Phylogenetic Analysis of Aquaspirillum ...
INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, Apr. 1991, p. 324-325 0020-7713/91/020324-02$02.OO/O Copyright 0 1991, International Union of Microbiological Societies
Vol. 41, No. 2
Phylogenetic Analysis of Aquaspirillum magnetotacticum Using Polymerase Chain Reaction-Amplified 16s rRNA-Specific DNA PETER A. EDEN,’ THOMAS M. SCHMIDT,2t RICHARD P. BLAKEMORE,’ AND NORMAN R. PACE2* Department of Microbiology, University of New Hampshire, Durham, New Hampshire 03824’ and Department of Biology and Institute for Molecular and Cellular Biology, Indiana University, Bloomington, Indiana 4740.5= The 16s rRNA gene of the magnetotactic magnetogenAquaspirillum magnetotacticum MSl was amplified by a polymerase chain reaction, using two eubacterial consensus oligodeoxynucleotide primers flanking the majority of the 16s rRNA gene, cloned, and sequenced. Phylogenetic analysis revealed that A . magnetotacticum MS1 belongs to the a-group of proteobacteria. This assignment offers perspective on the biochemical properties of A. magnetotacticum, since this organism is expected to have the general properties that are common to this phylogenetic group.
extension at 72°C for 2 min (first cycle) or more; 5 s was added to the extension time per cycle. Amplified DNA was extracted with phenol-chloroform (1:1) and precipitated from 2.5 M ammonium acetate with ethanol. The DNA was treated with T4 DNA polymerase and polynucleotide kinase (Boehringer Mannheim) and ligated as a 1.5-kilobase bluntended fragment into Bluescript KS vector (Stratagene) cleaved with SmaI restriction endonuclease (New England BioLabs) by using standard procedures (6). E. coli DHSa cells were transformed (8) with the recombinant vector and plated onto YT agar supplemented with ampicillin (100 Fg/ml). Plasmid DNA was isolated from transformants following alkaline lysis (3). One strand of the 16s rRNA gene was sequenced by using the dideoxynucleotide chain termination method (2). The sequence obtained by reverse transcription (9) of 16s rRNA from A . magnetotacticum MS1 verified that the cloned, amplified DNA originated from strain MS1. The A . magnetotacticum 16s rRNA sequence was aligned with the sequences of other 16s rRNAs on the basis of secondary structure and conserved sequences. A phylogenetic tree was constructed by using a least-squares, distance matrix method (12). The relationship shown in Fig. 1 is robust; bootstrap analysis (7) grouped the strain MS1 sequence with the sequences of other members of the a-group of proteobacteria in all 50 trees examined. The sequence also contained all of the “signature” features of the a-group of proteobacteria (14). The inferred secondary structure of the A . magnetotacticum 16s rRNA exhibited no unusual structural elements or variations in length compared with other members of the a-group of proteobacteria. Our data provide the first 16s rRNA-based phylogenetic classification of a magnetogen. The general applicability of the PCR, cloning, and sequencing techniques to such organisms in naturally occurring populations is appealing. Many morphologically distinctive forms of magnetotactic magnetogens have been observed in nature; however, few such organisms have been isolated in pure culture. rRNA sequence analyses of these bacteria would aid in evaluating the extent to which this interesting physiological and behavioral adaptation is phylogenetically distributed. The GenBank accession numbers for the organisms used in this analysis are as follows: Proteus vulgaris, J01874; E . coli, V00348; Vibrio harveyi, M58172; Chromatium v i m sum, M26629; Pseudomonas testosteroni, M11224; Chromatium violaceum, M22510; Neisseria gonorrhoeae, X07714; Rickettsia rickettsii, M21293; A . magnetotacticurn, M58171; Hyphomicrobium vulgare, X53182; Agrobacterium tume-
The magnetogens (4) are diverse microorganisms which produce intracellular (1) or extracellular (10) , nanometersized magnetic particles. When deposited intracellularly as membrane-enveloped magnetosomes, as in Aquaspirillurn magnetotacticum, these crystals comprise a geomagnetic navigational apparatus (4). However, since not all magnetogens are magnetotactic, magnetic particle biomineralization may have additional, as-yet-unknown significance in cell biology. Unique properties common to biogenic magnetic particles in different organisms suggest that there is a common genetic basis for magnetogenesis. However, the phylogenetic distribution of magnetogenesis among prokaryotes is unknown. To begin to address the question of evolutionary relatedness among magnetogens, we performed a sequence analysis of the 16s rRNA gene of A . magnetotacticurn MS1. rRNA sequences, because of their high degree of conservation, are useful for establishing phylogenetic relationships among organisms (14). Amplification of rRNA genes by the polymerase chain reaction (PCR) (13) provides convenient access to these genes for sequence analysis. A . magnetotacticurn MS1 (=ATCC 31632) was cultured in chemically defined medium ( 5 ) at 25°C. Cells in exponential growth were concentrated with a Pellicon filtration system (Millipore Corp., Bedford, Mass.) and pelleted by centrifugation at 1,200 x g for 20 min at 4°C. Genomic DNA was isolated by using standard techniques (11). The 16s rRNA gene was selectively amplified by the PCR, using oligodeoxynucleotide primers designed to anneal to conserved regions of the eubacterial16S rRNA. The forward primer corresponded to positions 8 to 27 of Escherichia coli 16s rRNA (5’-AGAGTTTGATCCTGGCTCAG-3’), and the reverse primer corresponded to the complement of positions A . mag1510 to 1492 (5’-GGTTACCTTGTTACGACTT-3’). netotacticum MS1 DNA (100 ng) was mixed with 2 U of Taq DNA polymerase in 100 pl of reaction buffer and subjected to the PCR (13) by using a Perkin-Elmer DNA Thermal Cycler. The PCR buffer contained 50 mM KCI, 10 mM Tris-HC1 (pH 8.3), 1.5 mM MgCl,, 0.2 pg of each oligonucleotide primer, 200 p M dATP, 200 pM dCTP, 200 pM dGTP, and 200 FM dTTP. The mixture was subjected to 40 cycles, with each cycle consisting of denaturation for 1.5 min at 92”C, primer annealing at 37°C for 1 min, and chain
* Corresponding author.
7 Present address: Department of Microbiology, Miami Univer-
sity, Oxford, OH 45056.
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VOL. 41, 1991
NOTES
325
Proteus vulgaris 1 Eschericftia coli Vibrio harveyi Chromatiuni vinosum Pseudomonas t e s t o s i x l 7 Chrornobacterium violaceurn I P Neisseria gonorrhoeae Rickettsia nckettsii Aquaspirillum magnetotacticurn Hyphomicrobium vulgare a Agrobactenurn turnefaciens Rochalimaea guintana Rhodopseudomonas marina Caulobacter crescentus
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Evolutionary Distance (Fixed mutations per nucleotide) FIG. 1. Phylogenetic relationships of selected proteobacteria based on 16s rRNA sequences. The proteobacterial tree was rooted with Anacystis nidulans (data not shown).
faciens, M11223; Rochalimaea quintuna, M11927; Rhodopseudomonas marina, M27534; Caulobacter crescentus, X52281; and Anacystis nidulans, X52281. This research was supported by Office of Naval Research contracts N14-87-K-0813 (to N.R.P.) and N00014-90-J-1053 (to R.P.B.) by grant GM34527 from the National Institutes of Health (to N.R.P.), and by grant DMB85-15540 from the National Science Foundation (to R.P.B.). REFERENCES 1. Balkwell, D. L., D. Maratea, and R. P. Blakemore. 1980. Ultrastructure of a magnetotactic spirillum. J. Bacteriol. 141: 1399-1408. 2. Biggin, M. D., T. J. Gibson, and G. F. Hong. 1983. Buffer gradient gels and 35Slabel as an aid to rapid DNA sequence determination. Proc. Natl. Acad. Sci. USA 80:3963-3965. 3. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7: 1513-1523. 4. Blakemore, R. P., and R. B. Frankel. 1989. Biomineralization by magnetogenic bacteria, p. 85-98. In R. K. Poole and G. M. Gadd (ed.), Metal-microbe interactions. IRL Press, New York. 5 . Blakemore, R. P., D. Maratea, and R. S. Wolfe. 1979. Isolation and pure culture of a freshwater magnetic spirillum in chemi-
cally defined medium. J. Bacteriol. 140:720-729. 6. Davis, L. G., M. D. Dibner, and J. F. Battey. 1986. Basic methods in molecular biology, p. 220-232. Elsevier, New York. 7. Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 37:783-791. 8. Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166557-580. 9. Lane, D. L., B. Pace, G. J. Olsen, D. A. Stahl, M. L. Sogin, and N. R. Pace. 1985. Rapid determination of 16s ribosomal RNA sequences for phylogenetic analyses. Proc. Natl. Acad. Sci. USA 82:6955-6959. 10. Lovely, D. R., J. F. Stolz, G. L. Nord, Jr., and E. J. P. Phillips. 1987. Anaerobic production of magnetite by a dissimilatory iron-reducing microorganism. Nature (London) 330:252-254. 11. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N . Y . 12. Olsen, G. J. 1987. The earliest phylogenetic branchings: comparing rRNA-based evolutionary trees inferred with various techniques. Cold Spring Harbor Symp. Quant. Biol. 52:825-838. 13. Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn, K. B. Mullis, and H. A. Erlich. 1988. Primerdirected enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487491. 14. Woese, C. R. 1987. Bacterial evolution. Microbiol. Rev. 51:221271.
Vol. 41, No. 2
Phylogenetic Analysis of Aquaspirillum magnetotacticum Using Polymerase Chain Reaction-Amplified 16s rRNA-Specific DNA PETER A. EDEN,’ THOMAS M. SCHMIDT,2t RICHARD P. BLAKEMORE,’ AND NORMAN R. PACE2* Department of Microbiology, University of New Hampshire, Durham, New Hampshire 03824’ and Department of Biology and Institute for Molecular and Cellular Biology, Indiana University, Bloomington, Indiana 4740.5= The 16s rRNA gene of the magnetotactic magnetogenAquaspirillum magnetotacticum MSl was amplified by a polymerase chain reaction, using two eubacterial consensus oligodeoxynucleotide primers flanking the majority of the 16s rRNA gene, cloned, and sequenced. Phylogenetic analysis revealed that A . magnetotacticum MS1 belongs to the a-group of proteobacteria. This assignment offers perspective on the biochemical properties of A. magnetotacticum, since this organism is expected to have the general properties that are common to this phylogenetic group.
extension at 72°C for 2 min (first cycle) or more; 5 s was added to the extension time per cycle. Amplified DNA was extracted with phenol-chloroform (1:1) and precipitated from 2.5 M ammonium acetate with ethanol. The DNA was treated with T4 DNA polymerase and polynucleotide kinase (Boehringer Mannheim) and ligated as a 1.5-kilobase bluntended fragment into Bluescript KS vector (Stratagene) cleaved with SmaI restriction endonuclease (New England BioLabs) by using standard procedures (6). E. coli DHSa cells were transformed (8) with the recombinant vector and plated onto YT agar supplemented with ampicillin (100 Fg/ml). Plasmid DNA was isolated from transformants following alkaline lysis (3). One strand of the 16s rRNA gene was sequenced by using the dideoxynucleotide chain termination method (2). The sequence obtained by reverse transcription (9) of 16s rRNA from A . magnetotacticum MS1 verified that the cloned, amplified DNA originated from strain MS1. The A . magnetotacticum 16s rRNA sequence was aligned with the sequences of other 16s rRNAs on the basis of secondary structure and conserved sequences. A phylogenetic tree was constructed by using a least-squares, distance matrix method (12). The relationship shown in Fig. 1 is robust; bootstrap analysis (7) grouped the strain MS1 sequence with the sequences of other members of the a-group of proteobacteria in all 50 trees examined. The sequence also contained all of the “signature” features of the a-group of proteobacteria (14). The inferred secondary structure of the A . magnetotacticum 16s rRNA exhibited no unusual structural elements or variations in length compared with other members of the a-group of proteobacteria. Our data provide the first 16s rRNA-based phylogenetic classification of a magnetogen. The general applicability of the PCR, cloning, and sequencing techniques to such organisms in naturally occurring populations is appealing. Many morphologically distinctive forms of magnetotactic magnetogens have been observed in nature; however, few such organisms have been isolated in pure culture. rRNA sequence analyses of these bacteria would aid in evaluating the extent to which this interesting physiological and behavioral adaptation is phylogenetically distributed. The GenBank accession numbers for the organisms used in this analysis are as follows: Proteus vulgaris, J01874; E . coli, V00348; Vibrio harveyi, M58172; Chromatium v i m sum, M26629; Pseudomonas testosteroni, M11224; Chromatium violaceum, M22510; Neisseria gonorrhoeae, X07714; Rickettsia rickettsii, M21293; A . magnetotacticurn, M58171; Hyphomicrobium vulgare, X53182; Agrobacterium tume-
The magnetogens (4) are diverse microorganisms which produce intracellular (1) or extracellular (10) , nanometersized magnetic particles. When deposited intracellularly as membrane-enveloped magnetosomes, as in Aquaspirillurn magnetotacticum, these crystals comprise a geomagnetic navigational apparatus (4). However, since not all magnetogens are magnetotactic, magnetic particle biomineralization may have additional, as-yet-unknown significance in cell biology. Unique properties common to biogenic magnetic particles in different organisms suggest that there is a common genetic basis for magnetogenesis. However, the phylogenetic distribution of magnetogenesis among prokaryotes is unknown. To begin to address the question of evolutionary relatedness among magnetogens, we performed a sequence analysis of the 16s rRNA gene of A . magnetotacticurn MS1. rRNA sequences, because of their high degree of conservation, are useful for establishing phylogenetic relationships among organisms (14). Amplification of rRNA genes by the polymerase chain reaction (PCR) (13) provides convenient access to these genes for sequence analysis. A . magnetotacticurn MS1 (=ATCC 31632) was cultured in chemically defined medium ( 5 ) at 25°C. Cells in exponential growth were concentrated with a Pellicon filtration system (Millipore Corp., Bedford, Mass.) and pelleted by centrifugation at 1,200 x g for 20 min at 4°C. Genomic DNA was isolated by using standard techniques (11). The 16s rRNA gene was selectively amplified by the PCR, using oligodeoxynucleotide primers designed to anneal to conserved regions of the eubacterial16S rRNA. The forward primer corresponded to positions 8 to 27 of Escherichia coli 16s rRNA (5’-AGAGTTTGATCCTGGCTCAG-3’), and the reverse primer corresponded to the complement of positions A . mag1510 to 1492 (5’-GGTTACCTTGTTACGACTT-3’). netotacticum MS1 DNA (100 ng) was mixed with 2 U of Taq DNA polymerase in 100 pl of reaction buffer and subjected to the PCR (13) by using a Perkin-Elmer DNA Thermal Cycler. The PCR buffer contained 50 mM KCI, 10 mM Tris-HC1 (pH 8.3), 1.5 mM MgCl,, 0.2 pg of each oligonucleotide primer, 200 p M dATP, 200 pM dCTP, 200 pM dGTP, and 200 FM dTTP. The mixture was subjected to 40 cycles, with each cycle consisting of denaturation for 1.5 min at 92”C, primer annealing at 37°C for 1 min, and chain
* Corresponding author.
7 Present address: Department of Microbiology, Miami Univer-
sity, Oxford, OH 45056.
324
VOL. 41, 1991
NOTES
325
Proteus vulgaris 1 Eschericftia coli Vibrio harveyi Chromatiuni vinosum Pseudomonas t e s t o s i x l 7 Chrornobacterium violaceurn I P Neisseria gonorrhoeae Rickettsia nckettsii Aquaspirillum magnetotacticurn Hyphomicrobium vulgare a Agrobactenurn turnefaciens Rochalimaea guintana Rhodopseudomonas marina Caulobacter crescentus
IY
-I
I
t
'i
I
t
I
0.0
I
I
0.04
I
I
0.08
I
0.12
Evolutionary Distance (Fixed mutations per nucleotide) FIG. 1. Phylogenetic relationships of selected proteobacteria based on 16s rRNA sequences. The proteobacterial tree was rooted with Anacystis nidulans (data not shown).
faciens, M11223; Rochalimaea quintuna, M11927; Rhodopseudomonas marina, M27534; Caulobacter crescentus, X52281; and Anacystis nidulans, X52281. This research was supported by Office of Naval Research contracts N14-87-K-0813 (to N.R.P.) and N00014-90-J-1053 (to R.P.B.) by grant GM34527 from the National Institutes of Health (to N.R.P.), and by grant DMB85-15540 from the National Science Foundation (to R.P.B.). REFERENCES 1. Balkwell, D. L., D. Maratea, and R. P. Blakemore. 1980. Ultrastructure of a magnetotactic spirillum. J. Bacteriol. 141: 1399-1408. 2. Biggin, M. D., T. J. Gibson, and G. F. Hong. 1983. Buffer gradient gels and 35Slabel as an aid to rapid DNA sequence determination. Proc. Natl. Acad. Sci. USA 80:3963-3965. 3. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7: 1513-1523. 4. Blakemore, R. P., and R. B. Frankel. 1989. Biomineralization by magnetogenic bacteria, p. 85-98. In R. K. Poole and G. M. Gadd (ed.), Metal-microbe interactions. IRL Press, New York. 5 . Blakemore, R. P., D. Maratea, and R. S. Wolfe. 1979. Isolation and pure culture of a freshwater magnetic spirillum in chemi-
cally defined medium. J. Bacteriol. 140:720-729. 6. Davis, L. G., M. D. Dibner, and J. F. Battey. 1986. Basic methods in molecular biology, p. 220-232. Elsevier, New York. 7. Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 37:783-791. 8. Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166557-580. 9. Lane, D. L., B. Pace, G. J. Olsen, D. A. Stahl, M. L. Sogin, and N. R. Pace. 1985. Rapid determination of 16s ribosomal RNA sequences for phylogenetic analyses. Proc. Natl. Acad. Sci. USA 82:6955-6959. 10. Lovely, D. R., J. F. Stolz, G. L. Nord, Jr., and E. J. P. Phillips. 1987. Anaerobic production of magnetite by a dissimilatory iron-reducing microorganism. Nature (London) 330:252-254. 11. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N . Y . 12. Olsen, G. J. 1987. The earliest phylogenetic branchings: comparing rRNA-based evolutionary trees inferred with various techniques. Cold Spring Harbor Symp. Quant. Biol. 52:825-838. 13. Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn, K. B. Mullis, and H. A. Erlich. 1988. Primerdirected enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487491. 14. Woese, C. R. 1987. Bacterial evolution. Microbiol. Rev. 51:221271.
