short se-quence stretches. Given that the groups are defined onthe basis of the full information content of rRNA se-quences, that means a number of positions or stretcheswhich are informative for the particular level of relat-edness, the existence of comprehensive signatures forthe particular group based on one or a few diagnosticresidues cannot be generally expected. Furthermore,evolution is progressing and signatures may be lostas a result of single base changes, while the phylo-
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Table 4. Alignment of 16S rRNA regions homologous to the target sites of probe RHI1247from a selection ofα-subclass proteobacteria. The solid line separates target (rhizobiagroup; upper part) and non-target (organisms which are not members of the rhizobia groupbut contain the probe target site and a selection of organisms with one or two base differ-ences within the homologous sequence region; lower part) organisms. Only base differencesare shown. Identitites are indicated by =
Abbreviations:ATCC – American Type Culture Collection, Rockville, Maryland, USA;IAM – Institute of Applied Microbiology, University of Tokyo, Bunkyo-ko, Tokyo, Japan;IFO – Institute for Fermentation, Osaka, Japan; DSM – Deutsche Sammlung von Mikror-ganismen und Zellkulturen, Braunschweig, Germany; LMG – Culture Collection of theLaboratorium voor Microbiologie, Universiteit Gent, Gent, Belgium; USDA – United StatesDepartment of Agriculture, Beltsville, USA.
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genetic position of the organisms is maintained. Thereare well defined phylogenetic groups for which singleprobes cannot be designed, e.g. the rhizobia group.Frequently, it is not possible to design single probesthat are specific for the complete group or which ex-clude all non-group organisms. The rhizobia probe(RHI1247, Table 3) represents an example of a groupspecific probe that detects all organisms of the rhizobiatarget group but fails to discriminate a few non-targetgroup organisms (Table 5). In many cases this prob-lem can be resolved by the combined application ofprobes with overlapping or supplementary specifici-ties. A second 16S rRNA targeted rhizobia groupspecific probe (RHI820, Tables 3, 5) was designedwhich matches 16S rRNA targets of the majority ofthe members of the group, but does not hybridizeto some of them (Blastobacter, Ochrobactrum, Phyl-lobacterium, Mesorhizobium huakuii, M. loti andM.mediterraneum). Sub-group specific probes (BLA999,Blastobacter aggregatusandB. capsulatus; RLM820,Mesorhizobium huakuii, M. mediterraneumand M.loti strains; RLP820,Phyllobacteriumand Mesorhi-zobium loti strains; Table 5) have been designed.The successful hybridization of probe RHI1247 andone of the complementary probes RHI820, BLA999,RLM820, and RLP820 allows reliable identifcation oforganisms as members of the rhizobia group and theirrespective sub-groups.
A similar problem concerns the reliability of probespecificity. Modern probe design is based on large andfairly comprehensive sequence data sets. However, theprobes can only be optimized with respect to the avail-able sequence data, and experimental evaluation ofprobe specificity is restricted by experimental facilitiesand the availability of reference strains. Given that asingle oligonucleotide probe targets a short sequencestretch often containing only one or two diagnos-tic positions, it is possible that the same target mayoccur within unknown rRNAs of organisms whichphylogenetically are not members of the specificitygroup. This is especially the case with probes specificfor lower taxonomic units (e.g. species). Nucleotideswhich are diagnostic at these phylogenetic levels oc-cupy rapidly changing sequence positions. Thus, thereis considerable risk that ‘false’ identities occur withinrRNAs of unrelated organisms resulting from multiplebase changes during the course of evolution. Conse-quently, the use of single specific probes may resultin misidentification. The multi-probe concept outlinedabove for group specific probes targeted to more con-served regions can also be applied to validate highly
specific probes. It is highly unlikely that ‘false’ identi-ties evolved at multiple non-homologous rRNA sitesin phylogenetically distant organisms. Using multi-ple probes of identical specificities targeting differentsites, successful hybridization of all probes of the setensures correct identification.
Given the availability of rapidly growing sequencedatabases and improved software (Strunk et al., 1998),the risk of improper probe design is substantiallyreduced. New probes should still be evaluated exper-imentally against targets from a representative selec-tion of reference organisms however. This is of majorimportance whenin situ techniques will be used forthe analysis of complex environmental samples. Thepotentially vast number of so far uncultured bacteriain such samples (Amann et al., 1995; Brock, 1987;Staley and Konopka, 1985) holds many possibilitiesof potential false positive identification. Table 6 showsthe results of probe evaluation using the broad rangerhizobia probe (RHI1247) in combination within situcolony and whole cell hybridization techniques and aselection of target and non-target organisms.
Hybridization techniques
A variety of solid and solution phase techniques forspecific probe hybridization to various nucleic acidpreparations are in use (Schleifer et al., 1993). Tar-get nucleic acids may originate from pure cultures,mixed cultures, or complex samples. For several rea-sons rRNAs are the preferred target molecules. Incomparison with 1–12 rRNA genes per cell, the highcopy number of rRNA molecules provides a naturaltarget amplification resulting in a higher sensitivity ofthe hybridization approach (Amann et al., 1995). Fur-thermore, potential non-specific binding to genomicDNA not encoding rRNA is eliminated by using puri-fied rRNA. Most hybridization techniques can be usedto determine the relative abundance of specific targets.Universal probes which are complementary to invari-ant or highly conserved rRNA sequence stretches andconsequently hybridize to rRNAs and/or the corre-sponding genes of essentially all organisms are usedas standards for hybridizations with specific probes(Stahl et al., 1988). Results obtained from such quanti-tative measurements do reflect the relative abundanceof rRNA molecules or genes rather than relative cellnumbers however. The number of rRNA moleculesand therefore the number of specific targets varies,e.g. in relation to the physiological status of the cell
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Figure 7. A: In situ hybridization of rhizobia within root nodules ofTrifolium sp. Cryosections of root nodules were fixed in an increasingethanol series and treated with methanol/H2O2 to block endogenous peroxidases (Amann et al., 1995). The peroxidase-labeled oligonucleotideprobe RHI1247 was detected with diaminobenzidine which formed brownish precipitates in the cells. Left panel shows phase-contrast micro-graph, right panel bright-field micrograph from one microscopic field. B: Autofluorescence of cyanobacteria and the surrounding plant materialin cryosections of the waterfernAzolla filiculoides. Identical microscopic fields are shown in the phase-contrast (left) and epifluorescencemicrographs (right, using a fluorescein-specific filter set). C: Cross-section of a dorsal leaf lobe fromAzolla caroliniana. Cyanobacteria livingin leaf cavities were hybridized with the eubacterial probe Eub338 labeled with peroxidase and detected as indicated in A. Phase-contrast (left)and bright-field (right) micrographs are shown. Bars represent 10µm.
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Table 5. Nested or supplementing specificities of probes RHI1247, RHI820, BLA999, RLM820and RLP820. The complete data set of available small subunit RNA sequences was screened forthe presence of the respective probe target sites. Only organisms which contain the target sites arelisted. All other organisms which are represented in the database contain at least one base differencewithin the homologous sequence parts. The solid line separates target (rhizobia group; upper part)and non-target (organisms which are not members of the rhizobia group but contain the RHI1247probe target site; lower part) organisms. Organisms for which only partial sequences are availablelacking the target region (e.g.Mesorhizobium cicerior Rhizobium tropici) are not included
(Schaechter et al., 1958). Also the number of rRNAgenes may differ even between closely related species.
Classical hybridization formats, e.g. dot-, slot-,or spot-blot techniques, typically rely on membranebound target nucleic acids. Alternatively, other solidsupports such as micro-titer plates can be used for tar-get immobilization. More sophisticated variations arethe capture techniques (Morrissey and Collins, 1989;Schleifer et al., 1993). Two probes of different oridentical specificities are used for immobilization ofthe target nucleic acids (capture probe) and for de-tection and quantification of targets (detector probe).
The hybrids of capture probe and target nucleic acidsmay be immobilized on solid supports such as para-magnetic beads by different techniques (Morrissey andCollins, 1989; Schleifer et al., 1993). One possibilityis the hybridization of capture probe tails and comple-mentary nucleic acids bound to the support, anotherthe interaction of capture probe associated haptens(e.g. biotin, digoxigenin) and binding proteins (e.g.avidin or antibodies) which are fixed on the support.In comparison with the direct hybridization methods,the capture probe assays provide higher sensitivity byspecific enrichment of the targets prior to detection.
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Table 6. Results ofin situcolony and/or cell hybridization of probe RHI1247to a selection of target and non-target organisms of theα-subclass of thePro-teobacteria. The dotted line separates target (rhizobia group; upper part) andnon-target (organisms which are not members of the rhizobia group; lowerpart) organisms.In situ colony hybridizations with peroxidase-conjugatedprobe RHI1247 were carried out in 5×SSC containing 35% (vv) formamideat 37◦C. In situcell hybridizations with fluorescent or peroxidase-conjugatedprobe RHI1247 were performed in 0.9M NaCl containing 45% formamide at46 ◦C
1Pinero et al., 1988;2Centro de Investigacion sobre Fijacion de Nitrogeno, Cuernavaca,Mexico;Abbreviations:CECT – Collection Espanola de Culturas Tipicos, Va-lencia, Spain; IBÖ – Institut für Bodenökologie, Forschungszentrumfür Umwelt und Gesundheit, Neuherberg, Germany; WS – Bakte-riologisches Institut, Süddeutsche Versuchs- und Forschungsanstaltfür Milchwirtschaft, Weihenstephan, Freising, Germany; nd – notdetermined. Other abbreviations are as specified for Table 4.
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The major advantage of reverse hybridization tech-niques (Ehrmann et al., 1994) is the simultaneous useof sets of probes of different or nested specificitiesbound to separate locations on solid supports such asmembranes or micro-titer plates. The target nucleicacids are simultaneously hybridized to all probes ofthe set. If pure cultures are analyzed, the resultinghybridization patterns allow the rapid identification ofisolates at various taxonomic levels, if complex mix-tures of organisms, e.g. environmental samples areinvestigated, the composition of that part of micro-bial communities can qualitatively be elucidated forwhich specific probes are available. In the latter case,for rapid and sensitive screening of complex samplesrDNA fragments containing the target or equivalentregions are amplifiedin vitro as potential hybridiza-tion partners. The principle of reverse hybridization isalso the basis of a promising technique which is underdevelopment (Lipschutz et al., 1995). High densityoligonucleotide probe arrays are prepared by light-directed chemical synthesis. Currently 20,000 sites onan area of 1.28 cm2 can be used forin situ probesynthesis. The target nucleic acids are labeled withfluorescent reporter groups and hybrids are detectedby epifluorescence confocal scanning.
The hybridization methods described above donot allow direct determination of viable or total cellcounts. This can only be achieved by applyingin situprobing techniques.In situ colony hybridization wasused to evaluate the rhizobia specific probe (RHI1247)with a selection of rhizobial and non-rhizobial culturecollection strains and isolates (Table 6). The bacterialstrains of interest were first grown to small colonieson suitable nucleic acid binding membranes placed onthe top of solid nutrient media. The cells were lysedin situ applying combinations of chemical (e.g. al-kaline), enzymatic (e.g. lysozyme) and physical (e.g.micro wave) treatments (Schleifer et al., 1993). Thereleased nucleic acids were fixed on the membrane andhybridized. Probes of different specificities markedwith different labels for different detection systemscan be used simultaneously. Alternatively, differentprobes can be applied in subsequent cycles of hy-bridization detection and denaturation. Depending onthe growth and lysis behaviour of the strains, up to2×103 minicolonies can be analyzed on one filter ofthe diameter of standard plates (9 cm) (Brockmannet al., 1996). Using appropriately diluted samplesand properly designed specific probes, the bacteria ofcomplex samples can be differentiated and enumer-ated as colony forming units. The advantages of this
technique are the rapid detection, enumeration andidentification of bacteria at defined taxonomic levelsamong a large number of non-target organisms. Themajor drawbacks are the demand for cultivation of thetarget organisms and the variability of growth and ly-sis behaviour. Fast growing organisms may inhibit ormask slow growing ones. This problem can be reducedby applying differently diluted samples and incuba-tion of membranes in parallel at different conditionsfor bacterial growth. Nucleic acids of cells resistant tothe lysis remain inaccessible for the probes. Excessiveproduction of extracellular polymers may hinder im-mobilization of the released target nucleic acids or thehybridization procedure itself. This seems to be a com-mon problem with a number of rhizobial strains. Falsenegative results of specific probe hybridization can berecognized as such by performing hybridizations witha universal probe as a positive standard however.
Currently, the most attractive technique for spe-cific probe based identification and detection isin situwhole cell hybridization. The procedure is based onspecific probe hybridization to intra-cellular targets.Again rRNA has advantages as a potential target nu-cleic acid due to its natural amplification. An averagebacterial cell contains somewhere between 103 and105 ribosomes and as many copies of the 5S, 16S and23S rRNA. These molecules can be made available inwhole fixed cells for hybridization with specific probesby permeabilization of the cell periphery with aldehy-des or alcohols. These compounds stabilize the cellstructure and kill the cells (Amann et al., 1995). Mi-croscopic identification of individual microbial cells isroutinely achieved with fluorescently labeled rRNA-targeted oligonucleotide probes (Amann et al., 1990;DeLong et al., 1989). The technique allows identifi-cation of individual cells within the microscopic fieldand provides high spatial resolution. Even complexenvironmental material can be analyzed within fewhours after sampling. As the technique is not rely-ing upon prior cultivation of the cells of interest itdifferentiates not directly between viable and nonvi-able cells. Morphological integrity and the presenceof high amounts of intra-cellular rRNA is a goodindication for cell viability, however. As the ma-jority of bacterial species has not yet been isolatedin pure culture, the combination of direct rRNA se-quence retrieval and fluorescentin situ hybridizationallows completely cultivation-independent single cellidentification and phylogenetic analysis (Amann etal., 1991; Spring et al., 1992). Fluorescentin situhybridization with rRNA-targeted probes is a most
16
useful tool for microbial ecology since it allows todirectly determine cell numbers of the target organ-isms in complex samples and to monitor strains withvariable morphology. In case of immobilized com-munities, as in symbioses of nitrogen fixing rhizobiaor cyanobacteria with plants, the spatial distributionscan be determined. Furthermore, rRNA-contents ofindividual cells may be measured which may yieldestimates on general metabolic activity of individ-ual cells (DeLong et al., 1989; Poulsen et al., 1993;Wallner et al., 1993). Some drawbacks of this excel-lent technique have to be taken into account. Giventhat successful probe hybridization depends on per-meabiltiy of the cell envelope and hybrid detection onthe ribosome content, cells may escape their detection.Fixation inevitably causes shrinking or deformation ofthe sample material. Thus the spatial distribution ofthe detected cells cannot correctly reflect the situationin the original sample.
An example of single cell identification of rhi-zobia using fluorescently labelled oligonucleotideprobes is given in Figure 6. An artificial mix-ture of Rhizobium etliand Escherichia coliwas si-multaneously hybridized with a fluorescein-labelledprobe specific forγ -subclass Proteobacteria (Manz etal., 1992) and carboxytetramethyrhodamine-labelledprobe RHI1247. After hybridization the two speciescould be clearly differentiated by the specific red (R.etli) and green fluorescence (E. coli) conferred bythe two probes. On examination of the symbioses ofnitrogen-fixing rhizobia with plants, the strong aut-ofluorescence of the plant material interfered with thedetection of fluorescent probes, however. This prob-lem could be avoided by the use of a nonfluorescenthybridization assay in which horseradish peroxidaselabelled oligonucleotides are used. The hybrids aredetected by the formation of a coloured precipitate(Amann et al., 1992). Using this technique, rhizobiacould be identified, e.g. in ethanol fixed cryosectionsof root nodules ofTrifolium sp. with probe RHI1247(Figure 7, panel A).
Autofluorescence is even more pronounced for theidentification of nitrogen fixing cyanobacteria in thedorsal leaves of the water fernAzolla caroliniana.Here, not only the plant material but even the tar-get bacteria strongly auto-fluorescence due to theirchlorophyll content (Figure 7, panel B). Again spe-cific detection of the cyanobacteria was achieved withthe enzyme-linked assay using the bacterial probeEUB338 (Amann et al., 1995). This technique is ad-vantageous not only for its ability to identify cells in
fluorescent environments but also since it does not re-quire an expensive epifluorescence microscope. Theformation of precipitates can be seen under phase con-trast (Figure 7, left side of panels A and C) but is bestdetected in a regular light microscope with brightfieldillumination (Figure 7, right side of panels A and C). Itshould be noted, however, that the spatial resolution isinferior to that achieved with fluorescent probes so thatdense formations of target cells are seen only as brownareas which makes enumeration almost impossible.
Limitations
The usefulness of highly variable positions for thedesign of highly specific probes needs further criti-cal evaluation. Most bacteria contain multiple rRNAgenes. Reports on base differences in multiple rRNAgenes of the same organisms are available (Boroset al., 1979; Branlant et al., 1981; Carbon et al.,1979; Dryden und Kaplan, 1990; Fleischmann et al.,1995; Gazumyan et al., 1994; Moriya et al., 1985;Ogasawara et al., 1993; Shen et al., 1982), but nosystematic investigations have been performed. Re-cent investigations (Nübel et al., 1996) indicate thatinteroperon differences exist at least in some phy-logenetic groups at highly variable rRNA sequencepositions. Various operons seem to be transcribed atdifferent frequencies in closely related strains. The po-tential problems for single probe based identificationare obvious: closely related strains missing the genevariant containing the correct target site may escapedetection.
Another limitation of rRNA based identificationand detection methods concerns their ability to resolvelower taxonomic levels and originates from the overallconserved character of the rRNA corresponding genes.Usually, strains are too closely related to contain diag-nostic sequence differences in their rRNAs. In mostcases, it is therefore not possible to gain phylogeneticinformation at this level or to design strain specificprobes. Even if there are a few odd base differencesthat might be used for differentiation, these are of lowsignificance for phylogenetic inference or probe basedidentification.
Another type of problem occurs when the cur-rent taxonomy does not agree with phylogeny andtherefore taxon specific probes cannot be designed.
The quantification of microbial cells with specificprobes is hampered by the demand for cultivation us-ing in situcolony hybridization and possible problems
17
of cell permeability and ribosome content applyinginsitu whole cell hybridization.
In general, rRNA targeted probes are of only lim-ited value when a phenotypic trait such as nitrogenfixation is of major interest. Application of rRNAtargeted probes combined with probes specific forgenes involved in the expression of the phenotypictrait might correlate phylogenetic identification withphysiological capacity (see Perret and Broughton, thispublication).
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