Table of Contents
Taxonomic Characterization of a Novel Rhizobial Strain Isolated from
Root Nodules of Entada phaseoloides and Phylogenetic Analysis of
Photosynthetic Rhizobia
KARLO DANTE TAPALES NATIVIDAD*, MASANORI OHASHI, JOSE JASON
LAUDATO CANTERA, HIROKO KAWASAKI-NAKAGAWA and TATSUJI SEKI
Laboratory of Applied Microbiology, The International Center for Biotechnology, Osaka
University, 2-1Yamada-oka, Suita-shi, Osaka 565-0871, Japan
Two experiments on rhizobial taxonomy were conducted and gave results
that could lead to the proposal of a new rhizobial genus and a new
taxonomic concept on photosynthetic rhizobia.
Characterization of Strain MAFF 210191: Preliminary studies on the
diversity of rhizobia in Okinawa, southern part of Japan, revealed that strain
MAFF 210191 exhibited phylogenetic character distinct from other rhizobia.
In this study, further taxonomic characterization of the strain was done.
Results showed that strain MAFF 210191 belong to the slow-growing group
of rhizobia and showed some characteristics (biochemical, physiological and
chemotaxonomic) quite different from other rhizobia. Phylogenetic analysis
based on 16S ribosomal DNA sequences showed that the isolate formed a
separate node far from other root-nodulating bacteria, while the nod A gene
sequence, encoding an acyl transferase involved in nod factor biosynthesis
showed high homology with that of Rhizobium tropici CFN 299. These
results suggest that MAFF 210191 posses a unique taxonomic position,
distinguished from other rhizobia, and probably a new genus under the
rhizobia group.
Phylogenetic Analysis of Photosynthetic Rhizobia: Several Bradyrhizobia
from various Aeschynomene species are of special interest because of their
ability to produce photosynthetic pigment. In this study, phylogenetic
relationship of four strains of photosynthetic rhizobia isolated from different
Aeschynomene species was analyzed. The 16S rDNA phylogeny showed that
photosynthetic rhizobia are mainly monophyletic and closely related to
Bradyrhizobium japonicum and Rhodo- pseudomonas palustris. The nif H
phylogeny placed the phototrophic rhizobia in a significant monophyletic
Research work in the “International Post-Graduate University Course in Microbiology”
supported by the Japanese Government, UNESCO and ICRO.
*
Present Address: BIOTECH, University of the Philippines at Los Banos, College, Laguna
4031, Philippines
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group with strains of B. japonicum, but far from R. palustris strains. On the
other hand, the nod A sequences from the photosynthetic rhizobia was found
to be highly conserved and was phylogenetically distant from nod A
sequences in the database. These results suggest that Bradyrhizobium
japonicum and photosynthetic rhizobia may have risen from a common
ancestor and that the nod A gene of photosynthetic rhizobia might have been
acquired in the latter part of their evolution or might have co-evolved with
its host plant.
Keywords: rhizobia, nod A, nif H, 16S rDNA, phylogeny.
Characterization of a Novel Rhizobial Strain Isolated from Root Nodules
of Entada phaseoloides
Introduction
Symbiosis between leguminous plants and soil bacteria are of considerable
environmental and agricultural importance since they are responsible for most of the
atmospheric nitrogen fixed on land. Members of the Leguminosae form the largest plant
family, with approximately 18,000 species and their success can be largely attributed to
their ability to form symbiotic relationship with nitrogen fixing bacteria known as rhizobia
[1].
Rhizobia are classically defined as symbiotic bacteria capable of eliciting and
invading root and stem nodules on leguminous plants, where they differentiate into
nitrogen-fixing bacteroids [2] and reduce atmospheric nitrogen to ammonia to the benefit of
the plant.
These bacteria were originally assigned to species on the basis of their host
specificity, a practice that has been seriously criticized [3]. Rhizobial taxonomy and
systematics has notably progressed in the past decade mainly due to the characterization of
new isolates from hosts that had not been previously studied, together with the generalized
use of 16S rDNA sequencing and polyphasic approach [4, 5]
Based on their 16S ribosomal DNA sequences, these nodule endosymbionts
constitute a polyphyletic assemblage of bacteria grouped in four major phylogenetic
branches of the �-2 subclass of Proteobacteria. Rhizobial strains are currently placed in the
following genera: Rhizobium, Mesorhizobium and Sinorhizobium, which constitute one of
the rhizobial clades; Azorhizobium, Bradyrhizobium and Methylobacterium which are
separately found in a well-resolved phylogenetic branch [2, 6]. However, recently, IRD
researchers have just identified two bacterial strains belonging to another group of
Proteobacteria that are capable of forming nodules in leguminous plants. Ribosomal DNA
sequencing of these two strains has shown that they belong to the Burkholderia genus, a
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member of the � -Proteobacteria subclass, phylogenetically distant from the rhizobia
described to date [1].
It is considered that the knowledge of bacterial species is still very limited and, for
this reason, bioprospection to identify new groups is encouraged. Initial studies on rhizobial
diversity on the southern part of Japan revealed that Strain MAFF 210191 isolated from
root nodules of woody legume Entada phaseoloides exhibited phylogenetic character
distinct from other rhizobia. Symbionts of legumes exhibiting ecological and agronomic
importance should be properly characterized prior to their use to sustainable agriculture and
environmental management. Thus, the aim of this study is to further characterize and
examine the taxonomic position of the said strain using polyphasic approach. This study
suggests that characterization of the symbionts of the yet unexplored legumes may reveal
rhizobial nature of additional members and possibly other taxonomic classes. Such study
may significantly contribute to the understanding of the origin and evolution of the
rhizobia-legume symbiosis, and may open new perspectives for environmental and
agricultural applications.
Materials and Methods
Bacterial strain and growth conditions
The strain used in this study was obtained from the Ministry of Forestry and
Fisheries, Japan designated as MAFF 210191, was isolated from root nodules of woody
legume Entada phaseoloides. The strain was grown on Yeast-Extract Mannitol Agar
(YMA)(yeast extract , 0.5 g; mannitol, 10 g; NaCl, 0.2 g; CaCl2, 0.2 g; MgSO4, 0.1 g;
K2HPO4, 0.5 g; agar, 15 g/ L). The strain was grown at 28°C unless otherwise stated.
Morphological characterization
Colony morphology (color, size, form, border, elevation, mucoidy) of the isolate
was evaluated by streaking the Isolate on YMA and allowing the bacteria to grow at 28°C
for 5-7 days. Gram staining was performed and cell morphology was observed using a
phase-contrast microscope.
Biochemical and physiological characterization
The following characteristics of Strain MAFF 210191 were analyzed: (i) utilization
of different sugars as sole carbon source; (ii) utilization of different amino acids as sole
nitrogen source. To test for substrate utilization, the different C and N sources were added
at a final concentration of 0.2% and 0.1% respectively to the basal medium (YMA). When
substrates were used as C source, mannitol was omitted, and when used as N source, yeast
extract was omitted from the basal medium. All of the substrates were filter sterilized
before they were added to the basal medium. The culture was incubated at 28°C and growth
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was recorded after 5-10 days. For the utilization of sugars as C-source, the amount of
extracellular polysaccharide and alkalined/acid production were noted.
Physiological characteristics such as tolerance to antibiotics, tryptone and NaCl at
various concentrations and pH and temperature range for growth were examined using
YMA as medium. Other tests such as test for catalase, hydrolysis of starch, gelatin, casein,
vitamin requirement, citrate utilization, and 3-ketolactose production were performed by
the methods described in the Manual of Methods for General Bacteriology [7].
Chemotaxonomic characterization
Quinone analysis
The organism was cultured in 100 ml yeast mannitol broth, harvested by
centrifugation, washed once with distilled water and freeze dried. Quinone fractions were
extracted with chloroform-methanol (2:1 v/v) from the lyophilized cells. The quinone
fractions were then separated by thin-layer chromatography in hexane-diethylether solution
(85:15 v/v). After separation the bands were viewed by UV illumination and recovered by
scraping. The quinones were extracted from the silica gel powder and dissolved in
acetonitrile and analyzed using High Performance Liquid Chromatography (HPLC)
(Hitachi LaChrom L-7100).
Fatty acid analysis
Cellular fatty acids were extracted and harvested from the cells according to the
method described by Hirashi et al. (1992) [8]. The analysis was based on the conversion of
fatty acids to methyl esters by mild acidic methanolysis. Cellular fatty acids were then
analyzed by gas-liquid chromatography based on their methyl ester derivatives.
G+C content of DNA
DNA base composition was determined using the method described by Tamaoka
and Komagata (1984) [9]. Twenty µg of purified DNA was denatured by incubating at
100°C for 2 minutes. Acetate buffer containing 2 mM ZnSO4 and P1 nuclease was added
to the denatured DNA and incubated at 37°C for 2 hrs. The solution was treated with
alkaline phosphatase (0.35 U/µl) and again incubated at 37°C overnight. G+C content of
genomic DNA was measured using HPLC (Hitachi LaChrom L-7100).
Plant nodulation test
Plant cultivar and seedling
Seeds of Macroptilium artropurpureum were surface sterilized by immersion in
concentrated sulfuric acid for 5 minutes and washed 3X with approximately 20 ml sterile
distilled water. The seed were allowed to germinate on 1% water agar for 3 days.
Cross nodulation test
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Three-day old seedlings were transferred to sterile germination tubes containing
nitrogen free medium. Different dilutions of bacterial suspension was inoculated and grown
on the nitrogen-free medium in a controlled environment cabinet at 25°C with 16 hours
photoperiod. The plants were examined periodically for 8 weeks for nodule formation.
Molecular characterization
DNA isolation
Chromosomal DNA was prepared by the method of Ausubel et al. (1995) [10].
Bacterial cells were lysed and proteins were removed by digestion with Proteinase K
(Sigma Chemical Co., MO, USA). Non-DNA components were removed by selective
precipitation with cetyltrimethylammonium bromide (CTAB), and DNA was recovered by
isopropanol precipitation.
16S rDNA amplification and sequencing
The primers used for DNA amplification and sequencing are described in Table 1.
Nearly full-length 16S ribosomal DNA was amplified using universal primer 20F forward
primer and 1500R reverse primer. The amplification was carried out with the following
conditions: initial denaturation at 94°C for 5 min; 25 cycles at 94°C for 30 sec, 55°C for
30 sec, and 72°C for 30 sec; and then final extension at 72°C for 7 min. The PCR product
was purified using QIAGEN Gel Extraction Kit and sequenced directly using overlapping
primers (Table 1) by the dideoxy-chain termination method (Sanger et al., 1977)[11] with a
BigDye™ Terminator cycle sequencing kit (PE Applied Biosystems) applied to ABI
PRISM 310 Genetic analyzer (Perkin-Elmer Co.).
nod A gene amplification and sequencing
Degenerate primers nod A-1 (5’-TGC RGT GGA ARX TRX XCT GGG AAA-3’)
and nod A-2 (5’-GGX CCGTCR TCR AAW GTC ARG TA-3’) with primer positions of
14-37 of nod A and 66-88 of nod B respectively. A touchdown PCR (annealing temperature
55-45°C in 25 cycles and additional 10 cycles at 45°C) was performed. PCR product was
purified using QIAGEN Gel Extraction Kit and cloned to E.coli using QIAGEN Cloning
Kit. Plasmids containing the insert (nod A gene fragment) were extracted using QIAprep
Spin Miniprep Kit and cycle sequenced using M13F and M13R primers. DNA sequence
was determined using the method mentioned previously.
Phylogenetic analysis
Sequenced data were analyzed by the ABI PRISM (Perkin-Elmer Co.) sequence
analysis program and assembled using the ABI Auto Assembler (Perkin-Elmer Co.).
Homology searches were performed via BLAST either at the National Center for
Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/) or at the DNA Data
Bank of Japan (DDBJ, http://www.ddbj.nig.ac.jp/). Phylogenetic trees were constructed by
neighbor-joining method using CLUSTAL X program [13].
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Table 1. Primers for 16S r DNA sequencing
Primer Name
Sequence (5’-3’)
Target gene
Reference
20F
520F
800F
920F
1240F
520R
920R
1500R
GAG TTT GAT CCT GGC TCA G
CAG CAG CCG CGG TAA TAC
GAT TAG ATA CCC TGG TAG
AAA CTCAAA TGA ATT GAC GG
TAC ACA CGT GCT ACA ATG
GTA TTA CCG CGG CTG CTG
CCG TCA ATT CAT TTG AGT TT
GTT ACC TTG TTA CGA CTT
Eubacteria 16S r DNA
Eubacteria 16S r DNA
Eubacteria 16S r DNA
Eubacteria 16S r DNA
Eubacteria 16S r DNA
Eubacteria 16S r DNA
Eubacteria 16S r DNA
Eubacteria 16S r DNA
12
12
12
12
12
12
12
12
Southern hybridization
Genomic DNA was digested with restriction enzymes and electrophoresed on TAE
agarose gel (0.8%). Digested DNA fragment were transferred to Hybond�-N+ nylon
membranes (Amersham Pharmacia Biotech, Buckinghamshire, England) by vacuum
transfer. The DNA probe was labeled with DIG labeling and detection kit (Roche
Diagnostics, Mannheim, Germany) and hybridization was carried out at 65°C. Detection of
hybridization signals using the DIG luminescent detection Kit (Roche Diagnostics)
according to the manufacturer’s instructions.
Results and Discussion
Rhizobial taxonomy has notably progressed for the past decade mainly due to the
generalized use of 16S rDNA as basis for classification. In this study, we characterized
bacterial strain MAFF 210191 using polyphasic approach.
16S rDNA sequencing and analysis
In order to determine the phylogenetic position of Strain MAFF 210191, nearly fulllength sequence of 16S rDNA was determined. Initially a BLAST search was used to find
close relations through sequence similarity. BLAST analysis revealed that the most closely
related sequences to that of strain MAFF210191 is that of Mesorhizobium sp. with 91%
identity. The sequenced data was analyzed and a phylogenetic tree was constructed.
Comparative analysis of 16S rDNA sequences of MAFF 210191 and species of rhizobia
and related genera revealed that Strain MAFF 210191 is distantly related to other symbiotic
nitrogen-fixing bacteria. It forms a separate node which appears to be distinct from the
other rhizobia as shown in Fig. 1. Strains from related genera (Phyllobacterium, Brucella,
Bartonella and Rhodopseudomonas) in the �-Proteobacteria were represented and found to
be interspersed between the members of the rhizobia group [14]. Such low sequence
similarity suggests that strain MAFF 210191 belongs to a different genus, and most
probably a member of the rhizobia group.
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Bartonella grahamii Z31349
Bartonella vinsonii U26258
100
Bartonella taylorii Z31350
88
Brucella melitensis L26166
90
Brucella ovis L26168
100
Brucella canis L37584
46
Rhizobium tropici U89832
100
Rhizobium rhizogenes D14501
100
Rhizobium leguminosarum U29386
87
94
Rhizobium etli U28916
89
Agrobacterium vitis AJ389910
80
Agrobacterium tumefaciens AF5317
Sinorhizobium terangae X68387
100
100 Sinorhizobium saheli X68390
88
Sinorhizobium meliloti AF533685
Mesorhizobium ciceri UO7934
100
Mesorhizobium loti X67229
85
Mesorhizobium tianshanense AF041447
99
89
Mesorhizobium meditteraneumL38825
Phyllobacterium myrsinacearumD12789
100
100
Phyllobacterium rubiacearum D12790
Rhodopseudomonas palustrisVA297
87 Rhodopseudomonas palustris 99D
100
Bradyrhizobium japonicum U69638
Bradyrhizobium elkani U35000
51
Methylobacter nodulans AF220762
55 30
Azorhizobium caulinodans D11342
50
Rhodobacter sphaeroides IFO1220
Rhodobacter azotoformans KA25
MAFF210191
0.1
Fig. 1. Phylogenetic tree based on nucleotide sequence of 16S rDNA showing the phylogenetic
position of Strain MAFF 210191 and its relationship among the rhizobia and closely related genera.
Phylogenetic trees were constructed using the neighbor-joining method; bootstrap values derived from
100 replicates are shown as percentages.
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Table 2. Phenotypic characteristics of Strain MAFF 210191
Characteristics
MAFF 210191
Colony morphology
Whitish, semi-translucent; irregular form; raised with entire
margin
Extracellular polysaccharide
Cell morphology
Gram staining
Motility
Spore formation
Mode of reproduction
Growth on YMA
Growth at pH
4.0
5.0
6.0
7.0
8.0
9.0
Growth at Temp
5C
15C
22C
28C
37C
45C
Growth at NaCl conc
0.02%
0.10%
0.25%
0.50%
0.75%
Oxidase test
Catalase test
Growth at tryptone conc
0.05%
0.10%
0.25%
0.50%
Growth on NA medium
Growth on TGYA medium
Growth on LBA medium
Growth on SPA medium
Vitamin requirement
3-Ketolactose production
Citrate utilization
Gelatin hydrolysis
Starch hydrlolysis
Casein hydrloysis
Mol % G+C of DNA
Major quinone content
Major fatty acid
+
Short rods to spherical; 1-1.2X1.8-2.2 µm
+
budding
+ slow
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
61.5%
Ubiquinone 10 (Q10)
18:1; 19 cyc
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Table 2 (continued)
Characteristics
MAFF 210191
Utilization of sugars as carbon source
Glucose
Sucrose
Fructose
Maltose
Galactose
Trehalose
Rhamnose
Sorbitol
Mannitol
Xylose
Dulcitol
Arabinose
Adonitol
Xylitol
Melezitose
Salicin
Erythritol
Inositol
Cellubiose
Ability to grow on basal medium containing
DL-�-alanine
L-arginine
L-cysteine
L-lysine
L-methionine
L-valine
L-phenylalanine
DL-iso-Leucine
L-histidine
L-proline
L-threonine
L-tryptophan
Glycine
L-glutamic acid
Resistance to
Kanamycin (10 µg/ml)
Tetracycline (10µg/ml)
Chloramphenicol (10 µg/ml)
Penicillin G (20 units/ml)
Erythromycin (10µg/ml)
Streptothricin (10µg/ml)
* Alkaline production
+
+*
+
+*
+*
+
+
+*
+*
+
+
+
+
+*
+*
+
+
+*
+
+
+
+
+
+
+
+
+
+
+
+
-
Morphology
Strain MAFF 210191 was characterized as a slow-growing rhizobia producing
copious exopolysaccharide slime on YMA. Exopolysaccharide slime production is a
characteristic of the rhizobia group, which protects them from drying out and also helps the
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bacterium stick to root hairs during various stages of its life cycle. Colonies are circular,
semi-translucent, raised and mucilaginous, reaching approximately 2mm in diameter within
5-7 days on YMA medium. The cells are gram-negative, rod shaped to spherical (11.2X1.8-2.2 µm), non-spore forming, motile and reproduce by budding.
Physiological and biochemical characteristics
Optimum temperature for growth of strain MAFF 210191 was 22-28°C and cells
could grow at 15°C but not at 37°C. The pH range for growth was 5-8. It can tolerate salt
concentration of 0.5% and less. It was able to utilize a wide array of sugars as carbon
source which includes; glucose, sucrose, fructose, maltose, galactose, trehalose, rhamnose,
sorbitol, mannitol, xylose, dulcitol, arabinose, adonitol, xylitol, salicin, erythritol, inositol
and cellubiose. The strain was able to grow on minimal salt medium, thus, carbon sources
supporting visibly more growth than control slants (basal medium) were scored positive.
Dubious cases showing slight traces of growth was not scored positive. It was able to
produce alkaline condition upon utilization of most of the sugars (Table 2), a characteristic
shared with the slow growing Bradyrhizobium. Growth of the strain was inhibited by
glycine and various amino acids (Table 2). Growth was inhibited with tryptone
concentration of more than 0.1% which is the reason why it was not able to grow on
complete media like NA, TGYA, SPA and LBA. Growth inhibitions by certain amino acids
and tryptone might be the factor why this bacterium was not isolated and characterized
before; in addition, very few studies have been conducted on the characterization of
endosymbionts of woody legumes like Entada. No requirements for vitamins and other
factors for growth were observed. Additional physiological and biochemical characteristics
are shown in Table 2.
Chemotaxonomic characteristics
The major respiratory quinone of strain MAFF 210191 was ubiquinone 10. The
major fatty acid in the cell is 19:0 cyclopropanic acid (26.97%). This result was quite
unique since the major fatty acids in the rhizobia group was 18:1 and 19 cyc, but in this
case, only small amounts of C 18:1 was detected for strain MAFF 210191. G+C content of
DNA was found to be 61.5%.
Plant infection test
To ensure that strain MAFF 210191 was indeed a rhizobium, we checked its ability
to re-nodulate a leguminous plant. Cross-nodulation experiment was done because of the
unavailability of the original host plant during the time of study. Macroptilium
artopurpureum was used as the test plant since it is a host legume to many rhizobia. Results
showed tha strain MAFF 210191 was not able to nodulate the host plant probably due to
host specificity. In the rhizobium-legume symbiosis, compatible bacteria and host plants
interact through an exchange of signals. Host specificity is mainly controlled by Nod
factors recognized by the host plant. All Nod factors are chitin oligomers mono N-acylated
at the non-reducing end and diversely substituted at the non-reducing ends of the molecules.
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Rhizobium tropici CFN299
100
MAFF210191
41
Mesorhizobiumplurifarium ORS1255
44
Sinorhizobium terangae ORS1009
100
14
Sinorhizobium sp. ORS1230
Methylobacter nodulans ORS2060
100
Burkholderia sp.STM678
14
Mesorhizobium ciceriUSDA3383
100
Mesorhizobium mediterraneum USDA3392
100
Mesorhizobium tianshanense USDA3592
18
100
81
81
25
100
Bradyrhizobium sp.ORS301
Bradyrhizobium sp.ORS304
Bradyrhizobium sp.ORS336
Bradyrhizobium japonicum USDA110
Bradyrhizobium elkanii USDA94
70
Sinorhizobium fredii USDA257
Rhizobium galegae HAMBI1174
41
Sinorhizobium meliloti 042B
Sinorhizobium saheli ORS609
Azorhizobium caulinodansD11342
0.1
Fig. 2. Phylogenetic tree based on nodA gene sequence showing the phylogenetic position of
Strain MAFF 210191. Phylogenetic trees were constructed using the neighbor-joining method;
bootstrap values derived from 100 replicates are shown as percentages.
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These various substitutions, which confer plant specificity, are encoded by host-specific
nod genes. The synthesis of the N-acylated oligo saccharide core of the Nod factor is
controlled by nod ABC genes, which are present in all rhizobia [15].
nod A gene sequencing and analysis
The nod A gene, involved in the transfer of an acyl chain to the chitin
oligosaccharide backbone Nod factor, has been shown to be a good nodulation marker [16].
We determined almost full sequence of nod A gene of strain MAFF 210191. nod A
sequence of strain MAFF 210191 revealed very high similarity with rhizobial nod gene of
Rhizobium tropici CFN 299 (99%). Because of the very high similarity and to ease the
notion of contamination, we did southern hybridization using the sequenced fragment as
probe. We were able to get intense signal and we re-sequenced the digested fragment.
Results obtained were similar to that of the previously sequenced 16S rDNA.
Based on nod A gene phylogeny, the strains representing the genus Sinorhizobium,
Mesorhizobium and Rhizobium were not tightly clustered and fragmented as seen in Fig. 2.
The nod genes are unique to rhizobia, and the phylogenies of nod A, nod B, nod C and nod
D, which are found in all rhizobia resemble each other but differ from the phylogeny of 16S
r DNA [17]. Indeed it has been suggested that the phylogenies of nod genes may correlate
with the host plant [18].
Mainly due to its distinct phylogenetic position based on 16S rDNA analysis and
several supporting phenotypic characters, we conclude that strain MAFF 210191 posses a
unique taxonomic position, suggesting that strain MAFF 210191 might be a new genus
under the root-nodulating bacteria.
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15. Chaintreuil, C., Biovin, C., Dreyfus, B., and Giraud, E. (2001). Characterization of
common nodulation genes of the photosynthetic Bradyrhizobium sp. ORS 285 reveals
the presence of a new insertion sequences upstream of nodA. FEMS Microbiology
Letters 194: 83-86.
16. Biovin, C. and Giraud, E. (1999). Molecular symbiotic characterization of rhizobia:
towards a polyphasic approach using Nod factors and nod genes. In: Highlights of
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Plenum Press, New York.
17. Haukka, K., Lindstrom, K. and Young, J. (1998). Three phylogenetic groups of nod A
and nif H genes in Sinorhizobium and Mesorhizobium isolates from leguminous trees
growing in Africa and Latin America. Appl. Envn. Microbiol. 64: 419-426.
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of rhizobia by PCR-based sequencing of a nod C segment. J. Bacteriol. 177: 468-472.
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Phylogenetic Analysis of Four Photosynthetic Rhizobium Strains based
on 16S rDNA, nif H and nod A Genes
Introduction
The soils in tropical regions are often poor in nitrogen, an element essential for
plant fertility. Some vegetation like leguminous plants can fix gaseous nitrogen present in
the atmosphere thru its symbiotic association with the bacteria rhizobia. The rhizobia
isolated from the stem nodules of Aeschynomene are of special interest because of their
ability to produce photosynthetic pigment, bacteriochlorophyll (bchl a) [1], aptly termed as
photosynthetic rhizobia. These photosynthetic rhizobia belong to the �-2 Proteobacteria
and are phylogenetically related to Bradyrhizobium species and the purple non-sulfur
Rhodopseudomonas palustris based on sequence analysis of the 16S rRNA [2]. The big
question is that whether this group of bacteria is related to known photosynthetic bacteria,
to known symbiotic bacteria, to both or to neither. Their very unique biological characters,
possessing the nodule forming and the photosynthetic abilities of B. japonicum and Rps.
palustris, respectively, have led us to think that they are the “missing link” between the two.
This group of bacteria is currently assigned under the Bradyrhizobium genus because of its
relatedness in many aspects, but it origin and evolution remains to be elucidated. As such,
these bacteria are good models for studying the evolution of nodulation, nitrogen fixation
and photosynthesis in the �-Proteobacteria. We thus performed a phylogenetic analysis of
the genes involved in nitrogen fixation (nifH) and nodulation (nodA) in comparison with
that of the 16S rRNA on four strains of photosynthetic rhizobium to investigate their
evolutionary relationship.
Materials and Methods
Bacterial strains
The photosynthetic rhizobia (BTAi 1, IRBG 2, IRBG 228 and IRBG 230) were
grown aerobically in tryptone-glucose yeast extract agar at 28°C [3]. E. coli strains for
recombinant DNA manipulations were grown overnight in Luria-Bertani (LB) medium at
37 �C.
PCR amplification and sequencing
DNA was extracted using the method of Ausubel et al. [4]. PCR reactions for 16S
rDNA and nif H were performed using standard procedures. Sequences of nifH was
determined using primers nifH-1c (5’-CARATCGCVTTYTACGG-3’) and nifH-GEM-R
(5’ADNGCCATCATYTCNCC-3’). The 16S rDNA gene sequences were determined as
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Table 1. Origin and host of bacterial strains.
Strain
BTAi1
IRBG 2
IRBG 228
IRBG 230
Host plant
Aeschynomene
indica
Aeschynomene
afraspera
Aeschynomene
pratensis
Aeschynomene
nilotica
Origin
United States
Source
BTI
Laguna, Philippines
IRRI
Iloilo, Philippines
IRRI
Laguna, Philipppines
IRRI
described by Lisdiyanti et al. [5]. Amplification and sequencing of nodA were done
as follows: A touchdown PCR (annealing temperature 55-45°C in 25 cycles and additional
10 cycles at 45°C ) was performed. Sequence of nod A was determined using degenerate
primers nod A-1 (5’-TGC RGT GGA ARX TRX XCT GGG AAA-3’) and nod A-2 (5’GGX CCGTCR TCR AAW GTC ARG TA-3’) with primer positions of 14-37 of nod A
and 66-88 of nod B respectively. DNA sequences were determined by the dideoxy-chain
termination method [6].
Sequence alignments and phylogenetic trees
The nucleotide sequences obtained in this work were converted to amino acid
sequences using Genetix-Win (version 3.1.0). BLAST search [7] was performed at the
National Center for Biotechnology Information (NCBI) databases. Phylogenetic analyses
were performed using the neighbor joining method [8] of the CLUSTAL X program [9].
Graphic representations of the resulting trees were made using the TreeView .
Results and Discussion
Phylogenetic analysis
In the 16S rDNA tree (Fig. 1), the photosynthetic rhizobia were placed in a monophyletic
group with the non-phototrophic Bradyrhizobium and Rps. palustris, suggesting that these
three species are closely related although they have different phenotypes. The same
topology was observed in the tree derived from the partial nif H sequences (Fig. 2), with
regards to the photosynthetic rhizobia, such that the nif H tree placed the photosynthetic
rhizobia in a highly supported monophyletic group with the B. japonicum strains, although
separated from the phototrophic Rps. palustris that clustered with other anoxygenic
phototrophic bacteria belonging to different phylogenetic lineages. Amino acid sequence of
the nod A of the photosynthetic rhizobia (IRBG 2, IRBG 230, IRBG 228 and
Photorhizobium thompsonianum BTAi 1) was combined with other available sequences of
Photosynthetic rhizobia (ORS 352, ORS 287, ORS 285 and ORS
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Bradyrhizobium sp. IRBG 2
Bradyrhizobium sp. IRBG 230
100
Bradyrhizobium sp. IRBG 228
‘P. thompsonianum’ BTAi1
Bradyrhizobium sp. MAFF 210318
98
B. japonicum USDA
110
B. japonicum IAM 12608
Rps. palustris VA2-2
95
Rps. palustris ATCC 17001
100
100
Rps. palustris 99D
Rps. palustris HMD 89
100
Rps. palustris HMD 88
B. elkanii USDA 76
Rhi. etli CFN-42
S. meliloti 1021
100
M. loti MAFF 303099
Rhodobacter sp. AP-10
Rba. capsulatus ATCC 11166
Rba. blasticus NCIMB 11576
94
100
100
Rba. sphaeroides ATCC 11166
Rba. azotoformans KA25
Rdv. strictum JCM 9220
100
Rdv. strictum JCM 9221
96
Rdv. sulfidophilum W4
Rhodovulum sp. CP-10
Rsp. rubrum
Nostoc (Anabaena) sp. PCC 7120
0.1
Fig. 1. Phylogenetic tree based on 16S rRNA showing the phylogenetic positions of strains of the
phototrophic rhizobia (written in bold letters).
Phylogenetic trees were constructed using the neighbor-joining method; bootstrap values derived
from 1000 replicates are shown as percentages; bold lines indicate branches with more than 90%
bootstrap value. The lengths of the horizontal lines are proportional to the evolutionary distances,
while the lengths of the vertical lines are meaningless. Bar represents 0.1 changes per nucleotide.
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Rhodopseudomonas sp. HMD 88 (ABO79624)
80
Rhodopseudomonas sp. HMD 89 (ABO79625)
Rhodopseudomonas palustris VA2-2 (ABO79623)
90
Rhodopseudomonas sp. 99D (ABO79627)
Rhodopseudomonas palustris ATCC 17001T (ABO79626)
91
Rhodobacter capsulatus ATCC 11166T (X07866)
Rhodospirillum rubrum (M33774)
Rhodobacter sphaeroides ATCC 11167T (AF031817)
83
Rhizobium etli CFN-42T (M15942)
Mesorhizobium loti MAFF 303099 (AP003007)
95
Sinorhizobium sp. NGR 234 (AE000105)
Bradryrhizobium sp. IRBG 2 (AB079615)
99
Bradryrhizobium sp. IRBG 230 (AB079617)
Bradryrhizobium sp. IRBG 228 (AB079616)
“Photorhizobium thompsonianum” BTAi 1 (AB079618)
Bradyrhizobium sp. MAFF 210318 (AB079620)
97
Bradyrhizobium japonicum IAM 12608T (AB079619)
100
81
Bradyrhizobium japonicum USDA 110 (K01620)
Bradyrhizobium elkanii USDA 76T
Anabaena sp. PCC 7120 (AF012326)
0.1
Fig. 2. Phylogenetic tree based on partial amino acid sequences of nif H showing the
phylogenetic positions of the phototrophic rhizobia.
Sequence from cyanobacterial species was used as outgroup. Species under study are
written in bold. Phylogenetic trees were constructed using the neighbor-joining method;
bootstrap values derived from 100 replicates are shown as percentages; bold lines indicate
branches with more than 80% bootstrap value. The lengths of the horizontal lines are
proportional to the evolutionary distances, while the lengths of the vertical lines are
meaningless. Sequence accession numbers are given in parenthesis; species sequenced in
this study are indicated. Bar represents 0.1 changes per amino acid.
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Bradyrhizobium sp. ORS287
Bradyrhizobium sp. IRBG 230 (This study)
Bradyrhizobium sp. ORS 364
Bradyrhizobium sp. ORS 352
Bradyrhizobium sp. ORS 285
100
Bradyrhizobium sp. IRBG 228 (This study)
93
Bradyrhizobium sp. IRBG 2 (This study)
P. thompsonianum BTAi 1 (This study)
Bradyrhizobium sp. ORS 301
100
Bradyrhizobium sp. ORS 304
100
Bradyrhizobium elkanii USDA 94
Bradyrhizobium japonicum USDA 110
Methylobacter nodulans ORS 2060
Burkholderia sp. STM 678
Sinorhizobium fredii USDA 257
994
Mesorhizobium ciceri USDA 3383
Mesorhizobium tianshanense USDA 3592
Rhizobium tropici CFN 299
Sinorhizobium terangae ORS 1009
Rhizobium galegae HAMBI 1174
Sinorhizobium meliloti 042B
Azorhizobium caulinodans D11342
0.1
Fig. 3. Phylogenetic tree based on partial amino acid sequences of nod A showing the
phylogenetic position of the phototrophic rhizobia.
Species under study are written in bold. Phylogenetic trees were constructed using the
neighbor-joining method; bootstrap values derived from 100 replicates are shown as
percentages; bold lines indicate branches with more than 80% bootstrap value. The lengths
of the horizontal lines are proportional to the evolutionary distances, while the lengths of the
vertical lines are meaningless. Sequence accession numbers are given in parenthesis; species
sequenced in this study are indicated. Bar represents 0.1 changes per amino acid.
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364) (Fig. 3). Phylogenetic analysis showed that the nod A sequences from the
photosynthetic rhizobia were highly conserved, regardless of the geographical location and
host species from which they were isolated. The nod A genes of these photosynthetic
rhizobia formed a highly supported monophyletic cluster separated from those of B.
japonicum and other known nod A gene.
The topology of the nod A phylogeny is different to those of the 16s rRNA and nif
H with regards to the position of the photosynthetic rhizobia. Based on 16S rDNA and nif
H phylogeny, it places the photosynthetic rhizobia very close to Bradyrhizobium japonicum,
but based on nod A phylogeny, it forms a tightly clustered monophyletic group
phylogenetically distant from already described nod A genes. This result suggests that the
nod A genes of the phototrophic rhizobia may have arisen from ancestral species different
from that of the B. japonicum although these species are closely related based on the 16S
rDNA and nifH phylogenies. Phylogeny of nod genes resemble each other but differ from
the phylogeny of 16S rDNA, suggesting that it might correlate with host plant [10].
Possible evolutionary implications
The close relationship between the nodule-forming Bradyrhizobium and the
photosynthetic rhizobia was revealed by the 16S rRNA [2] and nif H data. In addition, nif H
shares a common evolutionary history with the 16S rRNA [11, 12], and thus used as
molecular marker in diversity studies [13, 14]
Based on the fact that the 16S rRNA phylogeny reflects the species phylogeny, the
aberrant position of the phototrophic rhizobia in the nod A tree strongly suggests that the
phototrophic rhizobia may have acquired the nod A genes from ancestral donor species
different from that of the typical Bradyrhizobium before their diversification, probably by
lateral gene transfer. Transfer and/or acquisition of the symbiosis island region containing
the nodulation genes of rhizobia have already been described [15]. Moreover, it is also
possible that the nod A genes of the phototrophic rhizobia might have co-evolved with their
host plants, such that interaction of the phototrophic bacteria inside the stem nodules of
their hosts may have restricted the diversification of their nod A genes. Our results also
suggest that nod A genes of photosynthetic rhizobia were acquired before they were
geographically separated. Nevertheless, this study has provided a molecule that would
further distinguish the phototrophic rhizobia from the typical Bradyrhizobium species.
Acknowledgment
The authors would like to thank Mr. Rolando So of the International Rice Research
Institute, Philippines and Douglas K. Jones of the National Rhizobium Germplasm
Collection Center, ARS/USDA for the photosynthetic IRBG strains and the BTAi1 strain,
respectively.
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