System. Appl. Microbiol. 23,107-114 (2000) SYSTEI\I1AllC AND © Urban & Fischer Verlag _htt-,-p_:llw_w_w_.ur_ba_nf_is_ch_er_.de--.:./i_ou_rn_als_/s_am ____________ APPLIED MICROBIOLOGY

165 rRNA-Targeted Oligonucleotide Probes for the in situ Detection of Members of the Phylum Cytophaga­Flavobacterium-Bacteroides

ROLAND WELLER, FRANK OUVER GLOCKNER, and RUDOLF AMANN

Max-Planck-Institute for Marine Microbiology, Bremen, Germany

Received November 22, 1999

Summary

Bacteria of the Cytophaga-Flavobacterium-Bacteroides phylum (CFB-phylum) are numerically impor­tant members of many microbial communities. A suite of five 165 rRNA-targeted oligonucleotide probes for members of this group is described which was designed to dominantly target bacteria of the CFB-phylum that are found in particular habitats. For this we initially performed a literature survey for the sources and sites of isolation of hitherto described members of the CFB-phylum. Probe CFB286 is mostly complementary to the 165 rRNA of species originally isolated from freshwater habitats, howev­er, detects some marine and soil isolates and is the only probe which includes some food isolates. Probe CFB563 detects marine as well as animal-associated isolates. Probe CFB719, which also detects some environmental isolates, and probe CFB972 are mostly targeting animal-associated isolates. All probes are complementary to a variety of human-associated species within the CFB-phylum which, in the data set investigated (October 1998), made up 46% of all 165 rRNA sequences from the CFB-phylum. We conclude that it is difficult to find habitat-specific probes for members of the CFB-phylum and that the design of probes for monophyletic groups should remain the standard approach. Applicability of the probes for fluorescence in situ hybridization and specificity for single cell detection were evaluated for the four mentioned probes whereas the fifth, probe CFB1082, which almost exclusively targets human­associated species, was not further characterized. The new probes are of limited determinative value and should be used together with the already established probes for the CFB-phylum. It is the hybridization pattern observed for a given cell or culture with the enlarged probe set that is suggestive for its affiliation with a defined group within the CFB-phylum.

Key words: rRNA-targeted oligonucleotide probes - in situ hybridization - CFB-phylum

Introduction

A large fraction of the currently known gram-negative bacterial species and their phenotypic diversity is con­tained within two phyla. These are the former "purple bacteria" , now classified in the class Proteobacteria (STACKEBRANDT et al., 1988), and a large grouping origi­nally described as the flavobacter-bacteroides phylum (WOESE, 1987). The latter encompasses besides many other genera the well known gliding bacteria of the genus Cytophaga, the aerobic pigmented bacteria of the genus Flavobacterium, and anaerobic bacteria of the genus Bac­teroides. This group is therefore here referred to as the Cy­tophaga-Flavobacterium-Bacteroides- or CFB-phylum.

Like the Proteobacteria the CFB-phylum has devel­oped a truly surprising breadth of adaptations to all kinds of environments (REICHENBACH, 1991). Members

of the CFB-phylum can be found in habitats such as the human gut (FRANKS et al., 1998), polluted to seemingly pristine river water (KENZAKA et al., 1998) and in ex­treme habitats like Antarctic marine waters (MCGUIRE et al., 1987), to just list a few examples. Accordingly broad is their physiological repertoire. In this group we find anaerobic human pathogenic organisms, fermentative forms, and aerobic and microaerophilic organisms with the ability to synthesize a variety of either cell-bound or extracellular degradative enzymes like cellulases, chiti­nases or proteases. These properties make them very im­portant members of microbial food chains (REICHEN­BACH, 1991). It is therefore also not surprising that mem­bers of the CFB-phylum have been found in high abun­dance in various habitats by tools of molecular microbial

0723-2020100/23/01-107 $ 12.0010

108 R. WELLER et al.

ecology such as fluorescence in situ hybridization (FISH) (e.g. MANZ et al., 1996; LLOBET-BROSSA et al., 1998; GLOCKNER et al., 1999). Probes for the CFB-phylum are consequently of high importance for a fairly complete initial description of the composition of the bacterial fraction of complex communities with the top-to-bottom approach applying group-specific, rRNA-targeted oligo­nucleotides (AMMAN et al., 1995).

Since the first attempt to design rRNA-targeted oligo­nucleotide probes for this important group by MANZ and coworkers (MANZ et al., 1996) the 16S rRNA databases have expanded considerably. This is in part due to the di­rect retrieval of 16S rRNA sequences by culture-indepen­dent cloning from environmental nucleic acids (for ex­ample see WELLER & WARD, 1989; WARD et al., 1990; GIOVANNONI et a1.1990; DELONG et al., 1993). Conse­quently, the coverage of the CFB-phylum by probes like, e.g., CF319a (MANZ et al. 1996) is becoming more and more incomplete.

The aim of this study was to design oligonucleotide probes for use in FISH studies which target additional members of this important grouping. With the ever in­creasing 16S rRNA sequence database and the great phy­logenetic depth within the CFB-phylum it is impossible to describe the whole phylum with a single group-specific probe. We therefore investigated here the potential to construct a small set of probes that focus on the members of the CFB-phylum which had been originally isolated from specific habitats, e.g., a probe that would detect most of the members of the CFB-phylum originally ob­tained from marine samples. For that we set out in sum­mer 1998 to obtain as much information as possible on the origin of those members of the CFB-phylum for which a 16S rRNA sequence had been published by that time. Here, we report a suite of five oligonucleotide probes that preferentially target members of the CFB­phylum from specific habitats.

Materials and Methods

Organisms and culture conditions All organisms were purchased from DSMZ (Deutsche

Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). Flavobacterium ferrugineum (DSM 30193), Flexibacter polymorphus (DSM 9678), Pedobacter heparinus (DSM 2366) (formerly Cytophaga heparina) and Flavobacterium johnsoniae (DSM 2064) were grown in the media recommended by DSMZ at 25°C shaking at low speed to densities of about 0.5 at A60o •

Design and evaluation of probes Oligonucleotide probes were designed with the PROBE_DE­

SIGN tool of the ARB software package (STRUNK et al., 1999). All oligonucleotides probes were purchased labeled with the monofunctional, hydrophilic sulfoindocyanine dye Cy3 (Inter­activa, Vim, Germany) at the 5'-end. In situ hybridizations were performed as described by GLOCKNER and coworkers (GLOCK­NER et al., 1996). Hybridization and washing buffers were those described by MANZ et al. (MANZ et al., 1992). Optimal hy­bridization conditions were established by monitoring the hy­bridization to target- and nontarget-bacteria. Formamide con-

centrations were increased with increments of 10% until the nontarget organisms with one or more mismatches were no longer hybridizing. We furthermore determined the formamide concentration at which even the target organism was no longer detected to fully characterize the probe. The recommended hy­bridization stringency was then defined as the concentration at which the target-organism was still detected with a bright sig­nal, while the nontarget~organisms were not hybridizing.

Integration of habitat information into the phylogenetic tree Information concerning the site and source of original isola­

tion was taken from either the DSMZ catalog (http;//www. dsmz.de/dsmzfind.htm) or the organism-search page of the ATCC (http;l/phage.atcc.orgisearchengine/ba.html).

Results and Discussion

Probe design

The design of new group-specific probes for members of the CFB-phylum turned out to be difficult. Good group-specific probe which were complementary to most of the members of the CFB-phylum and no or only few outgroup organisms could not be found. CF319a is, at least for the Cytophaga/Flavobacterium branch, still the most general probe available. There are, of course other signatures (WOESE et al., 1985) for the CFB-phylum on the 16S rRNA as potential probe target sites. However, the larger the number of available sequences is, the more deviations from those idiosyncrasies are found. This problem also applies to already published group-specific probes like those for the alpha-, beta-, gamma-subclass of the class Proteobacteria (MANZ et al., 1992). For ex­ample, it has recently been shown that GAM42a, a probe targeting the gamma-proteobacteria, does not detect Xanthomonas sp. (BUCHHOLZ-CLEVEN et al., 1997) and Nevskia ramosa (GLOCKNER et al., 1998) which are both deep-branching gamma-subclass proteobacteria.

The problem of reliance on individual signatures has been realized from the beginning of the probe technology and it had been stated that a group-specific probe should only be used together with other probes (MANZ et al., 1992; AMANN et al., 1995; AMANN, 1995). It is the appli­cation and quantification of several group-specific probes that makes oligonucleotide hybridization a reli­able tool to examine community composition.

Since our intention was to supplement the set of group-specific probes with respect to the CFB-phylum our strategy had to be changed. It would have been pos­sible to select smaller, yet monophyletic groups for probe design, but given the depth of this phylum this would have resulted in a set of >20 probes which would have been fairly unpractical for FISH. It was subsequently at­tempted to design probes that should at least target most of the members of the CFB-phylum that had been isolat­ed from specific habitats. On this level group coverage became more complete and a total of five probes were designed. Sequences and positions as well as the recom­mended hybridization conditions for each probe are shown in Table 1. One probe, CFB1082, was not charac­terized in detail since it targets mainly human-associated

Probes for the CFB-Phylum 109

Table 1. Group-specific probes for members of the CFB-phylum and recommended formamide concentrations.

Probe Sequence (5'-3') 16S rRNA Target Position':· %Formamide Reference

CFB286 CFB563 CFB719 CFB972 CFB1082 CF319a CF319b BAC303

TCCTCTCAGAACCCCTAC GGACCCTTTAAACCCAAT AGCTGCCTTCGCAATCGG CCTTGGTAAGGTTCCTCG TGGCACTTAAGCCGACAC TGGTCCGTGTCTCAGTAC TGGTCCGTATCTCAGTAC CCAATGTGGGGGACCTT

,. According to BROSIUS et aI., 1981 + N.D. - not determined

286-304 563-580 719-736 972-989 1082-1100 319-336 319-336 303-319

organisms and our set of group-specific probes is intend­ed to be used in environmental microbiology. We do, however, report here its sequence and habitat distribu­tion to allow interested groups a further characterization for application in medical microbiology.

Before we go into detail we would like to shortly dis­cuss the consequences of a habitat-targeted strategy for probe design. Usually probes are designed for coherent phylogenetic groups. The clear affiliation of a probe-pos­itive cell to a well defined target group is the normal basis for the interpretation of FISH results in environ­mental microbiology. We are well aware that the habitat­based strategy it is not compatible with easy determina­tive use of the new probes in samples of unknown origin and composition. Ultimately, however, we hope that the pattern of hybridization/lack of hybridization with a whole set of probes will be suggestive for a quite narrow, potentially coherent target group very much like other molecular fingerprints.

Probes were designed in a way to yield a maximal number of internal mismatches to nontarget sequences (STAHL & AMANN, 1991). In the difference alignments (Fig. 1) the basis for the discrimination of target and nontarget sequences is visualized on a arbitrary selection of two target sequences (one of them used for determin­ing the dissociation temperature), few nontarget se­quences of members of the CFB-phylum used as negative controls, and representative of other bacterial groups (Rhodothalassium salexigens, a-subclass of Proteobacte­ria; Burkholderia cepacia, ~-subclass of Proteobacteria; Escherichia coli, y-subclass of Proteobacteria; Leptospira bif/exa, spirochetes).

The distribution of the site/source of isolation over the 16S rRNA-based reconstruction of the phylogenetic tree of the CFB-phylum taken from the ARB-database (STRUNK et al., 1999) is shown in Fig. 2. There are few direct links between phylogenetic position and isolation from a certain habitat. However, some clustering can be seen. The PrevotellalBacteroideslPorphyromonas group contains only human- and animal-associated organisms as does the genus Capnocytophaga. Also the group in­cluding the mostly human-associated Chryseobacte­riumlEmpedobacteriWeeksella species is close to animal­associated bacteria including a tight cluster of insect

50 20 30 20 N.D.+ 35 35 o

this study this study this study this study this study MANZ et aI., 1996 MANZ et aI., 1996 MANZ et aI., 1996

endosymbionts. There also seem to be groups that solely contain environmental isolates. However, soil, freshwa­ter and marine water isolates are found intermingled in these groups.

Since rRNA-targeted oligonucleotide probes have au­tomatically a phylogenetic component in their specificity, the newly designed probes show preferences to certain phylogenetic groupings (see Fig. 2). Typical examples of this feature are the tight cluster of nine insect endosym­bionts, which are all targeted by probe CFB972 or the detection of the PrevotellalBacteroideslPorphyromonas group by probe CFB1082. Given the habitat distribution over the phylogenetic tree it is also not surprising that we could not come up with probes for freshwater or soil­members of the CFB-phylum. Interestingly, we faced the same problem as MANZ and coworkers (MANZ et al. 1996). None of the newly designed probes targeted Flavobacterium ferrugineum which branches apart from the other flavobacteria deeply in the CFB-phylum. Hence, probe FFE8b (MANZ et al. 1996) remains the only probe for this species.

In situ hybridization

The applicability of three of the newly designed probes for in situ hybridizations was tested on a mixture of Flavobacterium johnsoniaelPedobacter heparinus. The results are shown in Fig. 3. Probes CFB563 (Fig. 3A) and CFB972 (Fig. 3C) bind only to the long rods of F. johnso­niae and discriminate against the short rods of P. hepari­nus. This discrimination is based on one terminal mis­match and two internal mismatches (see Fig. 1 for exact location) of the 16S rRNA sequence of P. heparinus, re­spectively. Probe CFB719 on the other hand targets P. heparinus and discriminates against F. johnsoniae based on two internal mismatches (Fig. 3B). Probe CFB286 also showed strong and specific hybridization (data not shown). With their good specificity and sensi­tivity those four probes should be suitable for environ­mental applications.

The probe target sites starting at positions 286, 563, 719 and 972 were accessible in the members of the CFB­phylum tested. The target sites had been selected based on a recently completed in situ accessibility map of

110 R. WELLER et al.

CFB286 Target Flexibacter polymorphus ATCC27820 Bacteroides splanchnicus NCTCI0825 Flexibacter canadensis ATCC29591 Pedobacter heparinus DSM2366 Flavobacterium johnsoniae DSM2064 Flavobacterium ferrugineum ATCC13524 Rhodothalassium salexigens ATCC35888 Burkholderia cepacia ATCC25416 Escherichia coli ATCCl1775 Leptospira biflexa ATCC23583

CFB563 Target Flavobacterium johnsoniae DSM2064 Flavobacterium flevense ATCC27944 Pedobacter heparinus DSM2366 Flavobacterium mizutai ATCC33299 Flavobacterium ferrugineum ATCC13524 Rhodothalassium salexigens ATCC35888 Burkholderia cepacia ATCC25416 Escherichia coli ATCCl1775 Leptospira biflexa ATCC23583

CFB719 Target Pedobacter heparinus DSM2366 Flexibacter canadensis ATCC29591 Psychroflexus gondwanensis ATCC51278 Flavobacterium ferrugineum ATCC13524 Flavobacterium johnsoniae DSM2064 Rhodothalassium salexigens ATCC35888 Burkholderia cepacia ATCC25416 Escherichia coli ATCCl1775 Leptospira biflexa ATCC23583

CFB972 Target Flavobacterium johnsoniae DSM2064 Flavobacterium aqua tile ATCCl1947 Flavobacterium ferrugineum ATCC13524 Pedobacter heparinus DSM2366 Flexibacter canadensis ATCC29591 Rhodothalassium salexigens ATCC35888 Burkholderia cepacia ATCC25416 Escherichia coli ATCCl1775 Leptospira biflexa ATCC23583

CFB1082 Target Bacteroides vulgatus ATCC8482 Bacteroides thetaiotaomicron ATCC29148 Flexibacter canadensis ATCC29591 Flavobacterium ferrugineum ATCC13524 Pedobacter heparinus DSM2366 Flavobacterium johnsoniae DSM2064 Rhodothalassium salexigens ATCC35888 Burkholderia cepacia IHCC25416

Escherichia coli ATCdl1775 Leptospira biflexa ATCC23583

3'CATCCCCAAGACTCTCCT5' 5' GUAGGGGUUCUGAGAGGA3'

A ...... A ......... . C ...... A ......... . .N .....• C ........ G · .. ACU. G. G ...... C. A ... CU.G. U ....... . .... CUNG ......... . C ... CU.G ......... . · ... CC. GC ........ G

3'TAACCCAAATTTCCCAGG5' 5' AUUGGGUUUAAAGGGUCC3'

................ G.

................ G.

.C ............. AG. · C .... CG ..... C . CG . .C .... CG ..... C .. G. .C .... CG ..... C.CA. ...... CG ........ GU

3'GGCTAACGCTTCCGTCGA5' 5'CCGAUUGCGAAGGCAGCU3'

................ A. · .A .............. . · .A ............. G. · .AG.G ........ G .. . .•.•. G •....••.... C ... G.G ........ G .. C ..... G ...•..... A •.

3' GCTCCTTGGAATGGTTCC5' 5'CGAGGAACCUUACCAAGG3'

.............. U ...

.............. CG ..

.............. CG .. · . CA ........... GCU ... AA ......... U.CC .•. A .......... UG. U ... AA ......... U •••

3'CACAGCCGAATTCACGGT5' 5' GUGUCGGCUUAAGUGCCA3'

.... U .. G ...•.. C .. G

.... U .. G •....• C .• G

...• U .. G ...•.. C .. G

..... A.G ...... C. U. A ... U .. G •..... C •• G A ..• 0 •• G ...... C •. G

A .•• U .. G ...... C .. G A ... U .. G .••... C .• G

.... Fig. 1. Sequences of the five de­signed probes shown on top of difference alignments of selected members of the CFB-phylum and nontarget bacteria (Rhodothalas­sium salixigens, Burkholderia cepacia, and Escherichia coli are members of the alpha-, beta-, and gamma-group of Proteobacteria, respectively, Leptospira biflexa is a spirochete). Dots indicate iden­tity to the probe target sequence, only mismatches to the target are stated. The ordering within the 4-5 selected members of the CFB-phylum is according to number of mismatches.

~

Fig. 2. 165 rRNA-based phylo­genetic tree of the CFB-phylum with color-coded information on the source/site of isolation. Num­bers after the group or species name refer to probes listed in Table 1 (e.g. 1082 to CFB1082 or 303 to BAC303) and indicate full complementarity of the respec­tive probe to the 16S rRNA of the species or to members of the group.

Prevotella group [10821719J1J19aJJOJI286!

Bacteroides fraglfis group [10821719/JOJJ

La ••••• C ===::::===-P porphyrOmonasiBacteroides group [10821719/J19aJJOJJ

'------ CDC group DF-J [1082/JI9a) '-------- Bacteroides splanchnicus [303I286J

Cytophaga xylanolyt/ca (286)

.-----"'- Marim/abt/ia group [JI9a) '----"'------ Cytophaga sp. [2861719J

Cytophaga sp. [719J Cytophaga fermentans [719J

n----- str PAOJO

Bacterial species L ___ -{====~R~ikellella microfuslIs [719/319a)

Bacteroides pu/redinis [719/319a)

Capnocytophaga group [563/9721319aJ319bJ

Cytophaga uliginosa [563/319aJ

Isolate volker [563J '--_....:L. ___ Cytophaga marinonava group [563/319aJ

4 ........... CytophagaiCeltulophaga group (563J

L--t •• ". Flexibacter man/imus group [719/563/319a J

Flavobacterium group (97215631319a~

'----- Myroides odora/us (972J

r--f! ••• -=z::::L~==--- Chryseobactenum group (972/563/319aJ

.--""""----- Empedobacter breVIs [9721563/319a) Weekselta virosa [563J

'----- OmithobacteriulIl rhino/rac/leale [972/563/319a)

insect endosymbionts [9721319a/319bJ

L-_____ "Microscilia aggregans subsp catalanica"

'-------- strclone BOAI-14 "Anaeroflexus maritimus· [719J

r -liD •• CZ::D =--- Sphingobactenum /Pedobacter group [719/319a!

'------ Flex/bacter canadensis [719/319b)

i--IJ •• [7::"J?" Flex/bac/er elegans group (319bJ

'--_____ ---=2=--___ Cilia-associated respiratory tract bacteria

,.-----'''------------ Saprosplra sp.IHaltscomenobacter sp. (319a)

'------- str clone aggregate agg32

,..-----'=----- Flex/bacter liIora/is/F. polymorpha [286)

F/ammeovirga apriea (286J Flexibacter aggregans [319aJ286J

'------"'-- Persicobacter diffluens (319a)

,-----=--- Flex/bacter /ractuosus [319aJ286J

"Microscilla furvescens· (286)

,--=--- Cytophaga hutchmsoni; (319b)/C. auran/iaca!

Sporocytophaga myxococcoldes (319b) ,..----=------ Microscilla sp .lFlexlbacler e/egans

F/ex/bacler ruber [319b)

r---[=======~ Sprrosoma Imguate (319b/286) Runella sllthyformis

Bactenallsolate '------ Cyclobae/erium mBrinus

Flexibaeler nexilis Flectobacillus major

Flex/bacter roseo/us

-C:=J

-Legend:

human-assoc.

animal-assoc.

in marine waters

in freshwater

in soil

variety of subdominant habitats

10%

112 R. WELLER et al.

A

Fig. 3. In situ hybridizations of mixtures of Flavobacterium johnsoniae and Pedobacter heparinus. Phase contrast (left) and epifluo­rescence micrographs (right) of identical microscopic fields are shown after in situ hybridization with probe CFB563 (A), CFB719 (B), and CFB972 (C). Bar, 10 pm.

Escherichia coli 16S rRNA (FUCHS et al., 1998). This in­dicates that the map has, at least for the relatively con­served sites targeted here, predictive power that extends from the class Proteobacteria to other bacterial phyla.

Environmental applications of the new probes

Together with probe CF319a probes CFB286 and CFB719 were used in preliminary tests to assess the mi­crobial community structure of Wadden Sea sediments. CFB286 which targets e.g. the marine Flexibacter litoralis, F. polymorpha and Flammeovirga aprica did not detect cells in the sample examined, while CFB719 detect­ed cells with good signal strength (data not shown). In the same samples morphologically similar small rods were detected by probe CF319a. Referring to the Fig. 2 with our knowledge of the habitat (marine, blue) and the prob­ing result CF319a+/CFB719+/ CFB286- which is in terms of the nomenclature used in Fig. 2 [719/319a] this sug­gests that these bacteria might belong to the Flexibacter maritimus group. Fig. 2 also indicates that this group should also be positive for probe CFB563. This has not yet been checked but our preliminary data suggest that the newly developed probes generally work fine for envi­ronmental applications. A slightly modified form of probe CFB563 was recently used in investigations of ma­rine sediments by ROSSELLO-MORA and coworkers (ROSSELLO-MoRA et al., 1999). This probe targets a group of marine bacteria encompassing Cytophaga uliginosa, C. marinoflava, C. lytica, and Flexibacter maritimus which is in part also covered by CF319a (Fig. 2).

Conclusions

We have added several new probes for members of the CFB-phylum to the set of group-specific probes for the characterization of environmental samples. Their users should always keep in mind that the development of such a set can not be finished as long as we have only analyzed part of the extant microbial diversity. In order to obtain reliable results the new probes should be used in the con­text of the full rRNA cycle (AMANN et al., 1995) which means that the individual result has be viewed in the light of additional hybridizations, patterns and sequencing data. The new probes do not replace but supplement older probes like CF319a which still remains the most encompassing probe for the CFB-phylum.

Probe sets should be considered working tools subject to continued refinement. Given the difficulties encoun­tered in our study for the design of probes for larger phy­logenetic assemblages it is likely that in the future the highly parallel application of multiple probes with more narrow specificity with technologies like DNA chips or probe microarrays may substitute in certain applications for the relatively small sets of group-specific probes used in FISH for a rapid initial analysis of complex microbial communities. However, it must be kept in mind that these new techniques are based on extracted nucleic acids and that there will likely also remain a niche for the es­tablished technique of FISH with group-specific probes.

Probes for the CFB-Phylum 113

Acknowledgements We would like to thank two anonymous referees for their

comments and Jorg Wulf for excellent technical assistance. Funding for this study came from the Max-Planck Society and the Fonds der chemischen Industrie.

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Corresponding author (current address): R. WELLER, Division of Microbiology, GBF, Mascheroder Weg 1, D - 38124 Braunschweig