APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2005, p. 7556–7558 0099-2240/05/$08.00⫹0 doi:10.1128/AEM.71.11.7556–7558.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 71, No. 11

Evidence for Horizontal Transfer of 1-Aminocyclopropane-1-Carboxylate Deaminase Genes N. Hontzeas,1* A. O. Richardson,2 A. Belimov,3 V. Safronova,3 M. M. Abu-Omar,1 and B. R. Glick4 Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 479071; Department of Biology, Indiana University, 1001 East Third Street, Bloomington, Indiana 47405-37002; Laboratory for Ecology of Symbiotic and Associative Rhizobacteria, All-Russia Research Institute for Agricultural Microbiology, Sh. Podbelskogo 3, Pushkin 196608, Saint Petersburg, Russian Federation3; and Department of Biology, University of Waterloo, 200 University Ave. West, Waterloo, Ontario N2L 3G1, Canada4 Received 12 April 2005/Accepted 5 July 2005

PCR was used to rapidly identify and isolate 1-aminocyclopropane-1-carboxylate (ACC) deaminase genes from bacteria. The Shimodaira-Hasegawa test was used to assess whether phylogenetically anomalous gene placements suggestive of horizontal gene transfer (HGT) were significantly favored over vertical transmission. The best maximum likelihood (ML) ACC deaminase tree was significantly more likely than four alternative ML trees, suggesting HGT. YVMGSAAGCTYGA) and DegACC3⬘ (5⬘-TTDCCHKYRT ANACBGGRTC) were designed based upon stretches of conserved base pairs towards the N terminus of the protein and around the putative pyridoxal phosphate cofactor binding domain of the protein (20), whereas for 3⬘ primer design, a conserved region close to the carboxyl terminus of the protein was utilized. This allows for the amplification of a fragment of approximately 750 bp. Thus, by using this PCR method, bacterial colonies can be quickly screened for the presence of the ACC deaminase gene. Nucleotide sequences were aligned using MUSCLE v3.52 (4, 16) and refined by eye. Phylogenetic analyses were performed using PAUP* v4.10b via an automated script (courtesy of D. W. Rice, Indiana University). Appropriate evolutionary models for maximum likelihood (ML) were chosen using Modeltest 3.6 (16). A starting topology was generated using parsimony analysis, from which substitution rates and gamma shape parameters were estimated, as appropriate for the nucleotide substitution model selected. Node support for the resultant best trees was evaluated by performing 100 bootstrap replicates. The best ML tree for ACC deaminase was compared to several constrained topologies using the Shimodaira-Hasegawa (SH) test (19) as implemented by PAUP*. The topology of the 16S rRNA tree follows a predicted taxonomy where species of the order Burkholderiales (Variovorax, Acidovorax, and Achromobacter) are grouped together and, likewise, members of the class Gammaproteobacteria (Pseudomonas, Enterobacter, and Serratia) are also grouped together, all belonging to the phylum Proteobacteria. More distant are the Rhizobium spp. which belong to the phylum Proteobacteria but to the class Alphaproteobacteria. The grampositive Rhodococcus spp. belong to an altogether separate phylum, Actinobacteria, and also display the furthest genetic distance to the gram-negative bacteria belonging to the Gammaproteobacteria class. The topology of the bacteria was confirmed by constructing ML trees that included the 16S rRNA sequences from the bacteria identified in this study as well as 190 16S rRNA sequences of other related bacterial species available in the Ribosomal Database Project II (http://rdp.cme

Plant growth-promoting bacteria include a diverse group of free-living soil bacteria that can stimulate the growth of plants by different direct or indirect mechanisms (5). Relatively recently, it was discovered that many plant growth-promoting bacteria contain the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase and that this enzyme can cleave the ethylene precursor ACC to ␣-ketobutyrate and ammonia and thereby lower the level of ethylene in developing or stressed plants (5, 6, 7, 9, 11, 13). The gene encoding this enzyme has been isolated from a few strains of Pseudomonas spp., Rhizobium leguminosarum of the yeast Hansenula saturnus, and the fungus Penicillium citrinum. The crystal structure has been determined for the yeast (15) and bacterial (12) ACC deaminase enzymes; the biochemical and thermodynamic properties of the ACC deaminase from Pseudomonas putida UW4 have been measured (10). Here, using PCR with degenerate primers, we have successfully isolated ACC deaminase genes from a range of both gram-positive and gram-negative bacterial species. Using the biochemical assay procedure, it was ascertained that all of these putative genes encoded functional ACC deaminase. Furthermore, we propose that ACC deaminase genes did not evolve vertically but instead have undergone horizontal gene transfer (HGT). The bacterial strains used in this work are listed in Table 1. The bacteria, except for rhizobial strains, were routinely maintained on Bacto Pseudomonas F as described previously (1, 2, 3). For genus and species identification, a colony PCR was performed with live cells cultured on solid Bacto Pseudomonas F or M79 medium as described previously (17). Species assignment was confirmed by submitting the 16S rRNA sequences to the Ribosomal Database Project II (http://rdp.cme.msu.edu) and comparing them with their nearest phylogenetic relatives. Degenerate primers DegACC5⬘ (5⬘-GGBGGVAAYAARM

* Corresponding author. Mailing address: Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907. Phone: (765) 494-5463. Fax: (765) 494-0239. E-mail: nhontzea@purdue .edu. 7556

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TABLE 1. List of bacteria tested for ACC deaminase Accession no. Strain

16S rRNA gene

ACC deaminase PCR products

Achromobacter xylosoxidans A551 Achromobacter xylosoxidans Bm1 Achromobacter sp. strain CM1 Acidovorax facilis 4P-6 Azospirillum brasilense Cd1843 Enterobacter aerogenes CAL3 Pseudomonas putida UW4 Pseudomonas syringae GR12-2 Pseudomonas brassicacearum Am3 Pseudomonas putida BM3 Pseudomonas marginalis DP3 Rhizobium leguminosarum 128C53K Rhizobium hedysari ATCC 43676 Rhizobium leguminosarum 99A1 Rhodococcus sp. strain Fp2 Rhodococcus sp. strain 4N-4 Serratia quinivorans SUD165 Variovorax paradoxus 3P-3 Variovorax paradoxus 5C-2 Variovorax paradoxus 2C-1 Escherichia coli JM109 Pseudomonas putida ATCC 17399

AY559500 AY556401 AF288728 AY197008

AY604539 AY604540 AAT35837 AY604529 None AY604544 AY823987 AY604545 AY604528 AY604533 AY604542 AF421376 AY604534 AY604535 AY604537 AY604538 AY604543 AY604532 AY604531 AY604530 None None

a

AY559494 AY559493 AY559495 AY007428 AF288727 AF311387 AY559496 AY559498 AY559497 AF288731 AY197005 AY559499 AY197002 AY197003 AY196950

ACC deaminase activity (nM ␣KB mg⫺1 h⫺1)a

Reference(s) or source

400 ⫾ 4 90 ⫾ 4 130 ⫾ 3 3,080 ⫾ 120 0 16 ⫾ 12 3,030 ⫾ 60 3,470 ⫾ 30 5,660 ⫾ 12 3,780 ⫾ 32 4,054 ⫾ 27 5⫾1 20 ⫾ 0.1 8⫾3 7,320 ⫾ 400 12,970 ⫾ 440 12 ⫾ 15 3,700 ⫾ 90 4,322 ⫾ 100 3,588 ⫾ 26 0 0

1, 2 1, 2 1, 2 1, 2 8 18 10 1, 2 1, 2 1, 2 1, 2 1, 2 14 14 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2 Promega, Madison, WI 18

KB, ketobutyrate.

.msu.edu). Another ML tree was constructed using the partial ACC deaminase DNA sequences from the 750-bp region amplified with the degenerate primers (Fig. 1B). The published and well-characterized ACC deaminase DNA sequence of the

bacterium Pseudomonas sp. ACP is shown in Fig. 1B. In this tree, Pseudomonas sp. and Achromobacter sp. ACC deaminase genes are distributed throughout the tree and not constrained to what would be predicted from a vertical transmission of the

FIG. 1. (A) ML phylogenetic tree with bootstrap values of 16S rRNA gene sequences (500 bp) of soil bacteria used to isolate ACC deaminase. (B) ML consensus phylogenetic tree with bootstrap values of partial ACC deaminase DNA (750 bp) sequences. 1, species of the order Burkholderiales; 2, members of the class Gammaproteobacteria; 3, species of the class Alphaproteobacteria; 4, gram-positive Rhodococcus spp. (phylum Actinobacteria).

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genes mirroring the bacterial 16S rRNA tree. The SH test (19) was used to assess whether phylogenetically anomalous gene placements suggestive of HGT are significantly favored over the hypothesis of vertical transmission. This test assigns a P value to the difference in likelihood between the best ACC deaminase ML tree constructed and the ML tree, based on the same data set, in which the ACC deaminase genes are constrained (e.g., as monophyletic groups), as would be expected in the case of vertical transmission of ACC deaminase genes. The ACC deaminase ML tree was compared to the following: (i) a tree assuming that all Achromobacter sp. ACC deaminase genes are monophyletic, (ii) a tree where the Enterobacteriaceae are clustered together, (iii) a tree where Pseudomonas sp. ACC deaminase genes are monophyletic, and (iv) an ACC deaminase tree in which the topology of the ACC deaminase genes mirrors the bacterial 16S rRNA genes. The P values for the SH tests for these four comparisons were ⬍0.0001, ⬍0.0076, ⬍0.0001, and ⬍0.0001, respectively. All four tests showed the best ML ACC deaminase tree to be significantly more likely than the alternative trees. As ACC deaminase genes have not evolved in the same manner as 16S ribosomal RNA genes, which we assume to be vertically transmitted, we propose that some ACC deaminase genes have evolved through horizontal transfer. Given that the pattern cannot be explained by a single transfer, there may have been multiple transfers. Determining precisely which bacterial species served as donors and which served as recipients of HGT is beyond the scope of this work. However, it seems likely that Pseudomonas spp. and Achromobacter spp. have evolved through HGT. Nucleotide sequence accession numbers. The 16S rRNA gene sequences of the studied strains have been submitted to the GenBank/DDBJ/EMBL databases under the accession numbers given in Table 1. We thank D. Rice for critically reviewing the article and assisting with phylogenetic analysis. N.H. was a recipient of a Natural Sciences and Engineering Research Council of Canada (NSERC) postgraduate scholarship. This work was supported by a National Science Foundation (CHE-0434637) grant to M.M.A.-O., a NATO (LST CLG 978202) grant to A.B. and B.R.G., and an NSERC discovery grant to B.R.G. REFERENCES 1. Belimov, A. A., V. I. Safronova, T. A. Sergeyeva, T. N. Egorova, V. A. Matveyeva, V. E. Tsyganov, A. Y. Borisov, I. A. Tikhonovich, C. Kluge, A. Preisfeld, K.-J. Dietz, and V. V. Stepanok. 2001. Characterization of plant growth promoting rhizobacteria isolated from polluted soils and containing 1-aminocyclopropane-1-carboxylate deaminase. Can. J. Microbiol. 47:642– 652.

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