| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Laboratory of Plant Biochemistry and Physiology, Department of Biology, Antwerp University, Campus Drie Eiken, Universiteitsplein 1, B-2610 Antwerp, Belgium
2 Professor Emeritus, Laboratory of Molecular Biology, Department of Biochemistry, Antwerp University, Campus Drie Eiken, Universiteitsplein 1, B-2610 Antwerp, Belgium
3 Laboratory of Microbiology, Faculty of Sciences, Ghent University, Ledeganckstraat, B-9000 Ghent, Belgium
4 National Botanic Garden, Domein van Bouchout, B-1860 Meise, Belgium
Correspondence
Sandra Van Oevelen
sandra.vanoevelen{at}ua.ac.be
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of ‘Candidatus Burkholderia calva’ and ‘Candidatus Burkholderia nigropunctata’ are AY277697 and AY277698, respectively.
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
), three genera placed in three different tribes of the family. The most visible aspect of this atypical symbiosis is the establishment of galls in the leaves of the host plant, harbouring the bacterial microsymbiont. The presence of the symbiotic partner is a prerequisite for normal development and survival of the plant (Gordon, 1963). Due to its obligate and cyclic nature, bacterial leaf symbiosis cannot be classified together with any other known type of plant–bacterium interaction.
The role these bacteria play in the development of their host is crucial. Their isolation, cultivation and identification are essential as a first step towards revealing the underlying mechanisms that support this intimate form of co-existence. In a previous study, sequence analysis of the small-subunit (16S) rRNA gene was used to identify the endophyte associated with Psychotria kirkii as a novel Burkholderia species. Due to its uncultured nature the provisional status Candidatus was proposed for this bacterial endosymbiont, i.e. ‘Candidatus Burkholderia kirkii’ (Van Oevelen et al., 2002a). In the current study, similar methods were used to identify the bacterial microsymbionts associated with two further members of the genus Psychotria.
Bacteria-rich galls were isolated from Psychotria calva Hiern (no. 19620512) and Psychotria nigropunctata Hiern (no. 19750521). Psychotria lucens var. lucens (no. 19610404), a species which is always devoid of bacterial galls (Petit, 1964), was used as an overall negative control. Acquisition numbers refer to the living collection of the National Botanic Garden of Belgium. All methods were as previously described (Van Oevelen et al., 2002a).
DNA extractions were performed using the DNeasy Plant Mini Kit (QIAgen) according to the manufacturer's instructions. Amplification of the 16S rRNA genes was performed and amplified fragments were cloned into a pGEM-Teasy vector. All clones were subjected to an EcoRI digest and partially sequenced, to allow selection of clones containing bacterial 16S rRNA genes. We selected seven and three ‘bacterial’ clones from P. calva and P. nigropunctata, respectively. Comparison of the partial sequences (minimum 600 bp) of all the bacterial 16S rRNA genes obtained from a single plant species showed that no differences occurred among them. This was shown for both plants involved, indicating that each species was hosting only a single bacterial strain in its leaf galls.
The complete 16S rRNA gene sequences for the symbionts were determined for each of the plant species. As the bacterial presence is limited to leaf galls and the stem apical region, we considered gall-free samples taken from in between galls as negative controls. If the obtained 16S rRNA genes belonged to contaminating bacteria they would not only be present in the leaf galls but also in any other part of the leaf, including the negative control samples. However, the absence of bacteria in gall-free samples was confirmed for both plant species, which proved that the retrieved 16S rRNA gene sequences represented the bacterial symbionts rather then any contaminants. Likewise, the gall-free species P. lucens var. lucens was shown to lack any bacterial DNA, as was expected. Additional proof came from the analysis of the apical region. Bacterial 16S rRNA genes obtained from the stem apex of P. calva and P. nigropunctata were sequenced. Comparisons revealed them to be identical to the bacterial 16S rRNA genes obtained from the leaf galls of the respective species. This indicates the presence of the same bacterial symbiont in both the stem apex and leaf galls. Sequences were submitted to the SSU rRNA database (Wuyts et al., 2004) and aligned with their closest relatives using DCSE software (De Rijk & De Wachter, 1993). Distance matrices were calculated using the Jukes & Cantor (1969) substitution model. Phylogenetic trees were constructed according to the neighbour-joining method (Saitou & Nei, 1987), using the TREECON program (Van de Peer & De Wachter, 1994). Bootstrap values were used as a measure of reliability (Felsenstein, 1985).
Phylogenetic analysis, based on their full-length 16S rRNA gene sequences, places the endosymbionts of P. calva and P. nigropunctata in the genus Burkholderia, as was shown earlier for the bacterial partner of P. kirkii, ‘Candidatus Burkholderia kirkii’ (Van Oevelen et al., 2002a). Intersequence similarities range from 95·9 to 97·9 %. Sequence similarities to the closest cultured neighbour, Burkholderia sp. NF100 (Hayatsu et al., 2000), are alike, ranging from 96·5 to 97·9 %, whereas similarities shared with the closest validly named species, Burkholderia glathei (Viallard et al., 1998), are slightly lower, ranging from 96·2 to 96·5 %.
A distance matrix was constructed containing 13 Burkholderia species. The evolutionary tree presented in Fig. 1 is based on this analysis. Even though all symbionts cluster closely together, the evolutionary distances separating the different microsymbionts are sufficiently large to recognize the endosymbionts of both P. calva and P. nigropunctata as distinct and novel Burkholderia species. In previous studies we included six different varieties of P. kirkii, a geographically widespread species that shows great morphological variation (Van Oevelen et al., 2002a, b). Despite the diverse nature of this species, our phylogenetic analysis showed that the microsymbionts of the six acquisitions of P. kirkii that were investigated are very closely related and represent a single Burkholderia species. Our current data suggest that all microsymbionts of Psychotria originate from a single Burkholderia species. They also indicate that bacterial leaf nodulation in Psychotria is the result of a single infection event in an ancestor to modern Psychotria, after which the closed nature of the symbiotic cycle allowed genetic differentiation during the subsequent co-evolution of plant and bacteria. The inter-isolate distances between microsymbionts of distinct host species are large enough to permit recognition of three different Burkholderia species, corresponding to the three investigated Psychotria species.
|
. Cross-sections through the bacterial galls were made from fresh leaves using razor blades. Sections were dehydrated by a treatment in 70 % ethanol (30 min) and methylal (2x30 min), and subsequently critical-point dried. Dried samples were mounted on aluminium stubs, sputter coated with gold and analysed using a JEOL 6400 scanning electron microscope. Bacterial rods are visible in the leaf galls of P. calva (Fig. 2a) and P. nigropunctata (Fig. 2b). No flagella are observed.
|
.
Descriptions of ‘Candidatus Burkholderia calva’ and ‘Candidatus Burkholderia nigropunctata’
‘Candidatus Burkholderia calva’ (calva, from the specific epithet of the host plant) [(
-Proteobacteria, genus Burkholderia); NC; G–; R; NAS (GenBank no. AY277697), oligonucleotide sequence complementary to unique region of 16S rRNA gene 5'-TCGGAACCCTGCTGAAAGGTGGGGGTGCTCGAAAGAGAACCGGT-3'; S (Psychotria calva, stem apex and leaf galls)]. Van Oevelen et al., this study.
‘Candidatus Burkholderia nigropunctata’ (nigropunctata, from the specific epithet of the host plant) [(-Proteobacteria, genus Burkholderia); NC; G–; R; NAS (GenBank no. AY277698), oligonucleotide sequence complementary to unique region of 16S rRNA gene 5'-CCCTGCTGAGAGGTGGGGTGCTCGGAAGAGAACCGTC-3'; S (Psychotria nigropunctata, stem apex and leaf galls)]. Van Oevelen et al., this study.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]
Gordon, J. F. (1963). The nature and distribution within the plant of the bacteria associated with certain leaf-nodulated species of the families Myrsinaceae and Rubiaceae. PhD thesis, Imperial College, University of London.
Hayatsu, M., Hirano, M. & Tokuda, S. (2000). Involvement of two plasmids in fenitrothion degradation by Burkholderia sp. strain NF100. Appl Environ Microbiol 66, 1737–1740.
Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In Mammalian Protein Metabolism, vol. 3, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.
Murray, R. G. E. & Stackebrandt, E. (1995). Taxonomic note: implementation of the provisional status Candidatus for incompletely described procaryotes. Int J Syst Bacteriol 45, 186–187.
Petit, E. (1964). Les espèces africaines du genre Psychotria L. (Rubiaceae). – I. Bull Jard Bot Etat Brux 34, 1–228.
Robbrecht, E. (1988). Tropical woody Rubiaceae. Characteristic features and progressions. Contributions to a new subfamilial classification. Opera Bot Belg 1, 1–272.
Saitou, N. & Nei, M. (1987). The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Van de Peer, Y. & De Wachter, R. (1994). TREECON for Windows : a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput Appl Biosci 10, 569–570.
Van Oevelen, S., De Wachter, R., Vandamme, P., Robbrecht, E. & Prinsen, E. (2002a). Identification of the bacterial endosymbionts in leaf galls of Psychotria (Rubiaceae, angiosperms) and proposal of ‘Candidatus Burkholderia kirkii’ sp. nov. Int J Syst Evol Microbiol 52, 2023–2027.[Abstract]
Van Oevelen, S., Prinsen, E., De Wachter, R. & Robbrecht, E. (2002b). The taxonomic value of bacterial symbiont identification in African Psychotria (Rubiaceae). Syst Geogr Pl 71, 557–563.
Viallard, V., Poirier, I., Cournoyer, B., Haurat, J., Wiebkin, S., Ophel-Keller, K. & Balandreau, J. (1998). Burkholderia graminis sp. nov., a rhizospheric Burkholderia species, and reassessment of [Pseudomonas] phenazinium, [Pseudomonas] pyrrocinia and [Pseudomonas] glathei as Burkholderia. Int J Syst Bacteriol 48, 549–563.
Wuyts, J., Perrière, G. & Van de Peer, Y. (2004). The European ribosomal RNA database. Nucleic Acids Res 32, D101–D103.
This article has been cited by other articles:
|
|
 |
|
  L. P. Partida-Martinez, I. Groth, I. Schmitt, W. Richter, M. Roth, and C. Hertweck Burkholderia rhizoxinica sp. nov. and Burkholderia endofungorum sp. nov., bacterial endosymbionts of the plant-pathogenic fungus Rhizopus microsporus Int J Syst Evol Microbiol, November 1, 2007; 57(11): 2583 - 2590. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Vandamme, K. Opelt, N. Knochel, C. Berg, S. Schonmann, E. De Brandt, L. Eberl, E. Falsen, and G. Berg Burkholderia bryophila sp. nov. and Burkholderia megapolitana sp. nov., moss-associated species with antifungal and plant-growth-promoting properties Int J Syst Evol Microbiol, October 1, 2007; 57(10): 2228 - 2235. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
