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Molecular Ecology (2010) 19, 28–30
NEWS AND VIEWS
PERSPECTIVE
Insights into the history of the legumebetaproteobacterial symbiosis
A N N E T T E A . A N G U S * and A N N M . H I R S C H * †
*Department of Molecular, Cell and Developmental Biology,
†Molecular Biology Institute, University of California-Los
Angeles, 621 Charles E. Young Drive South, Los Angeles, CA
90095-1606, USA
The interaction between legumes and rhizobia has been
well studied in the context of a mutualistic, nitrogen-fixing symbiosis. The fitness of legumes, including important agricultural crops, is enhanced by the plants’ ability
to develop symbiotic associations with certain soil bacteria that fix atmospheric nitrogen into a utilizable form,
namely, ammonia, via a chemical reaction that only bacteria and archaea can perform. Of the bacteria, members of
the alpha subclass of the protebacteria are the bestknown nitrogen-fixing symbionts of legumes. Recently,
members of the beta subclass of the proteobacteria that
induce nitrogen-fixing nodules on legume roots in a species-specific manner have been identified. In this issue,
Bontemps et al. reveal that not only are these newly
identified rhizobia novel in shifting the paradigm of our
understanding of legume symbiosis, but also, based on
symbiotic gene phylogenies, have a history that is both
ancient and stable. Expanding our understanding of
novel plant growth promoting rhizobia will be a valuable
resource for incorporating alternative strategies of nitrogen fixation for enhancing plant growth.
Keywords: Bacteria, Molecular Evolution, Microbial Evolution, Species Interactions
Received 3 November 2009; revision received 8 November 2009;
accepted 10 November 2009
Nitrogen fixation by bacteria is a process that promotes
plant growth under nitrogen-deficient or nitrogen-limiting
conditions. Nitrogen gas, which is abundant (c. 80% of our
atmosphere) but highly unreactive, is reduced to ammonia
through the action of the enzyme nitrogenase. Ammonia is
incorporated into the plant’s metabolic pathways to build
DNA and proteins for essential processes such as cellular
growth and development. By themselves, plants are unable
to fix nitrogen, but some plants benefit from their interaction with specialized bacteria that perform this energetically
expensive feat, while associated with or housed within
Correspondence: Ann M. Hirsch, Fax: (310) 206-5413;
E-mail: [email protected]
plant organs. Being housed within plant root nodules
greatly enhances nitrogen fixation by providing the bacteria
with an environment that not only supplies sufficient carbon, but also protects the oxygen-sensitive enzyme, nitrogenase. Molecular communication between plants and soil
bacteria initiates the early events of nodulation, including
recognition, aggregation ⁄ attachment of bacteria to plant
roots, and root hair curling. Bacterial nod genes, induced by
plant-derived compounds, are responsible for the synthesis
of Nod factor, which is specifically recognized by the host
legume. The nodulating bacteria often form biofilms on root
surfaces during these early stages, which may be critical for
successful nodule formation and nitrogen fixation in later
stages. Recognition leads to further molecular signalling
events, which ultimately lead to nodule development, bacterial entry, and ultimately differentiation of the bacteria
into bacteroids surrounded by plant membrane. It is within
these symbiosomes that rhizobia fix nitrogen. The nif genes,
which are responsible for nitrogen fixation, are found only
in prokaryote genomes and encompass both structural and
regulatory genes that encode a functioning nitrogenase.
In this issue, Bontemps et al. (2010) show that certain
Burkholderia species, recently recognized as nitrogen-fixing
symbionts of legumes and other crops such as coffee and
maize (Estrada-De Los Santos et al. 2001; Moulin et al. 2001)
have a much richer history than initially suspected. The
authors’ research complements the developing hypothesis
that the Burkholderia-legume symbiosis, although newly published, is in fact a well-defined and effective symbiosis that
is common in nature (Chen et al. 2003, 2005; Sawada et al.
2003; Martı́nez-Aguilar et al. 2008). These earlier studies,
which followed the initial publication of Burkholderia species
as new symbionts of legumes (Moulin et al. 2001), hypothesized that nif and nod genes originated by horizontal gene
transfer from alphaproteobacteria. Bontemps et al. (2010)
expand on this idea with a comprehensive and in depth
study of Mimosa-associated symbionts in the region native to
the legume (Fig. 1), a 1800-km area in central Brazil. The
authors found that Burkholderia species were the prevalent
isolates from root nodules. In total, 47 different species of
Mimosa were sampled, with 143 bacterial isolates originating
from individual nodules on plant roots; 98% of these isolates
were identified as Burkholderia (Fig. 2). It appears that these
betaproteobacteria have a specific association with Mimosa
given the abundance of Burkholderia isolates. Moreover, the
type strain B. phymatum establishes nodules on roots of 30 of
31 Mimosa species.
Sequencing of genes essential to symbiosis provides
strong evidence that nodulation is not a new function
among Burkholderia species. The breadth of the study
(including 67 sampling sites in three regions in Brazil
over various elevations) provided a solid foundation with
2010 Blackwell Publishing Ltd
NEWS AND VIEWS: PERSPECTIVE 29
Fig. 1 A representative of one of the preferred legume symbionts (Mimosa humivagans) of many Burkholderia species. Mimosa
species are rich in diversity in their native habitats in South
America.
Fig. 2 Light micrograph of a nodule section from Mimosa
blanchetii growing in the Cerrado (Brazil) that illustrates the
immunolocalization and identification of nitrogenase (dark-coloured cells) in Burkholderia strain JPY461 using a B. phymatum
STM815-specific antibody.
which phylogenetic comparisons were made. PCR amplification and sequencing of 16S rDNA, recA, nifH and
nodC provided the raw data for phylogenetic analyses.
The data showed that the isolated Burkholderia species
represented specific groups that formed seven distinct
clades. The majority of isolates belonged to clusters 5
and 6 (34% and 54%, respectively) and were spatially
distinct because the clusters mapped correlatively to specific sampling sites according to elevation. However, the
most exciting finding was the high congruence of
sequences observed from the analysis of the symbiotic
genes, which indicates a long and stable genetic history
of nodulation and nitrogen fixation for Burkholderia. Only
two alphaproteobacteria were isolated among the 141 isolates, and their symbiotic genes were very distant from
those of Burkholderia. The high congruence within the
clades of Burkholderia species and variance from the alphaproteobacteria provides strong evidence that horizontal gene transfer events occurred much longer ago than
previously predicted.
2010 Blackwell Publishing Ltd
By choosing both the nitrogen fixation-related gene nifH
and the nodulation-related gene nodC, Bontemps et al.
(2010) clearly separated the two symbiotic functions. Many
bacteria fix nitrogen without nodulating their host, and nif
genes are found in a number of unrelated bacterial groups.
However, nodulation genes are restricted to internalized
symbionts and confined to what traditionally have been
called ‘rhizobia’. Interestingly, the authors demonstrate by
phylogenetic analysis that Mimosa-associated Burkholderia
acquired the nodulation gene nodC through horizontal gene
transfer long ago. Furthermore, species from Mimosa and
Burkholderia show specificity for each other, as determined
by inoculation and coinoculation studies between various
plant and bacterial species. Together, this work supports
the hypothesis that the symbiosis is not only very old, but
also highly specific.
The study presented by Bontemps et al. (2010) provides
compelling evidence that Mimosa and Burkholderia species
have an ancient history of symbiotic coexistence. Not only
does Mimosa develop these symbioses with the betaproteobacteria in their local region, but in other parts of the
world, these legumes also prefer Burkholderia. In conclusion, this symbiosis has a history that goes further back
than expected, as evidenced by deeply branched clades of
genes involved in nodulation. It is perhaps an interesting
juxtaposition that the betaprotebacteria, which were only
recently reported as nodule-inducing rhizobia, have such a
deep history of symbioses with legumes. The recent finding of betaproteobacteria as nitrogen-fixing symbionts
along with the current study by Bontemps et al. (2010)
opens up many possibilities to uncover the true diversity
of Burkholderia species.
References
Bontemps C, Elliott GN, Simon MF et al. (2010) Burkholderia
species are ancient symbionts of legumes. Molecular Ecology, 19,
44–52.
Chen WM, Moulin L, Bontemps C et al. (2003) Legume symbiotic
nitrogen fixation by b-proteobacteria is widespread in nature.
Journal of Bacteriology, 185, 7266–7272.
Chen WM, de Faria SM, Straliotto R et al. (2005) Proof that Burkholderia strains form effective symbiosis with legumes: a study of
novel Mimosa-nodulating strains from South America. Applied
and Environmental Microbiology, 71, 7461–7471.
Estrada-De Los Santos P, Bustillos-Christales R, Caballero-Mellado J (2001) Burkholderia, a genus rich in plant-associated nitrogen fixers with wide environmental and geographic
distribution. Applied and Environmental Microbiology, 67, 2790–
2798.
Martı́nez-Aguilar L, Dı́az R, Peña-Cabriales JJ et al. (2008) Multichromosomal genome structure and conformation of diazotrophy
in novel plant-associated Burkholderia species. Applied and Environmental Microbiology, 74, 4574–4579.
Moulin L, Munive A, Dreyfus B (2001) Nodulation of legumes by
members of the b-subclass of proteobacteria. Nature, 411, 948–
950.
Sawada H, Kuykendall LD, Young JM (2003) Changing concepts in
the systematics of bacterial nitrogen-fixing legume symbionts.
Journal of General and Applied Microbiology, 49, 155–179.
30 N E W S A N D V I E W S : P E R S P E C T I V E
Dr Hirsch has worked on biological nitrogen fixation for over
25 years, investigating the involvement of both plant and
microbial partners in the symbiosis, especially bacterial attachment to the host. Dr Hirsch and her co-workers recently discovered that the core nodulation (nod) genes of Sinorhizobium
meliloti, the nitrogen-fixing endosymbiont of alfalfa, are
required for the establishment of mature biofilms. This result
defines a new role for Nod factor in addition to its significance
as a signaling molecule. The Hirsch lab also studies the beta-
rhizobial strain Burkholderia tuberum, recently reported as
inducing the development of nitrogen fixing on legume hosts.
Current research centers on the sequencing and annotation of
four plant-associated Burkholderia genomes as well as a search
for B. tuberum mutants affected in attachment and nodule
development ⁄ function.
doi: 10.1111/j.1365-294X.2009.04459.x
2010 Blackwell Publishing Ltd