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Characterization of amplified polymerase chain reaction glnB and nifH gene fragments of nitrogen-fixing Burkholderia species
2003; Oxford University Press; Volume: 36; Issue: 2 Linguagem: Inglês
10.1046/j.1472-765x.2003.01253.x
ISSN1472-765X
AutoresVictor Augustus Marin, K. R. S. Teixeira, José Ivo Baldani,
Tópico(s)Plant-Microbe Interactions and Immunity
ResumoLetters in Applied MicrobiologyVolume 36, Issue 2 p. 77-82 Free Access Characterization of amplified polymerase chain reaction glnB and nifH gene fragments of nitrogen-fixing Burkholderia species V.A. Marin, V.A. Marin Embrapa Agrobiologia, Laboratorio de Genética e Bioquímica, Seropédica, Rio de Janeiro State, BrazilSearch for more papers by this authorK.R.S. Teixeira, K.R.S. Teixeira Embrapa Agrobiologia, Laboratorio de Genética e Bioquímica, Seropédica, Rio de Janeiro State, BrazilSearch for more papers by this authorJ.I. Baldani, J.I. Baldani Embrapa Agrobiologia, Laboratorio de Genética e Bioquímica, Seropédica, Rio de Janeiro State, BrazilSearch for more papers by this author V.A. Marin, V.A. Marin Embrapa Agrobiologia, Laboratorio de Genética e Bioquímica, Seropédica, Rio de Janeiro State, BrazilSearch for more papers by this authorK.R.S. Teixeira, K.R.S. Teixeira Embrapa Agrobiologia, Laboratorio de Genética e Bioquímica, Seropédica, Rio de Janeiro State, BrazilSearch for more papers by this authorJ.I. Baldani, J.I. Baldani Embrapa Agrobiologia, Laboratorio de Genética e Bioquímica, Seropédica, Rio de Janeiro State, BrazilSearch for more papers by this author First published: 14 January 2003 https://doi.org/10.1046/j.1472-765X.2003.01253.xCitations: 7 Correspondence to: J.I. Baldani, Embrapa Agrobiologia, Laboratorio de Genética e Bioquímica, Seropédica, 23851-970, Rio de Janeiro, Brazil (e-mail: ibaldani@cnpab.embrapa.br). AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Aims: To clone and sequence polymerase chain reaction (PCR)-amplified glnB and nifH genes of the nitrogen-fixing bacteria Burkholderia brasilensis strain M130, B. tropicalis strain PPe8 and B. kururiensis strain KP23. Methods and Results: The glnB and nifH gene fragments were amplified by PCR using universal degenerated primers. A very high percentage of similarity for the nifH (100%) and glnB (96%) genes was observed between strains M130 and KP23. A similarity of 100% for the nifH gene was also observed between strains M130 and PPe8. However, the identity for the glnB gene was 98% and the similarity 88%. The phylogenetic tree of the nifH gene showed a very high degree of similarity to the 16S rDNA gene. Conclusions: The nitrogen-fixing bacteria of the Burkholderia genus formed a cluster separated from the other species of the genus mainly when the nifH rather than the glnB gene was used to construct the phylogenetic tree. Significance and Impact of the Study: Knowledge of the nifH and glnB gene sequences of B. brasilensis, B. tropicalis and B. kururiensis will support new studies on the diversity of these diazotrophs in natural environments. Introduction The genus Burkholderia comprises over 25 species; however, the species Burkholderia vietnamiensis is the only nitrogen-fixing bacterium officially described (Gillis et al. 1995). Two other new nitrogen-fixing bacteria belonging to the genus Burkholderia have been described and provisionally named B. brasilensis (strain M130) and B. tropicalis (strain PPe8) (Baldani 1996). Recently, a new species, named B. kururiensis (strain KP23), was identified as a trichloroethylene-degrading bacterium (Zhang et al. 2000) and its ability to fix nitrogen in a similar manner to strain M130 was demonstrated by De Los Santos et al. (2001). Two other strains (STM678 and STM815) of the genus Burkholderia have also been very recently described as possessing nitrogen fixation ability; furthermore, they could establish a symbiosis with legume plants (Moulin et al. 2001). The genus Burkholderia is very attractive because of its widespread ecology and physiological characteristics. It has been found in environments such as soil, water and the plant rhizosphere (Parke and Gurian-Sherman 2001). Minerdi et al. (2001) were the first to identify and report the characterization of nifHDK genes isolated from the arbuscular fungus spores of Gigaspora margarita but they strongly suggested that these genes belonged to the genome of the endosymbiont Burkholderia. The functional nifH gene has highly conserved regions as well as a great divergence in other regions; it can, therefore, be used to evaluate the phylogenetic relationships among nitrogen-fixing micro-organisms (diazotrophs) of several groups (Ueda et al. 1995). Since the nifH gene only occurs in nitrogen-fixing micro-organisms, it has been used to monitor the presence of these diazotrophs, e.g. in pure cultures (Franke et al. 1998), in soil (Widmer et al. 1999) as well as in plants (Ueda et al. 1995; Lovell et al. 2000). It has also been detected in arbuscular mycorrhizal fungus spores (Minerdi et al. 2001), marine environments (Zehr et al. 1998) and termite guts (Ohkuma et al. 1996; Kudo et al. 1998). The PII protein, encoded by the glnB gene, is involved in the regulation of glutamine synthetase activity and the global nitrogen regulation system. It also regulates adenylyltransferase (ATase) and the NifA protein activity in several nitrogen-fixing bacteria. In addition, its role has been demonstrated in nitrate utilization (Rhizobium leguminosarum and Azospirillum brasilense) and in ammonia and methyl-ammonia transport (A. brasilense) (Arcondéguy et al. 2001). Recently, a fragment of the glnB gene was cloned and sequenced from A. amazonense (Potrich et al. 2001). The objective of this work was to partially sequence the nifH and glnB genes in different diazotrophic Burkholderia species and compare them with those sequences of other nitrogen-fixing organisms present in the GenBank database, with a view to understanding the diversity of these genes in nitrogen-fixing bacteria of the genus Burkholderia. Materials and methods Bacterial strains The strains used in this study were M130 (B. brasilensis), PPe8 (B. tropicalis) and KP23 (B. kururiensis). The first two strains were obtained from the Embrapa Agrobiologia Culture Collection (Rio de Janeiro, Brazil). Dr Yoichi Kamagata (National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Japan) donated the third strain. Polymerase chain reaction amplification of the nifH and glnB genes The total DNA was extracted according to the hexadecyltrimethylammonium bromide (CTAB) method (Sambrook et al. 1989). For the nifH gene, the degenerated primers described by Ueda et al. (1995) were used. The reaction mixture contained: 1 × buffer, 2·5 mmol l−1 MgCl2, 200 µmol l−1 deoxynucleotide triphosphate (dNTPs), 100 pmol of each primer and 2 U of Taq DNA polymerase (Promega, Madison, WI, USA) in a final volume of 50 µl. The reaction conditions with 10 ng of DNA template were: 4 min at 94 °C, 1 min at 94 °C, 1 min at 50 °C and 1 min at 72 °C (for 35 cycles) and 4 min at 72 °C. The glnB gene primers were kindly provided by Dr Irene Schrank Universidade Federal do Rio Grande do Sul (UFRGS, Brazil): up 5′-GCCATCATTAAGCCGTTCAA-3′ and down 5′-AAGATCTTGCCGTCGCCGAT-3′. The reaction mixture contained: 1 × buffer, 2·5 mmol l−1 MgCl2, 200 µmol l−1 dNTPs, 100 pmol of each primer and 1 U of Taq DNA polymerase (Promega) in a final volume of 25 µl. The reaction conditions with 200 ng of DNA template were: 5 min at 95 °C, 30 s at 52 °C, 30 s at 72 °C and 30 s at 94 °C (for 36 cycles), 2 min at 52 °C, 5 min at 72 °C. The polymerase chain reaction amplification was performed in a thermal cycler (PTC-100; MJ Research, Waltham, MA, USA). Fragments of approx. 400 bp (nifH) and 200 bp (glnB) were obtained and cloned using the pGEM-T Easy Vector (Promega). The clones were sequenced using the PRISM Ready Reaction Dye Deoxy Terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA). Sequence alignment and phylogenetic analysis Sequence data were analysed using Genedoc (Nicholas and Nicholas 1997). Sequence multiple alignments were carried out with ClustalW (Thompson et al. 1994) and the phylogenetic trees constructed by the neighbour-joining method (Saito and Nei 1987). The MEGA package (Kumar et al. 1993), with the distance of Jukes and Cantor, was used (Jukes and Cantor 1969). The other program parameters were maintained unchanged. Bootstrap analyses of 1000 replicates were established (Felsenstein 1985). Nucleotide sequence accession numbers The nucleotide sequences of the nifH, glnB and 16S rDNA genes of several nitrogen-fixing bacteria were obtained from GenBank and used to construct the phylogenetic trees. Results In general, the amino acid sequences of the glnB gene of the Burkholderia species showed a similarity level higher than 82% when compared with the GlnB proteins of other nitrogen-fixing bacteria, such as Herbaspirillum seropedicae, R. leguminosarum, Bradyrhizobium japonicum, A. brasilense, Klebsiella pneumoniae and Azotobacter vinelandii(Table 1). A very high similarity level (96%) was verified between the amino acid sequences of the glnB gene for B. brasilensis strain M130 and B. kururiensis strain KP23. A much lower similarity level (88%) was observed between strains M130 and PPe8. On the other hand, a similarity level higher than 90% was observed between the species of the genus Burkholderia and H. seropedicae. In the phylogenetic tree of the nifH gene, the strains belonging to the genus Burkholderia formed a cluster with high bootstrap values (Fig. 1). The only exception was the Burkholderia strain isolated from Gigaspora (Minerdi et al. 2001), which showed a greater similarity to the species belonging to the α-Proteobacteria group. The phylogeny of the nifH genes was consistent with that obtained with the 16S rDNA gene (1, 2). Although the bootstrap values were low (Fig. 1), the phylogenetic trees obtained with the partial sequences were always consistent with those obtained for the complete nifH gene sequences (Young 1992; Ueda et al. 1995; Achouak et al. 1999). Table 1. Similarity (%) of the glnB gene amino acid sequences Organism B. kururiensis B. tropicalis H. seropedicae R. leguminosarum Br. japonicum A. brasilense Kl. pneumoniae Az. vinelandii Burkholderia brasilensis AY098587 96 88 94 86 88 86 90 90 Burkholderia kururiensis AY098589 85 91 83 85 84 88 86 Burkholderia tropicalis AY098591 90 82 88 86 88 86 Herbaspirillum seropedicae U86073 85 91 92 92 91 Rhizobium leguminosarum X04880 90 86 83 84 Bradyrhizobium japonicum M26753 94 88 86 Azospirillum brasilense X51499 88 91 Klebsiella pneumoniae AJ006531 86 Azotobacter vinelandii U91902 Figure 1Open in figure viewerPowerPoint Phylogenetic analysis of nifH gene amino acid sequences of several nitrogen-fixing bacteria constructed by the NJ method. The bootstrap values (1000 replicates) are indicated in the grouping base. The scale bar indicates the distance in substitutions by nucleotide Figure 2Open in figure viewerPowerPoint Phylogenetic analysis of 16S rDNA gene sequences of nucleotides constructed by the NJ method. The bootstrap values (1000 replicates) are indicated in the grouping base. The scale bar indicates the distance in substitutions by nucleotide Very high similarity (above 99%) was observed among the amino acid sequences of the nifH gene for the species of the genus Burkholderia tested (B. brasilensis, B. kururiensis, B. tropicalis and Burkholderia sp. strain STM678). Burkholderia tropicalis (strain PPe8), isolated from sugar cane (Baldani 1996; Baldani et al. 2000), showed high identity with B. brasilensis (98%), B. kururiensis (98%) and Burkholderia sp. strain STM678 (95%). An identity and similarity of 100% was observed between B. brasilensis and B. kururiensis. Due to the similarity between the nifH gene sequences of B. brasilensis and B. kururiensis strains, the nucleotide sequences of the 16S rDNA gene of both species were compared. An identity of 99·93% was observed between the species, differing in only one nucleotide. Discussion In this study, the partial nifH and glnB gene sequences of the nitrogen-fixing species B. brasilensis, B. tropicalis and B. kururiensis are reported. It is shown that the bacteria of the Burkholderia genus formed a cluster separated from the other species. Interestingly, the amino acid sequences of the glnB gene of B. tropicalis presented much higher similarity to the glnB sequences of H. seropedicae than to the glnB sequences of B. brasilensis and B. kururiensis. The amino acid sequence alignment demonstrated the presence of the residue Tyr-51 for all species from the glnB gene. This residue is a site for uridylylation, usually present in the PII protein when the bacteria are grown in low nitrogen concentrations. It is known to play a role in the deadenylylation of glutamine synthetase by ATase (Arcondéguy et al. 2001). Genes homologous to glnB have been found in A. amazonense, A. brasilense, H. seropedicae and other bacteria, suggesting the presence of two copies of glnB-like genes in these organisms (Arcondéguy et al. 2001; Potrich et al. 2001). The phylogenetic tree obtained with the amino acid sequences determined from the nifH gene of the Burkholderia species showed a similar topology to that described by other authors (Ueda et al. 1995; Ohkuma et al. 1996; Franke et al. 1998; Vermeiren et al. 1999). The observation that the Burkholderia sp. isolated from the fungus G. margarita is close to A. brasilense was also verified by Minerdi et al. (2001) who attributed this effect to the occurrence of lateral transfer of the gene. The similarity of the phylogenetic trees inferred from 16S rDNA and nifH genes supports the hypothesis that the nifH gene has descended vertically (Ueda et al. 1995; Ohkuma et al. 1996). These authors also suggested that the preservation of this gene in several organisms is due to evolution from a common ancestor, instead of a lateral transfer in recent times. However, other studies indicate a possible lateral transfer of nif genes, explaining why the operon nifHDK of H. seropedicae (β-proteobacteria) has a high similarity to Br. japonicum (α-proteobacteria) (Hurek et al. 1997; Vermeiren et al. 1999). Similarly, it has also been detected in Acidithiobacillus ferroxidans, a γ-proteobacteria that is closely related to Br. japonicum (Kelly and Wood 2000). The similarity values of the sequences from nifH (100%) and 16S rDNA (99%) genes suggest that the bacterium B. brasilensis strain M130 (Baldani et al. 1997; Cruz et al. 2001) is closely related to the species B. kururiensis described by Zhang et al. (2000). De Los Santos et al. (2001) also suggested this hypothesis, based on the amplified ribosomal DNA (rDNA) restriction analysis (ARDRA) profile and nitrogen-fixing ability of strains KP23 and M130. However, additional DNA : DNA studies showed that this is not the case (V. L. Baldani, personal communication). Similar high values of 16S rDNA (99·38%) were also observed for the species H. seropedicae (strain Z67) and H. rubrisubalbicans (strain M4) (Baldani et al. 1996), although they showed very low DNA : DNA hybridization values. The higher similarity for the nifH gene is probably due to a more conserved region that needs to be preserved during the evolution of the bacteria. 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