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Applied and Environmental Microbiology, October 1998, p. 4089-4092, Vol. 64, No. 10
Center for Microbial Ecology, Michigan State
University, East Lansing, Michigan 48823-1101
Received 6 April 1998/Accepted 3 August 1998
Small-subunit ribosomal DNA (SSU rDNA) from 20 phenotypically
distinct strains of 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria was partially sequenced, yielding 18 unique strains belonging to members of the alpha, beta, and gamma subgroups of the class Proteobacteria. To understand the origin of 2,4-D
degradation in this diverse collection, the first gene in the 2,4-D
pathway, tfdA, was sequenced. The sequences fell into three
unique classes found in various members of the beta and gamma subgroups
of Proteobacteria. None of the Bacteria capable of mineralizing
2,4-dichlorophenoxyacetic acid (2,4-D), a commonly used herbicide, are
found in many different phylogenetic groups (2, 3, 7, 11, 22,
23). Evidence suggests that numerous variants of 2,4-D catabolic
genes exist and that catabolic operons consist of a near-random mixing
of these variants (7). Interspecies gene transfer is a
well-documented phenomenon (13), and horizontal gene
transfer of the 2,4-D-degrading plasmid pJP4 has been shown (3,
5). However, not all 2,4-D catabolic operons are found on
plasmids (10, 11, 16, 20). The extent to which other 2,4-D
genes have been exchanged in nature is unknown. The aim of this
research was to assess the role of horizontal gene transfer in the
evolution of 2,4-D-degrading strains. This article summarizes the
results of two aspects of this work Mating experiments.
A collection of 2,4-D degraders containing
15 unique strains as determined by genomic fingerprinting
(7) was used as a source of donors in a series of mating
experiments (Table 1). Burkholderia
cepacia D5, lacking the ability to grow on 2,4-D and not
hybridizing to any tfd genes, was used as a recipient in
mating experiments. Strain D5 contains neomycin phosphotransferase genes (nptII) carried on transposon Tn5 and is
resistant to 50 µg each of kanamycin, carbenicillin, and bacitracin
per ml. All of the 2,4-D strains used were sensitive to these
antibiotics. Filter matings were performed with a donor-to-recipient
ratio of 1:10. Colonies which grew on selective medium (500 ppm of
2,4-D in mineral salts agar [MMO] [23] including 50 µg of kanamycin, carbenicillin, and bacitracin per ml) were subjected
to further tests. Their ability to catabolize 2,4-D was tested in
liquid medium (same composition as that described above).
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Evidence for Interspecies Gene Transfer in the
Evolution of 2,4-Dichlorophenoxyacetic Acid Degraders
ABSTRACT
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References
-Proteobacteria yielded
tfdA PCR products. A comparison of the dendrogram of the
tfdA genes with that of the SSU rDNA genes demonstrated
incongruency in phylogenies, and hence 2,4-D degradation must have
originated from gene transfer between species. Only those strains with
tfdA sequences highly similar to the tfdA sequence of strain JMP134 (tfdA class I) transferred all
the 2,4-D genes and conferred the 2,4-D degradation phenotype to a
Burkholderia cepacia recipient.
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the study of the transfer of the
entire 2,4-D pathway by using standard mating experiments and a
phylogenetic study of the tfdA gene. The tfdA
gene codes for an -ketoglutarate-dependent 2,4-D dioxygenase which
converts 2,4-D into 2,4-dichlorophenol and glyoxylate (6). This 861-bp gene was first sequenced from Ralstonia eutropha
JMP134 (19). Two more tfdA genes were cloned from
chromosomal locations in Burkholderia strain RASC and
Burkholderia strain TFD6 (16, 20). These proved
to be identical to each other and 78.5% similar to the original. An
alignment of the two variants allowed conserved areas to be identified
and primers to be designed for the amplification of
tfdA-like genes from other sources (24). Sequence
analysis of putative tfdA fragments and the small-subunit
ribosomal DNA (SSU rDNA) of the strains carrying them allowed us to
construct phylogenies of the genes and their hosts and to look for
congruency between them.
TABLE 1.
2,4-D-degrading strains, geographic origins, and GenBank
accession numbers
Phylogenetic analyses. Total genomic DNA was isolated from 20 unique 2,4-D-degrading strains (including all 15 used for mating experiments) grown on 500 ppm of 2,4-D mineral salts medium amended with 50 ppm of yeast extract. SSU rDNA was amplified by using fD1 and rD1 as primers (25). Putative tfdA fragments were amplified by using primers TVU and TVL as previously described (24). PCR products were purified with a Gene Clean kit (Bio 101, La Jolla, Calif.). Sequencing was done with an Applied Biosystems model 373A automatic sequencer (Perkin-Elmer Cetus) by using fluorescently labeled dye termination at the Michigan State University Sequencing Facility. The sequencing primer used for SSU rDNA fragments was 519R (5' GTA TTA CCG CGG CTG CTG G-3'). For tfdA fragments, the sequencing primers were the same as the amplification primers. GenBank accession numbers for these sequences are given in Table 1.
The SSU rDNA sequences were compared to sequences in GenBank by using the Basic Local Alignment Search Tool (BLAST) (1), and those strains with the highest maximal segment pair scores were retrieved from GenBank and included in the phylogenetic analysis. Sequences were aligned manually with the software SeqEd (Applied Biosystems) and with MacClade (14). Sites where nucleotides were not resolved for all sequences were deleted from the alignment, as were those nucleotides corresponding to the small loop in this region that is absent in the alpha subgroup of the class Proteobacteria. These deletions left 283 unambiguous sites for the construction of the SSU rDNA phylogenies. Phylogenetic trees were constructed by using the neighbor-joining analysis of pairwise Jukes-Cantor distances (4), and the topology was confirmed by using the maximum parsimony method PAUP (21). Desulfomonile tiedjei of the
-Proteobacteria was used as
an outgroup. Bootstrap analysis based on 100 replicates was used to
place confidence estimates on the tree. Only bootstrap values of
greater than 50 were used.
2,4-D degrader diversity.
The 2,4-D degraders in this study
were distributed throughout the alpha, beta, and gamma subgroups of the
Proteobacteria (Fig. 1). The
lack of representation of gram-positive bacteria is likely a reflection
of isolation methods, not of the lack of gram-positive 2,4-D degraders.
The majority of these strains were members of the beta subgroup of
Proteobacteria, five of which were most closely related to
the genus Burkholderia, having at least 92% sequence similarity with each other. Three were closely related to
Rhodoferax fermentans (close to the class
Comamonadaceae), three were related to Ralstonia
eutropha, and one was related to Alcaligenes
xylosoxidans. TFD39 falls outside any clear cluster. One member of
the 
-Proteobacteria, strain I-18, a haloalkaliphile, was found to be
closely related to the salt-loving genus Halomonas
(15). The remaining six strains all clustered in the alpha
branch of Proteobacteria (Fig. 1). Of this subgroup, five
were most closely related to the genus Sphingomonas.
One member of the -Proteobacteria, strain M1, which is the most
oligotrophic and slow growing of all the strains used in this study, is
97% similar to Rhodopseudomonas palustris. The character of
strain M1 correlates well with its phylogenetic placement near the
slow-growing genus Bradyrhizobium.
|
), class II
(
), and class III (
) types of tfdA genes are
indicated. Bootstrap confidence limits (percentages) are indicated
above each branch. Scale bar represents a Jukes-Cantor distance of
0.01.
tfdA gene fragments.
tfdA gene fragments
were successfully amplified and sequenced from 10 strains of
-Proteobacteria and 1 strain of -Protobacteria. None of the
strains from the -Proteobacteria gave any amplificates with these
primers. These 313 contiguous nucleotides were aligned with additional
tfdA sequences from JMP134 and from strain RASC (Fig.
2). Three distinct classes of
tfdA gene sequences with slight variations in each class
were found. Class I included fragments from JMP134, TFD39, TFD23, K712,
and TFD9 that differed from each other by 2 bp at the most. Class I
tfdA genes are probably plasmid encoded. All strains with a
class I tfdA gene examined so far contained
broad-host-range, self-transmissible plasmids containing 2,4-D genes
(2, 3, 11, 17). All of the strains with a class I
tfdA gene were able to transfer the 2,4-D phenotype in the
mating studies reported above. The class II tfdA sequences included identical fragments amplified from RASC, TFD6, and TFD2 which
were 76% similar to those in class I. Class III included identical
fragments from strains TFD31, B6-9, and I-18 which were 77% similar to
class I genes and 80% similar to class II genes. Both class II and III
tfdA genes differed from each other and from class I genes
in the same nine sites corresponding to the third base pair of the
codons. The tfdA phylogenetic tree is a simple one, with
three distinct branches that are incongruent with the SSU rDNA-derived
phylogeny (Fig. 3). Class I
tfdA sequences were found in Burkholderia-like
strains, in strains related to the Comamonas-Rhodoferax
group, and in the Ralstonia-Acaligenes group, all in the
-Proteobacteria. Class II sequences are less widely distributed,
found only in Burkholderia-like branches. However, even in
this subgroup, this tfdA variant is found in strains that
differ by 7% at the SSU rDNA level (RASC and TFD2). However, the class
III sequences were most interesting, being found both in the
Comamonas-Rhodoferax group and in a strain of the
-Proteobacteria, I-18, strains that differ by 24% at the SSU rDNA
level. Class III genes have since been found in a collection of
randomly isolated non-2,4-D degraders, including gram-positive bacilli,
as well as in various gram-negative bacteria, even though the gene is
not expressed (10).
|
|
-Proteobacteria yielded a PCR product when amplified
with the conserved tfdA primers. This finding complements our observation that none of these bacteria hybridized to the tfdA gene, even under conditions of low stringency,
indicating that any tfdA-like genes in the
-Proteobacteria are likely to be more divergent from the ones
sequenced here (7, 11). In addition, none of the
Sphingomonas strains in the study hybridized with a whole
pJP4 probe, and similarly, no Sphingomonas strains scored
positive for transfer of 2,4-D-degrading ability to recipient B. cepacia D5. Together these results suggest a reduced gene flow between members of the - and - or -Proteobacteria or poor gene expression of - or -derived genes by -Proteobacteria. Although plasmid pJP4 is a broad-host-range plasmid and has been known to
transfer to -Proteobacteria such as Rhizobium and
Agrobacterium species and to -Proteobacteria such as
Pseudomonas putida, Pseudomonas fluorescens, and
Pseudomonas aeruginosa, the 2,4-D pathway is not expressed
in these strains of the - or -Proteobacteria (3). Phylogenetically limited expression of plasmid-borne
3-chlorobenzoate-degradative genes has also been noted for the
pseudomonads (8). Subsequent studies have found divergent
but related sequences for the tfdB and tfdC genes
in 2,4-D-degrading Sphingomonas strains (7, 12,
24).
With the exceptions of the minor differences within the class I
pJP4-like tfdA sequences, there were no intermediate
tfdA sequences. The most likely explanation of this is that
the rate of horizontal transfer of the tfd genes is high
relative to the rate at which mutations can accumulate. Examination of
sequences of tfdA genes from a greater variety of organisms
may turn up more intermediate variation.
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ACKNOWLEDGMENTS |
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We thank Arturo Massol-Deya and Jizhong Zhou for primers and Tom Schmidt, Bonnie Bratina, and Jim Smith for advice and assistance with sequence analysis. We thank William Holben, Jong-Ok Ka, Nancy Tonso, Penny Amy, and Charles Greer for the use of their strains and Richard Lenski for valuable discussions in comparing phylogenies of tfdA and SSU rDNA. We also thank the people in the Research on Microbial Evolution Laboratory for their support.
This work was supported by the National Science Foundation (grant DEB9120006), a part of the Joint Project on Microbial Evolution with the Research and Development Corporation of Japan (JRDC).
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FOOTNOTES |
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* Corresponding author. Present address: University of Toronto at Scarborough, 1265 Military Trail, Scarborough, Ontario, Canada M1C 1A4. Phone: (416) 287-7221. Fax: (416) 287-7279. E-mail: fulthorpe{at}scar.utoronto.ca.
Present address: 1798 Colorado Drive, East Lansing, MI 48823.
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REFERENCES |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. | Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410[Medline]. |
| 2. |
Amy, P. S.,
J. W. Schulke,
L. M. Frazier, and R. J. Seidler.
1985.
Characterization of aquatic bacteria and cloning of genes specifying partial degradation of 2,4-dichlorophenoxyacetic acid.
Appl. Environ. Microbiol.
49:1237-1245 |
| 3. |
Don, R. H., and J. M. Pemberton.
1981.
Properties of six pesticide degradation plasmids isolated from Alcaligenes paradoxus and Alcaligenes eutrophus.
J. Bacteriol.
145:681-686 |
| 4. | Felsenstein, J. 1988. Phylogenies from molecular sequences: inference and reliability. Annu. Rev. Genet. 22:521-565[Medline]. |
| 5. | Friedrich, B., M. Meyer, and H. Schlegel. 1983. Transfer and expression of the herbicide-degrading plasmid pJP4 in aerobic autotrophic bacteria. Arch. Microbiol. 134:92-97[Medline]. |
| 6. |
Fukumori, F., and R. P. Hausinger.
1993.
Alcaligenes eutrophus JMP134 "2,4-dichlorophenoxyacetate monooxygenase" is an -ketoglutarate-dependent dioxygenase.
J. Bacteriol.
175:2083-2086 |
| 7. | Fulthorpe, R. R., C. McGowan, O. V. Maltseva, W. H. Holben, and J. M. Tiedje. 1995. 2,4-Dichlorophenoxyacetic acid-degrading bacteria contain mosaics of catabolic genes. Appl. Environ. Microbiol. 61:3274-3281[Abstract]. |
| 8. |
Fulthorpe, R. R., and R. C. Wyndham.
1991.
Transfer and expression of the catabolic plasmid pBRC60 in wild bacterial recipients in a freshwater ecosystem.
Appl. Environ. Microbiol.
57:1546-1553 |
| 9. |
Fulthorpe, R. R., and R. C. Wyndham.
1992.
Involvement of a chlorobenzoate catabolic transposon, Tn5271, in community adaptation to chlorobiphenyl, chloroaniline, and 2,4-dichlorophenoxyacetic acid in a freshwater ecosystem.
Appl. Environ. Microbiol.
58:314-325 |
| 10. | Hogan, D. A., D. H. Buckley, C. H. Nakatsu, T. M. Schmidt, and R. P. Hausinger. 1997. Distribution of the tfdA gene in soil bacteria that do not degrade 2,4-dichlorophenoxyacetic acid, 2,4-D. Microb. Ecol. 34:90-96[Medline]. |
| 11. |
Ka, J. O.,
W. E. Holben, and J. M. Tiedje.
1994.
Genetic and phenotypic diversity of 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria isolated from 2,4-D-treated field soils.
Appl. Environ. Microbiol.
60:1106-1115 |
| 12. | Leander, M., T. Vallaeys, and R. R. Fulthorpe. 1998. Amplification of putative chlorocatechol dioxygenase fragments from alpha and beta proteobacteria. Can. J. Microbiol. 44:482-486[Medline]. |
| 13. | Levy, S. B., and R. V. Miller. 1989. Gene transfer in the environment. McGraw Hill Publishing Company, New York, N.Y. |
| 14. | Maddison, W. P., and D. R. Maddison. 1992. MacClade: analysis of phylogeny and character evolution, version 3.0. Sinauer Associates, Sunderland, Mass. |
| 15. |
Maltseva, O.,
C. McGowan,
R. Fulthorpe, and P. Oriel.
1996.
Degradation of 2,4-dichlorophenoxyacetic acid by haloalkaliphilic bacteria.
Microbiology
142:1115-1122 |
| 16. |
Matheson, V. G.,
L. J. Forney,
Y. Suwa,
C. Nakatsu,
A. J. Sexstone, and W. H. Holben.
1996.
Evidence for acquisition in nature of a chromosomal 2,4-dichlorophenoxyacetic acid/-ketoglutarate dioxygenase gene by different Burkholderia spp.
Appl. Environ. Microbiol.
62:2457-2463[Abstract].
|
| 17. | Nakatsu, C. H., F. Fukumori, R. Fulthorpe, R. Hausinger, W. Holben, Y. Kamagata, R. Korona, R. Lenski, O. Maltseva, V. G. Matheson, C. McGowan, Y. Suwa, J. Tiedje, N. Tonso, E. Top, A. Wright, and L. Forney. 1997. Genetic diversity of 2,4-dichlorophenoxyacetic acid (2,4-D) catabolic genes, p. 197-206. In K. Horikoshi, M. Fukuda, and T. Kudo (ed.), Microbial diversity and genetics of biodegradation. Japan Scientific Societies Press, Tokyo, Japan. |
| 18. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 19. |
Streber, W. R.,
K. N. Timmis, and M. H. Zenk.
1987.
Analysis, cloning, and high-level expression of 2,4-dichlorophenoxyacetate monooxygenase gene tfdA of Alcaligenes eutrophus JMP134.
J. Bacteriol.
169:2950-2955 |
| 20. |
Suwa, Y.,
A. D. Wright,
F. Fukumori,
K. A. Nummy,
R. P. Hausinger,
W. E. Holben, and L. J. Forney.
1996.
Characterization of a chromosomally encoded 2,4-dichlorophenoxyacetic acid/-ketoglutarate dioxygenase from Burkholderia sp. strain RASC.
Appl. Environ. Microbiol.
62:2464-2469[Abstract].
|
| 21. | Swofford, D. L. 1993. PAUP: Phylogenetic Analysis Using Parsimony, version 3.1. Computer program distributed by the Illinois Natural History Survey, Champaign, Ill. |
| 22. | Tiedje, J. M., J. M. Duxbury, M. Alexander, and J. E. Dawson. 1969. 2,4-D metabolism: pathway of degradation of chlorocatechols by Arthrobacter sp. J. Agric. Food Chem. 17:1021-1025. |
| 23. | Tonso, N. L., V. G. Matheson, and W. E. Holben. 1995. Polyphasic characterization of a suite of bacterial isolates capable of degrading 2,4-D. Microb. Ecol. 30:3-24. |
| 24. | Vallaeys, T., R. R. Fulthorpe, A. M. Wright, and G. Soulas. 1996. The metabolic pathway of 2,4-dichlorophenoxyacetic acid degradation involves different families of tfdA and tfdB genes according to PCR-RFLP analysis. FEMS Microb. Ecol. 20:163-172. |
| 25. |
Weisburg, W. G.,
S. M. Barns,
D. A. Pelletier, and D. J. Lane.
1991.
16S ribosomal DNA amplification for phylogenetic study.
J. Bacteriol.
173:697-703 |
Applied and Environmental Microbiology, October 1998, p. 4089-4092, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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