Phylogeny and Diversity of Achromatium oxaliferum

SYSlEtv\4TIC AND

System. Appl. Microbiol. 22, 28-38 (1999)

© Urban & Fischer Verlag

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Phylogeny and Diversity of Achromatium oxaliferum FRANK OLIVER GLOCKNERl, HANS-DIETRICH BABENZIEN2, JORG WULF!, and RUDOLF AMANN! lMax-Planck-Institut fur marine Mikrobiologie, Bremen, Germany 2Institut fur Gewasserokologie und Binnenfischerei, Neuglobsow, Germany Received November 10, 1998

Summary Achromatium oxaliferum was first described in 1893 by Schewiakoff as an unusually large bacterium living in freshwater sediments. Up to now no pure culture is available. Physical enrichments of achromatia collected from the acidic Lake Fuchskuhle, which houses a peculiar, smaller variety, and the neutral Lake Stechlin were investigated by the cultivation-independent rRNA approach. peR in combination with cloning and sequencing was used for the retrieval of 24 partial and 4 nearly full-length 16S rRNA sequences that formed two distinct phylogenetic clusters. Fluorescence-in-situ-hybridization (FISH) with four 16S rRNA-targeted oligonucleotide probes unambiguously assigned the different sequences to either regular, large A. oxaliferum cells or to the smaller Lake Fuchskuhle population, tentatively named "A. minus". The two Achromatium sp. 16S rRNA sequence clusters form a stable deep branch in the gamma subclass of the class Proteobacteria. The closest cultivated relatives are Chromatium vinosum, Rhabdochromatium marinum and Ectothiorhodospira halophila with 16S rRNA similarities of 86.2 to 90.5%. Profound differences in the population structure of achromatia were revealed in the two lakes by FISH. In one sample from Lake Stechlin three genotypes could be visualized, and 49% of the cells were assigned to A. oxaliferum clone AST01, 28% to Achromatium sp. genotype AFKl92/AFK433 and 23% to Achromatium sp. genotype AFKl92/AST433. In contrast, a morphologically and phylogenetically homogeneous population of "A. minus". was present in Lake Fuchskuhle.

Key words: Achromatium oxaliferum - phylogeny - diversity - 16S rRNA - fluorescence-in-situ-hybridization

Introduction More than one century ago in 1891 Lauterborn (LAUTERBORN, 1915) observed a new organism which was described two years later by SCHEWIAKOFF (1893) as Achromatium oxaliferum. Since then achromatia have been reported in diverse freshwater and marine sediments and new aspects of these conspicuous colorless sulfur bacteria were described (BERSA, 1920; DE BOER et al., 1971; LA RIVIERE and SCHMIDT, 1989; LA RIVIERE and SCHMIDT, 1991; LACKEY and Lackey, 1961; SKUJA, 1948; STARR and SKERMAN, 1965). The cells of A. oxaliferum are spherical, ovoid or cylindrical and exhibit a variety of cell sizes, ranging between a minimum length of 5 ).1m and a maximum length of 125 ).1m (LA RIVIERE and SCHMIDT, 1991). Within the cells sulfur globules and massive CaC0 3 inclusions occur. Over the last 100 years only one additional species, A. volutans, was described by HINTZE (1903). It is consistently smaller (5 ).1m x 40 ).1m) than A. oxaliferum, restricted to saline waters and does not show calcite inclusions. All known Achromatium species live at the sediment-water interface close 0723-2020199/22/01-028 $ 12 .0010

to the transition zone characterized by H 2 S and O 2 gradients. This, together with the large sulfur and calcite inclusions has led many authors to the suggestion that A. oxaliferum may play an important role in the sulfur and carbon cycling of certain sediments (BABENZIEN, 1991; DE BOER et al., 1971; LA RIVIERE and SCHMIDT, 1991). From physically enriched preparations evidence was obtained, that A. oxaliferum can oxidize reduced sulfur species to sulfate (GRAY et al., 1997). Nevertheless, exact data on the physiology must await the availability of pure cultures. Cultivation independent molecular biological methods have been used by HEAD et al. (1996) to study the phylogeny of A. oxaliferum, assigning it to a deep branch of the gamma subclass of the class Pro-

teobacteria. It was the aim of this study to investigate the phylogeny of achromatia in two lakes in the Brandenburg-Mecklenburg lake district, Lake Stechlin and Lake Fuchskuhle, by the cultivation-independent rRNA approach. The acidic Lake Fuchskuhle contains a peculiar population

Phylogeny and Diversity of Achromatium oxaliferum characterized by a considerably smaller average size of 8 pm x 15 pm than the representatives described in all other lakes, including Lake Stechlin (BABENZIEN, 1991). Fluorescence-in-situ-hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes was used to study the population structure and diversity of achromatia in the two habitats investigated.

Materials and Methods Sampling: Achromatia were collected from the littoral zone of Lake Stechlin and Lake Fuchskuhle in May 1996 and luly 1998. Important data like trophic state (VOLLENWEIDER, 1968), total P-concentration [fIgl-1], pH, Ca 2+-concentration [mgl- 1] and conductivity [fIScm-1] are for Lake Stechlin, oligotrophic, 10, 7.2-8.5, 45.6, 305, and for Lake Fuchskuhle, mesotrophicl acidotrophic, 30, 4.2-4.6, 1.8, 45, respectively. Physical data were determined as described by DITTRICH et a!. (1997). Detailed description of the lakes can be found elsewhere (CASPER, 1985). Physical enrichments of achromatia were obtained from the upper layer of the lake sediments by the "gold-panning" method (DE BOER et a!., 1971) in the following way: Initially crude particles were removed by screening the samples with a 80 fIm mesh. From the remaining particulate material in the filtrate Achromatium cells were picked up with a Pasteur pipette and transferred to a small black glass bowl and repeatedly suspended in filtered (0.2 Jlm) lake water. Due to the fast sedimentation of the heavy achromatia, detritus could be removed with the supernatant and cells could be enriched on the bottom of the glass bowl. Crude preparations of the purified cells were used for FISH. Cells for DNA-extraction were washed excessively in sterile water to remove contaminating smaller bacteria. Purity was checked by epifluorescence microscopy of DAPI stained cells (5 min, 1 fig ml- 1). Oligonucleotide probes: CY 3-, carboxytetramethylrhodamine- and carboxyfluorescein-Iabeled oligonucleotides were purchased from Interactiva (Ulm, Germany). Probe sequences, hybridization conditions and references are given in Table 1. Fluorescence-in-situ-hybridization: For FISH physical cell enrichments were fixed overnight with 4% paraformaldehyde in phosphate-buffered saline (PBS: 130 mM sodium chloride, 10 mM sodium phosphate [pH 7.2]) and stored in 50% (vol/vol) ethanol-PBS at -20°C until further use. Cells were immobilized in the hybridization wells of gelatin-coated [0.1 % gelatin, 0.01 % KCr(S04lzl slides (Paul Marienfeldt KG, Bad Mergentheim, Germany) by air-dying at 46°C. To minimize cell loss each well was overlaid with 30 fIl 0.05% agarose-1x PBS solution. After air-drying at room temperature, each slide was immersed in 50, 80 and 96% (v/v) ethanol for 3 minutes each. For hybridization, each well was overlaid with 10 fIl of a hybridization solution containing 0.9 M NaC!, 20 mM Tris/HCl (pH 7.4), 35% formamide, 0.01 % SDS, 5 ng/fIl labeled oligonucleotide. Slides were incubated for 90 min at 46°C in an equilibrated humid chamber. Probes GAM42a, AST433 and AFK433 were used with competitor oligonucleotides (Table 1). Hybridization mixtures were removed and slides were transferred to a prewarmed (48°C) vial containing 50 ml of washing solution (80 mM NaC!, 20 mM Tris/HCI (pH 7.4), 5 mM EDTA, and 0.01 % SDS) and incubated without shaking for 15 min. The slides were briefly rinsed with distilled water, air dried and mounted in a Citifluor-DAPI mixture (1 fig ml- 1 final cone. Citifluor Ltd., London, United Kingdom). Slides were observed with a Zeiss Axioplan epifluorescence microscope (Zeiss, lena, Germany) equipped with a 100 W high

29

pressure mercury bulb and specific filter sets (DAPI: Zeiss 01, fluorescein: Zeiss 09; CT: Chroma HQ 41007, Chroma Tech. Corp., Brattleboro, VT, USA). Color photomicrographs were done on Kodak Elite 400 (Rochester, NY, USA). For interference-contrast micrographs the automatic exposure system was used. Furthermore, color photomicrographs were taken with a CCD color video camera (Diagnostic Instruments, Inc., Burroughs, Mi) and the Metamorph V3.51 (Metamorph Universal Imaging Corp., West Chester, Pal image analysis system was used for aligning and overlaying multicolor pictures. The different populations of achromatia present in Lake Stech lin in August 1998 were enumerated at a magnification of 160x and 400x. First, by phase-contrast and epifluorescence microscopy all cells tentatively belonging to Achromatium sp. were determined based on their morphology and DAPI staining. Subsequently, by switching to fluorescein and CT-specific filter sets, cells that showed green, red and green plus red excitation were counted. In total, 252 achromatia were scored in 15 independent fields. Composition of a 16S rDNA clone library: Total cellular nucleic acids were isolated by lysis with high-salt extraction buffer (1.5 M NaCI), extended heating (2 to 3 h), proteinase K and sodium dodecyl sulfate followed by phenol-chloroform extraction as described by ZHOU et al. (1996). For the gene library from the physical enrichment of achromatia from Lake Stechlin almost full-length bacterial 16S rRNA gene fragments were amplified from genomic DNA by PCR using two general bacterial SSU rDNA primers (SPRINGER et aI., 1993). The nucleotide sequence of the primers were 616F, 5'-AGAGTTTGATYMTGGCTCAG-3' (Escherichia coli 16S rDNA positions 8-27, BROSIUS et a!., 1981), and 630R, 5'-CAKAAAGGAGGTGATCC-3' (E. coli 16S rDNA positions 1529-1546, BROSIUS et aI., 1981). For the clone library from Lake Fuchskuhle a forward primer specific for Achromatium sp., ACH65F, 5'-AACGCGAAAGGGGGCA-3' (E. coli 16S rDNA positions 65-81, BROSIUS et aI., 1981), was combined with the universal reverse primer 630R. Amplification was performed using a capillary PCR cycler (Idaho Technologies, Idaho Falls, USA). In a final volume of 50 fIl, the reaction mixture contained 25 pmol each of the amplification primers, 200 11M each of the dNTPs, 50 mM Tris (pH 8.3), 250 fIg/ml BSA, 1 mM tartrazine, 2 mM MgCI 2, 5 fIl template, and 2.5 U of Taq DNA polymerase (Promega, Madison, Wis.). After initial heating to 94°C for 45 s, 35 cycles were performed consisting of 94°C (15 s), 49°C (20 s) and 72 °C (30 s). Following the final cycle, the reaction was extended at 72°C for 1 min. The amplification products were analyzed by agarose gel electrophoresis. The 1.5 kb-band was excised, DNA was purified with an extraction kit (Boehringer, Mannheim, Germany), and subsequently ligated into the pCRTM 2.1 vector using the TA Cloning® Kit (Invitrogen, Carlsbad, Ca, USA). Transformants were plated on dYT agar plates (1.6% tryptone, 1.0% yeast extract, 0.5% NaC!, 0.2% glucose, 1.5% bacto-agar) containing the antibiotic ampicillin (100 fig ml- 1), spread evenly with 40 fIl of X-Gal (5-chloro-4-bromo-3-indolyl-~-D-galac­ topyranoside) (40 mg ml- 1 ) and 40 fIl of IPTG (isopropyl-~-D­ thiogalactopyranoside) (100 mM), and incubated at 37°C overnight. For Lake Stechlin twenty white clones and for Lake Fuchskuhle ninety-six white clones were picked on dYT agar plates for further examination. Plasmid DNA was extracted using the QIAprep-spin kit (Qiagen, Hilden, Germany). Presence of inserts of the correct size was checked by linearization of recombinant plasmids with Nod followed by agarose gel electrophoresis. 16S rDNA sequencing: 16S rDNA clones were sequenced using an ABI automated sequencer (Perkin Elmer, Applied Biosystems Prism 377). Cycle sequencing protocols of the chain

30

F. O. GLOCKNER et al.

termination technique (CHEN and SEEBURG, 1985) were applied according to the manufacturer's instructions with dye-labeled dideoxynucleotides. Data analysis: Sequences were added to the 16S rRNA sequence database of the Technical University Munich using the program package ARB (STRUNK et al.). The tool ARB_ALIGN was used for sequence alignment. The alignment was checked by eye and corrected manually. 16S rRNA-based phylogenetic trees were reconstructed based on distance matrix analyses of all available 16S rRNA primary structures of members of the gamma, beta and alpha subclass of the class Proteobacteria. Tree topologies were evaluated by performing maximum parsimony, neighbor joining and maximum likelihood analyses. Only at least 90% complete sequences were used for the calculation of the different trees. Partial sequences were inserted into the reconstructed tree by applying the parsimony criteria without allowing for changes in the overall tree topology. Nucleotide sequence accession numbers: The four almost full-length 16S [RNA sequences obtained of Achromatium sp. have been deposited with EMBL, the accession numbers are A]010593 to A]010596.

Results Light microscopy of the sediment from Lake Stechlin showed that achromatia are abundant members of this habitat. Cells persist in the sediment throughout the year with highest numbers detected in the surface layers (max. lxl0 4 cells cm-3 ) (BABENZIEN and SASS, 1996). The frequency of detection decreased with depth and below 15 mm no cells could be found. In all layers a high variability of size was observed ranging from 10 11m x 30 11m to 35 11m X 125 11m (Fig. 1A). The achromatia found in Lake Fuchskuhle differed from those in Lake Stech lin in their size and size distribution. The population was morphologically quite homogeneous with coccoid cells of an average size of 10 11m x 15

11m (Fig. IB). The maximum abundance in the surface layers of the sediment was approximately 1 x 10 3 cells cm-J • Cells from both habitats showed the typical massive calcite inclusions (CaC0 3 ) and the sulfur globules.

Phylogenetic analysis of the 165 rONA clone libraries Eighteen 16S rDNA clones of the gene library constructed from Lake Stechlin were chosen for partial sequencing (400 to 700 bases). Seven of these clones affiliated with A. oxaliferum clone 5 (Rydal) (HEAD et ai., 1996). Two of these clones (ASTOI and ASTlO) were chosen for full sequencing. Nine of the remaining clones partially sequenced were affiliated with different phyla of the Bacteria. Interestingly, four out of these nine clones were related to the newly described order Verrucomicrobiales (WARD-REINEY et ai., 1995). From the Lake Fuchskuhle 16S rDNA gene library six clones were chosen for partial sequencing. All six clones affiliated in the Achromatium branch of the 16S rRNA tree close to the partial sequences of A. oxaliferum clone 7 and 8 (Rydal) (HEAD et ai., 1996). Clones AFK55 and AFK57 were chosen for determination of full-length sequences. Comparative sequence analysis between the two nearly full-length sequences ASTOI and ASTlO from Lake Stechlin showed 99.8% similarity. The two nearly fulllength sequences from Lake Fuchskuhle, AFK55 and AFK57 shared 99.9% similarity. Since with respect to the 16S rRNA secondary structure none of the few differences was associated with complementary changes across helices, they represent likely sequencing errors and/or PCR artifacts. All further analysis were done with the sequences ASTOI and AFK57. These sequences share a similarity of 93.6% and a comparison with the almost full-length sequence of A. oxaliferum clone 5 (Rydal)

Fig. 1. Interference-contrast micrographs of physical enrichments of achromatia, (A) from the littoral sediment of Lake Stechlin, (B) from the littoral sediment of Lake Fuchskuhle. Cells are filled with large calcite inclusions and smaller sulfur globules. Bar, 25 11m.

Phylogeny and Diversity of Achromatium oxaliferum

31

Table 1. Oligonucleotide probes used in this study. Probe sequence (5'-3')

Targeta site (rRNA positions)

[%) Reference FAb in situ

EUB338 Bacteria NON338 -

GCTGCCTCCCGTAGGAGT ACTCCTACGGGAGGCAGC

16S,338-355 16S,338-355

0-35 0-35

GAM42a c AST433 d AFK433' AST192 AFKI92

GCCTTCCCACATCGTTT TTCCCCCCCGAAAGTGC TTCCCCCCTGAAAGTGC AAGAGTCCCCCACTTTCCC TACGGTCCCCTGCTTTCCC

23S, 1027-1043 16S,433-450 16S,433-450 16S,I92-211 16S,I92-211

35 35 35 35 35

Specificity

Probe

Gamma subclass of Proteobacteria A. oxaliferum clone ASTOI (Stechlin) "A. minus" clone AFK57 (Fuchskuhle) A. oxaliferum clone ASTOI (Stechlin) "A. minus " clone AFK57 (Fuchskuhle)

(AMANN et aI., 1990) (WALLNER et aI., 1993) (MANZ et aI., 1992) This study This study This study This study

Escherichia coli numbering (BROSIUS et aI., 1981). bPercent formamide (FA) in in situ-hybridization buffer. cProbe used with unlabeled competitor BET42a (MANZ et aI., 1992) d Probe used with unlabeled competitor AFK433 eprobe used with unl.abeled competitor AST433 a

Table 2. Similarity between clones and selected organisms based on 349 sequence positions. %

1

2

3

4

5

6

7

8

9

10

11

1 2 3 4 5 6 7 8 9 10 11 12

89.2 91.8 90.0 89.3 87.6 88.7 89.0 89.3 89.0 90.8 89.7

90.7 86.4 88 .2 85.0 87.5 87.9 87.5 85.4 87.5 86.8

89.0 90.0 87.9 90.0 90.4 90.3 89.7 91.1 90.8

94 .7 92.2 94.7 95 .0 95.7 90.4 92.2 92.2

92.2 99.3 99.6 98.9 92 .2 92.2 93.6

92.2 92.6 91.8 91.5 92.9 90.8

99.6 98.9 92.2 92.2 93.6

99.3 92.6 92 .6 94.0

91.8 91.8 93.2

96.5 96.1

96.1

12

1 - Ectothiorhodospira halophila; 2 - Chromatium vinosum; 3 - Rhabdochromatium marinum; 4 - A. oxaliferum clone ASTOl(Stechlin); 5 - A. oxaliferum clone 5 (Rydal); 6 - A. oxaliferum clone 1 (Rydal); 7 - A. oxaliferum clone 3 (Rydal); 8 - A. oxaliferum clone 4 (Rydal); 9 - A. oxaliferum clone 6 (Rydal); 10 - A. oxaliferum clone 7 (Rydal); 11 - A. oxaliferum clone 8 (Rydal); 12 - "A. minus" clone AFK57 (Fuchskuhle)

showed 94.0% and 92.6% similarity for AST01 and AFK57, respectively. The closest cultured relatives to the Achromatium clade are Chromatium vinosum, Rhabdochromatium marinum and Ectothiorhodospira halophila with similarities ranging between 86.2 % and 90.5% determined for the full-length sequences. Comparative similarity values for the partial sequences from A. oxaliferum clone 1 and 3-8 (Rydal) to the sequences AST01, AFK57 and selected cultivated bacteria are given in Table 2. A phylogenetic tree was reconstructed showing the relationship of the newly determined sequences AST01 and AFK57 to selected sequences of members of the beta and gamma subclasses of the class Proteobacteria (Fig. 2). This tree was calculated only on nearly full-length sequences and was corrected by taking into consideration the different results of the various tree reconstruction al-

gorithms. Bifurcations indicate branchings which appeared stable and well separated from neighboring branchings in all cases. Multifurcations indicate tree topologies which could not be significantly resolved based on the available data set. Based on this tree the partial sequences of A. oxaliferum clone 1, 3-4 and 6-8 (Rydal) were inserted (Fig. 3). Two clusters were formed within the Achromatium clade. Cluster A with the A. oxaliferum clone 1 and 3-5 (Rydal) sequences and the sequence AST01. Cluster B containing A. oxaliferum clone 7, 8 (Rydal) sequences and the AFK57 sequence. Several trees were reconstructed either on the nearly full-length sequences and on a region of 349 bases, which was present in all sequences published so far, to confirm the stability of the two clusters (data not shown). In all cases they were stable and clearly separated with long internodal branches.

32

F. O.

GLOCKNER

et al. Achromatium oxaliferum clone AST01 (Stechlin) Achromatium oxafiferum clone 5 (Rydal)

Chromatlum vmosum Xylella fastldlosa

Methylomlcroblum ag"e Methylomonas methan/ca Cyc/oclasl/cus pugetii

Xanthomonas fragariae

Thlomicrosptra crunogena Thiomlcrospira pelophila

Thloploca ingrica - -_ _ _......;:::...._ _ _~~_

Oceanospirillum beijerinckii

Thiobacillus caldus

~-.....,,---

Pseudomonas aeruginosa

Pseudomonas fluorescens Shewanella putrefaciens Aeromonas hydrophila

Proteus vulgaris Ectothiorhodospira ha/ophila Ectothiorhodospira halochloris

Yersinia pestis

Serratia marcescens Escherichia coli

10%

ig. 2. Phylogenctic trce ba ed on omparative analy i of 16 ' rON from A. oxali(erum clone
"A. mil1l1s" clone

rRNA secondary structure The sequence of A. oxaliferum clone A5T01 had the same unusual secondary structure motif in the V6 region of the rRNA (helices 37/38) (Fig. 4C) and pentaloop structure in helix 11 (positions 208-211; E. coli numbering, BROSIUS et aI., 1981) originally described for A. oxaliferum clone 5 (Rydal) (HEAD et aI., 1996). In contrast, the sequence AFK57 is consistent with the currently ac-

cepted secondary structure model for bacterial 165 rRNA (GUTELL et aI., 1994) (Fig. 4A), neither showing the deletion of a helix in the V6 region (Fig. 4B) nor the pentaloop structure.

Fluorescence-in-situ-hybridization Probes EUB338, specific for members of the domain Bacteria, and GAM42a, specific for the gamma subclass

A. oxallferum clone 1 (Rydal) A. oxa/iferum clone AST01 (Stechlin)

A. oxaliferum clone 6 (Rydal) A. oxallferum clone 4 (Rydal)

Cluster A

A. oxaliferum clone 3 (Rydal) A. oxallferum clone 5 (Rydal) ·A. minus· clone AFK57 (Fuchskuhle) A. oxaliferum clone 7 (Rydal)

Cluster B

A. oxallferum clone 8 (Rydal)

R. marinum C. vinosum

E. halophila E. halochloris 10%

Fig. 3. Phylogenetic tree modified from Fig. 2 by adding A. oxali(erum clone 1, 3-4 and 6-8 (Rydal) partial sequences using parsimony criteria, without allowing changes in the overall topology of the tree. The bar indicates 10% estimated sequence divergence.

Phylogeny and Diversity of Achromatium oxaliferum

AG

A

G

A

A-U C-G U-A

B

U-A

U-A U-A

G· UG A UGCCU U A I II G_CAAGGGC 0

C-G 1000 - A - U E. coli

A-U

G· U U· G

U'G

G-C

AG G A

AG G A

C-G C-G C-GG A UGCCU U A I I I G_CAAGGGC A-U G· U

1000 - A - U

/lA. minus"

A-U C A-U U-A U-A C-G C-G C-G A

G

U

G

A A

U A

G • U

A-U

1000 -

U-U

U • G

A. oxalifernm

Fig. 4. Secondary structure model of the V6 region of (A) E. coli, (B) "A. minus" clone AFK57 (Fuchskuhle) and (C) A. oxaliferum clone ASTOI (Stechlin).

of the class Proteobacteria were used to adjust the FISH methodology to the unusually large cells of Achromatium sp. (Table 1). With the protocol described in Materials and Methods clear hybridization signals were obtained with probes EUB338 and GAM42a, while the fluorescence seen with the other group-specific probes applied was of the level of weak autofluorescence characteristic for freshly fixed cells of Achromatium sp. (data not shown). No treatment with 1 M HCl as described by HEAD et al. (1996) to remove the intracellular calcite was necessary, since calcite did not cause non-specific probe binding. However, it was important to process the fixed cells within one week after fixation with paraformaldehyde because extended storage in PBS/EtOH resulted in an significant increase of autofluorescence. Two probes targeting each 16S rRNA sequence were made to assign the two full-length sequences of clones AST01 and AFK57 to Achromatium populations in Lake Stechlin and Lake Fuchskuhle (Table 1). Hybridization conditions were optimized on whole fixed achromatia retrieved form the respective lakes. Formamide series (0-60%) were scored by eye for signal strength (data not shown). Stringencies were ultimately set to 35% formamide for all four probes, a concentration at which they fully discriminated the populations investigated in this study. Probes AFK192 and AFK433, constructed to be specific for clone AFK57, hybridized specifically to all achromatia found in Lake Fuchskuhle and no signals were obtained with probes ASTl92 or AST433, both designed for the clone sequence retrieved from Lake Stechlin. Based on differences with the other 16S rRNA sequences of A. oxaliferum of more than 6% and the differences in the cell morphology, the population found in Lake Fuchskuhle will be described according to MURRAY and SCHLEIFER (1994) as "Candidatus Achromatium minus" (BABENZIEN et al., in prep.). The potential to readily differentiate the Achromatium populations by FISH with, e.g., probes AFK192-CT and AST192-F is

33

shown in Fig. SA for an artificial mixture of Achromatium cells from Lake Fuchskuhle and Lake Stechlin. The smaller red population represents "A. minus". Unexpected results were obtained using samples from Lake Stechlin. Hybridization with a combination of probes AST192-F and AST433-CT showed besides the expected yellow cells, that bound both the red and the green probe, cells only emitting red signals and unhybridized cells. When probes AFK192-CT and AFK433-F were used similar results were obtained (Figs. 5B, 6A). Since all achromatia present in the sample hybridized with probes EUB338 and GAM42a, proving that they contained sufficient amounts of accessible rRNA for in situ-detection, Lake Stechlin obviously contains at least three genotypes of Achromatium sp. A second set of experiments was performed (Fig. 6B) combining in one well probes AFK433-F with AST433-CT and on a different well AST192-F and AFKl92-CT. On both wells all cells were either red or green and no unhybridized achromatia were detected (Fig. 5C). Based on the initial two sets of hybridizations (Figs. 6A, 6B) a third genotype was postulated expressing a 16S rRNA with target sites for probes AFKl92 and AST433. To prove this third Achromatium sp. genotype probes AFKl92-CT and AST433-F were applied simultaneously (Fig. 6C). The respective double-exposure micrographs (Fig. 5D) showed as expected green cells representing A. oxaliferum clone AST01, red Achromatium sp. cells with the signatures for the probes AFKl92 and AFK433 and a third Achromatium sp. genotype with the signatures for probe AFK192 and AST433 in which the addition of green and red signals results in yellow cells. In a sample taken from Lake Stechlin in August 1998 the three genotypes had a ratio of about 2:1:1, with the A. oxaliferum clone AST01 accounting for 49% (± 12%), the Achromatium sp. AFKl92/AFK433 genotype for 28% (± 9%) and the third genotype for 23% (± 12%) of the total Achromatium population.

Discussion In this study, new aspects of the phylogeny and diversity of the genus Achromatium were revealed. Although no pure culture of these widely distributed and locally abundant organisms are available these large and morphologically conspicuous bacteria can be studied in physically enriched samples (BABENZIEN and SASS, 1996; DE BOER et al., 1971; GRAY et al., 1997; HEAD et al., 1996; LA RIVIERE and SCHMIDT, 1989; LA RIVIERE and SCHMIDT, 1991). Here, two lakes from the Brandenburg-Mecklenburg lake district were investigated in which achromatia had been reported before (BABENZIEN et al., 1998; BABENZIEN and SASS, 1996). In accordance with published data (HEAD et al., 1996) FISH with group-specific probes unambiguously affiliated the achromatia in both lakes with the gamma subclass of the class Proteobacteria. Cultivation-independent retrieval of 16S rRNA sequences of Achromatium sp. was successful for both lakes. With an overall 16S rRNA similarity of 86.2% to 90.5% to the

A

B

o

Phylogeny and Diversity of Achromatium oxaliferum

next cultivated organisms the three full length sequences, A. oxaliferum clone 5 (Rydal), A. oxaliferum clone AST01 (Stechlin) and "A. minus" clone AFK57 (Fuchskuhle), form a distinct cluster in the deep branch of the gamma subclass of the class Proteobacteria. All tree reconstruction methods consistently group the Achromatium sp. sequences with those of Rhabdochromatium sp. and Chromatium sp. (Fig. 2). As expressed by the multifurcations in Fig. 2 the currently available data set does not yield a stable order of deep branches in the gamma subclass of the class Proteobacteria. Expanding the tree by the addition of the partial sequences published by HEAD et al. (1996) two distinct clusters became evident (Fig. 3). Cluster A, addressed in the following also as the A. oxaliferum cluster, is supported by the identical unusual 16S rRNA secondary structure in the V6 region (Fig. 4) and the pentaloop in Helix 11. The currently published partial sequences do not show whether the unusual secondary structure motif is really a common characteristic of all 16S rRNA sequences of the A. oxaliferum cluster, but it can also be found in the fulllengths sequence of A. oxaliferum clone 1 (Rydal) (HEAD, pers. communication). The sequences in cluster B, related to the 16S rRNA of the newly described "A. minus" with similarity values of above 96% (Table 2), are clearly separated from cluster A (Fig. 3). This is supported by the absence of the unusual structure motif in the V6 region in the full-length sequences of "A. minus" clone AFK57 and A. oxaliferum clone 7 and 8 (Rydal) (HEAD, pers. communication). It is interesting that only the "A. minus" clone AFK57 sequence did not show the pentaloop structure in Helix 11. This is a feature noted in all other Achromatium-derived 16S rRNA sequences whether they fell within cluster A or B. Nevertheless, all calculation methods used, placed cluster B next to cluster A, supporting the monophyly of the genus Achromatium. Remarkably, the Chromatium-RhabdochromatiumAchromatium branch supported by our data is characterized by a large size variation. For example Chromatium minutissimum is a quite regular-sized bacterium (1.0-1.2 J.lm x 2.0 J.lm), while C. okenii can reach a size of up to SJ.lm x 20 J.lm (PFENNIG and TROPER, 1991). Rhabdochromatium marinum is a large, long rod of 1.5-1.7J.lm x 16-32 J.lm (DILLING et aI., 1995) but also small species like R. minus have been reported by WINOGRADSKY, 1888. The length of Achromatium sp. ranges between up to 125 J.lm for large cells of A. oxaliferum and 15 J.lm for "A. minus" cells. Acknowledging the potential pitfalls of extrapolating to physiology from phylogenetic data (TESKE et aI., 1994), it is nevertheless tempting to speculate based on

35

the stable, monophyletic affiliation of Achromatium sp. with the cultured species of Chromatium sp. and Rhabdochromatium marinum on its physiology. The genera Chromatium and Rhabdochromatium encompass phototrophs which can fix carbon dioxide, oxidize sulfur compounds and deposit sulfur intracellularly. For physical enrichments of Achromatium evidence for a participation in the oxidation of reduced sulfur compounds has been gained (GRAY et aI., 1997), however it is still unclear whether Achromatium can fix CO 2 , Preliminary attempts to detect RuBisCO activity were not successful (BABENZIEN, unpublished results). The affiliation with phototrophs and the tendency of Achromatium to develop autofluorescence could be an indication of remnant photosynthetic pigments that were part of the light harvesting system of ancestors of todays Achromatium sp .. The studies necessary to further investigate these interesting questions can of course be initiated on physical enrichments of achromatia but would, especially in the light of the considerable diversity shown to exist for achromatia in this study, largely benefit from the availability of pure cultures. The oligonucleotide probes To prevent getting false positive results two specific probes were constructed for each of the 16S rRNA sequences from A. oxaliferum clone AST01 and "A. minus" clone AFK57. Evaluating the full-length and partial sequences published by HEAD et al. (1996) the probes AST433 and AFK433 can be used as general probes for cluster A and B, respectively, with the exception of A. oxaliferum clone 1 (Rydal) which shows 3 mismatches to the probe AST433. This clone is phylogenetically quite distant from the rest of cluster A (Fig. 3). The probes ASTI92 and AFKI92, in contrast, can currently serve as clone-specific probes with the exception of ASTI92 which also is complementary to clone 6 (Rydal). Therefore, we believe that the set of four probes for Achromatium would be useful to study the population structure of Achromatium sp. in freshwater sediments. In contrast to the homogeneous population of "A. minus" present in Lake Fuchskuhle the sediment of Lake Stechlin obviously houses quite a high diversity of achromatia. A natural 2:1:1 mixture of A. oxaliferum clone ASTOl, Achromatium sp. genotype AFKI92/AFK433 and an additional Achromatium sp. AFKl92/AST433 genotype can be distinguished by FISH. Based on the very limited molecular data available for the Achromatium sp. AFKl92/AFK433 and the Achromatium sp. AFKl921 AST433 genotypes, essentially only the target sequences of the probes, we can only speculate on the affiliation of

Fig. 5. FISH of enriched sediment samples. Phase contrast (left) and epifluorescence (right) micrographs are shown for identical microscopic fields. (A) Simultaneous hybridization with probes AFKl92-CT (red) and AST192-F (green) on an artificial mixture of samples from Lake Stech lin and Lake Fuchskuhle. (B) Simultaneous hybridization with probes AFK192-CT and AFK433-F on Lake Stechlin samples. (C) Simultaneous hybridization with probes AST433-CT and AFK433-F on Lake Stechlin samples. (D) Simultaneous hybridization with probes AFKl92-CT and AST433-F on Lake Stechlin samples. Bar, 20 pm, applies to all panels.

36

F. O.

GLOCKNER

et al.

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Phylogeny and Diversity of Achromatium oxaliferum these the two Achromatium spp. genotypes. Due to identical probe target sites a close 16S rRNA relationship of the Achromatium sp. AFKI92/AFK433 genotype with "A. minus" can be suggested. Also· the Achromatium sp. AFK192/AST433 genotype seems to be closer to "A. minus". To emerge the Achromatium sp. AFKI921 AST433 genotype from A. oxaliferum clone ASTOI 16S rRNA five base changes between positions 192 and 211 are required, three of which are in helices. The mutation of the "A. minus" clone AFK57 16S rRNA to the target site of AST433, however, only needs one base change at E. coli position 441 (Table 1, black boxes). In a current secondary structure model (GUTELL et al., 1994) this base is not involved in any base pairing and might therefore be switched easily without the need of a concurrent mutation across helices. The possibility that genetically distinct sub-populations might exist within an A. oxaliferum community was first introduced by HEAD et al. (1996). In that study several different sequences related to A. oxaliferum could be retrieved from a 16S rRNA gene library from a single sediment of Lake Rydal. Our results now demonstrate by FISH, that different genotypes of Achromatium sp. really coexists in the same sediment. Now, the ecological questions arise, what factors allow for the coexistence of different Achromatium sp. populations in Lake Stechlin and what causes the morphological and phylogenetical homogenity of "A. minus" in Lake Fuchskuhle. The most striking differences between the two lakes are the trophic state, the conductivity and the pH. It was shown before, that independent from the trophic state the morphologically smaller species, "A. minus", could only be found in Lake Fuchskuhle (BABENZIEN, 1991). Therefore, it is likely to conclude that the acidic pH in combination with the extreme low conductivity in Lake Fuchskuhle prevents the establishment of a diverse Achromatium sp. community in this habitat. Perhaps the general ecological rule applies that lower diversity is to be expected in more extreme environments. Whether the different Achromatium spp. populations in Lake Stechlin reall y live together in the same niche or are separated, e.g., in different depth layers, can not be determined with the bulk samples used in this study. Care should be taken in future investigations to conserve the natural stratification of the sediment layers. In Lake Stechlin different sized cells were visible but no correlation between the genotypes determined with the probes and the size could be made. Both large and small sized cells appeared for all three Achromatium genotypes examined. With the probes developed in this study it should now be possible to monitor discrete populations of Achromatium sp. in situ independent of mor phological differences. They can be used to examine whether the different genotypes in Lake Stech lin indeed dwell in different niches and whether the achromatia found in other locations resemble those found in Lake Fuchskuhle and Lake Stechlin. This should stimulate further research on the phylogeny, physiology and cultivation of achromatia.

37

Acknowledgements This work has been supported by grants Am73/2A and Ba1288/2-1 of the Deutsche Forschungsgemeinschaft. We thank Ian Head and Richard Howard for fruitful discussions and Wolfgang Ludwig for the excellent program package ARB. Some history about this paper: Over his scientific career Hans-Dietrich Babenzien repeatedly discovered achromatia in the sediments of various lakes in the Brandenburg-Mecklenburg lake district. After the German reunification he met Karl-Heinz Schleifer and convinced him by showing spectacular SEM-micrographs that a further investigation of Achromatium oxaliferum with molecular techniques would be worthwhile. The samples were forwarded to the group of Rudi Amann, to use the cultivation-independent rRNA approach to investigate the phylogeny of Achromatium. On the occasion of his 60th birthday we would like to dedicate this manuscript to Karl-Heinz Schleifer thanking him for his long-standing support and the excellent education two of us (RA and FOG) obtained in the Department of Microbiology at the TUM.

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STARR, M. P., SKERMAN, V. D.: Bacterial Diversity: the natural history of selected morphologically unusual bacteria. Annu. Rev. Microbiol. 19,420-422 (1965). STRUNK, 0., GROSS, 0.; REICHEL, B., MAY, M., HERMANN, S., STUCKMAN, N., NONHOFF, B., LENKE, M., GINHART, A., VILBIG, A., LUDWIG, T., BODE, A., SCHLEIFER, K.-H., LUDWIG, W.: ARB: a software environment for sequence data. http://www.mikro.biologie.tu-muenchen.de. Department of Microbiology, Technische Universitat Miinchen, Munich, Germany, TESKE, A., ALM, E., REGAN, J. M., TOZE, S., RITTMANN, B. E., STAHL, D. A.: Evolutionary Relationships among Ammoniaand Nitrite-Oxidizing Bacteria. J. Bacteriol. 176, 6623-6630 (1994). VOLLENWEIDER, R. A.: Scientific fundamentals of eutrophication of lakes and flowing waters with special reference to phosphorus and nitrogen. OECDIDAS/CSI. 68.27, 1-159 (1968). WALLNER, G., AMANN, R., BEISKER, W.: Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms. Cytometry. 14, 136-143 (1993). WARD-RAINEY, N., RAINEY, F. A., SCHLESNERR, H., STACKEBRANDT, E.: Assignment of hitherto unidentified 16S rDNA species to a main line of descent within the domain Bacteria. Microbio!. 141,3247-3250 (1995). WINOGRADSKY, S.: Beitrage zur Morphologie und Physiologie der Bakterien. Zur Morphologie und Physiologie der Schwefelbakterien. 1, 1-120 (1888). ZHOU, J., BRUNS, M. A., TI EDJE,]. M.: DNA recovery from soils of diverse composition. App!. Environ. Microbiol. 62, 316-322 (1996). ZWART, G., HUISMANS, R., VAN AGTERVELD, M. P., VAN DE PEER, Y., DE RUK, P., EENHOORN, H., MUYZER, G., VAN HANNEN, E. J., GONS, H. J., LAANBROEK, H. J.: Divergent members of the bacterial division Verrucomicrobiales in a temperature freshwater lake. FEMS Microbiol. Ecol. 25, 159-169 (1998). Corresponding author: RUDOLF AMANN, Max-Planck-Institut fiir marine Mikrobiologie Celsiusstr. 1, D-28359 Bremen, Germany Phone: +49 4212028-930; Fax: +494212028-790 e-mail: [email protected]