Applied and Environmental Microbiology, November 1999, p. 4935-4942, Vol. 65, No. 11
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Culture-Independent Characterization of a Gene
Responsible for Nitrogen Fixation in the Symbiotic Microbial Community
in the Gut of the Termite Neotermes koshunensis
Satoko
Noda,1,2
Moriya
Ohkuma,1,3,*
Ron
Usami,2
Koki
Horikoshi,2 and
Toshiaki
Kudo1,2
The Institute of Physical and Chemical Research
(RIKEN)1 and Japan Science and
Technology Corporation (JST),3 Wako, Saitama
351-0198, and Department of Applied Chemistry, Toyo University,
Kawagoe, Saitama 350-8585,2 Japan
Received 12 July 1999/Accepted 27 August 1999
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ABSTRACT |
Expression of the nitrogen fixation gene, nifH, in the
gut of the termite Neotermes koshunensis was characterized
without cultivation. nifH cDNA was directly amplified from
mRNA of the mixed microbial population in the gut by reverse
transcription (RT)-PCR. Analyses of the RT-PCR products revealed that,
among the diverse nifH sequences, only a few corresponding
to an alternative nitrogenase (encoded by the anf gene)
were preferentially transcribed in the termite gut. Expression of the
anf gene was further investigated quantitatively under
several termite feeding conditions by competitive PCR. The levels of
expression of the anf gene were largely congruent with the
nitrogen fixation activity displayed by the termite. The amounts of the
genomic anf gene in the population showed no significant
change, indicating that the level of expression was critical for
nitrogen fixation activity. Interestingly, no significant decrease in
the expression level was observed when the diet contained molybdenum
(Mo), which represses ordinary anf genes. A 3.6-kb DNA
region downstream of the anf gene was isolated and found to contain reading frames homologous to anfH,
anfD, and anfG of the Bacteria
domain which encode subunits of an alternative nitrogenase having no Mo
as a cofactor. This DNA region also contained reading frames encoding
glnB-like proteins, which is a common feature of the
nitrogenase genes of the Archaea domain. These results indicate that the anf group of nitrogenase genes is the
most important group of genes responsible for nitrogen fixation in
N. koshunensis and that the anf gene possesses
novel features with respect to the regulation of its expression and its
gene organization.
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INTRODUCTION |
Nitrogen fixation by the symbiotic
microorganisms inhabiting the gut of termites is important, since
termites thrive on a nitrogen-poor diet (3, 7, 8). The
identity of the microorganisms responsible has been investigated in
evolutionarily diverse termites based on comparisons of the sequences
of the nitrogen fixation gene, nifH, directly amplified by
PCR and cloned from the microbial community in the termite gut
(26, 27). These culture-independent studies have revealed
the presence of a phylogenetically diverse and hitherto unrecognized
population of potential nitrogen-fixing microorganisms in the gut of
termites. This kind of molecular approach, as opposed to conventional
microbiological approaches, is beneficial and is increasingly being
used to study natural microbial communities, to avoid the largely
unrepresentative nature of microbial cultivation (1).
However, since there are some differences in the efficiencies of DNA
extraction, PCR amplification, and cloning, the distribution of
nifH sequences as final clones may not reflect the real
distribution of nifH genes in the original microbial
community. Furthermore, it must be emphasized that the existence of
nifH sequences does not always mean that nitrogen-fixing
activity is being expressed by the corresponding microorganisms, since
the nitrogenase genes are regulated at the transcriptional and
posttranslational levels (4, 12, 19). In fact, in spite of
the existence of remarkably diverse nifH sequences in the
gut microbial community, some termite species exhibit only a slight
level of nitrogen fixation activity (26). In order to
understand the nature of nitrogen fixation in termites, it is necessary
to clarify the real distribution and contribution of the nitrogen
fixation genes and the corresponding microorganisms.
Sequence analysis of PCR products amplified from mixed communities has
proved time-consuming and is potentially biased due to the requirement
for cloning prior to analysis. However, methods of resolving the
diversity of the amplified products in a single electrophoretic
profile, such as fluorescently labeled terminal-restriction fragment
length polymorphism (FLT-RFLP) analysis (9, 18) and
denaturing gradient gel electrophoresis (23), have been successfully developed to study natural microbial communities. FLT-RFLP
analysis, especially, is expected to be beneficial once the sequences
of the amplified products have been determined, since the identity and
affiliation of the profiles obtained are predictable. FLT-RFLP analysis
of PCR-amplified nifH genes has already been applied to
comparing the diazotrophic inhabitants in the gut among several termite
species (26). As stated above, the existence of the
nif genes does not always mean that biological activity is
being expressed. An elegant method of monitoring microbial activity in
situ would be detection of the mRNA. For this purpose, the reverse
transcription (RT)-PCR method is advantageous because of its high
sensitivity and specificity, and it has been used to detect mRNA in
environmental samples (5, 20, 36). These molecular
approaches depend on the PCR technique, and conventional PCR serves to
amplify target DNA exponentially, making it difficult to use the
technique in a quantitative manner. However, competitive PCR (15,
33, 37), in which an internal DNA standard has been added as a
control to correct for the variation among reactions, allows reliable
PCR quantification and has been applied to environmental samples
(2, 14, 17, 20, 29).
Biological nitrogen fixation is catalyzed by a nitrogenase complex
(12). A typical nitrogenase is a molybdenum (Mo)-containing enzyme encoded by the nifHDK operon. Mo-independent
nitrogenases have cofactors that coordinate vanadium in place of Mo (V
nitrogenase), or they have neither Mo nor vanadium (alternative
nitrogenase); these nitrogenases are encoded by the
vnfH-vnfDGK operon and the anfHDGK operon,
respectively. There is an especially high degree of sequence
conservation in the nifH, vnfH, and
anfH genes, and for this reason, the nifH gene is
usually used to detect nitrogen fixation genes in natural environments
(6, 26, 27, 34, 35, 38, 39). The genes within the single
operon are regulated simultaneously, but the three operons,
nif, vnf, and anf, are regulated
differentially. Although the transcription of all three types of
nitrogenase operons is strictly regulated by the availability of fixed
nitrogen, the availability of Mo differentially affects the expression
of nitrogenase genes at the transcriptional level (4, 19).
In the absence of Mo, the nif operon is repressed, whereas
in the presence of Mo, the vnf and anf operons
are repressed.
The dry-wood termite Neotermes koshunensis (order, Isoptera;
family, Kalotermitidae) shows high nitrogen fixation activity (26), and stable isotope measurements have shown that more
than half of the fixed nitrogen in the case of this termite is derived from atmospheric N2 (31). The presence of
diverse nifH genes has been demonstrated in the gut of
N. koshunensis (26). In this study, we examined
the expression of the nitrogen fixation genes in the gut microbial
community of N. koshunensis by culture-independent molecular
methods. The mRNA of nifH genes was detected by RT-PCR, and
the RT-PCR products were analyzed. A preferentially transcribed nitrogen fixation gene was characterized with respect to the regulation of its expression and the gene organization.
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MATERIALS AND METHODS |
Collection and culture of termites.
The termite N. koshunensis was collected in the Okinawa prefecture, Japan, in
October 1997, January 1998, and August 1998. Fifty to 100 termites were
fed a sterile diet as follows. One gram of filter paper (Toyo-roshi no.
5) was moistened with 0.6 ml of sterile water with or without 2%
(NH4)2SO4. Sodium molybdate was
added to the water solution at 1 mM, when appropriate. After 5 days,
the worker-like larvae were removed for DNA and RNA extraction and for
measurement of nitrogen fixation activity. Nitrogen fixation activity
was measured by the acetylene reduction assay as described previously
(26).
DNA and RNA extraction.
Approximately 30 termites were
collected, and after their exterior surfaces had been washed with
distilled water, their entire guts were removed with forceps and gently
squeezed. DNA from the intestinal mixed population was extracted as
described previously (25). Alternatively, the DNA was
extracted by using a DNA purification system (Qiagen) according to the
supplier's instructions. Total RNA was extracted from the gut mixed
population as follows. The gut microflora was treated with a solution
(0.5% sodium dodecyl sulfate, 20 mM sodium acetate, 10 mM EDTA;
adjusted to pH 5.5) to induce cell lysis, an equal volume of phenol
(equilibrated with the above solution) was added, and then the mixture
was incubated at 60°C for 10 min. After centrifugation, the aqueous
phase was transferred to a new tube, and then the total RNA was
precipitated with ethanol. The RNA was further purified by
precipitation with 2.5 M LiCl. Purified RNA was incubated with
RNase-free DNase I (Boehringer Mannheim) and with anti-RNase (Ambion,
Inc.). Residual DNase activity was heat inactivated at 95°C for 5 min.
RT.
The total RNA (10 µg) and 10 pmol of YAA primer
(27) in a total volume of 9 µl was heat denatured at
70°C for 10 min. The reverse transcriptase (RTase) used was
SuperScriptII RNase H
RTase (Life Technologies). After
heat denaturation, an RT reaction mixture was prepared as specified by
the manufacturer's instructions concerning the RTase. The RT mixture
was preheated to 42°C for 2 min, and then 1 µl (200 U) of RTase was
added. After incubation at 42°C for 60 min, the RTase was heat
inactivated at 70°C for 10 min and the reaction products were
precipitated by ethanol. The resulting cDNA was used as a template for
the subsequent PCR amplification.
PCR amplification for FLT-RFLP analysis.
The nifH
genes were amplified from the cDNA and from DNA extracted from the
termite gut by PCR with Ex Taq DNA polymerase (Takara)
according to the manufacturer's instructions. The PCR primer
corresponded to amino acid positions 11 to 16 (Klebsiella pneumoniae nifH numbering) in the case of the forward primer IGK (27) and positions 132 to 137 in the case of the reverse
primer VCG (5'-GCRAANCCNCCRCANAC-3'). The forward primer was
labeled at the 5' end with a fluorescent dye, Cy5. All of the primers used in this study were synthesized by and purchased from Pharmacia. When the IGK-YAA primer combination was used for PCR with the cDNA as
template, nonspecific amplification of sequences other than
nifH was observed. Thus, we used the internal VCG primer instead of the YAA primer. The reaction conditions were 35 cycles of
94°C for 30 s, 50°C for 45 s, and 72°C for 2 min. The
PCR products were purified on a low-melting-point agarose gel by means
of the Wizard PCR prep DNA purification system (Promega). The purified PCR products were digested with HhaI (Takara), and the
lengths of the fluorescently labeled terminal restriction fragments of the PCR products were determined by electrophoresis by using an automatic sequencer, ALFred Express (Pharmacia), and analyzed by means
of Fragment Manager software (Pharmacia).
Cloning of RT-PCR products and nucleotide sequencing.
The
primers used for amplification of the nifH gene from the
cDNA were IGK and YAA. The reaction conditions were as follows: 35 cycles of 94°C for 30 s, 50°C for 45 s, and 72°C for 2 min. The PCR products of the expected size were purified on a
low-melting-point agarose gel by means of the Wizard PCR prep DNA
purification system (Promega). Purified PCR products were cloned into
the vector pGEM-T (Promega) according to the manufacturer's
instructions. The insertion of DNA fragments of the appropriate sizes
was confirmed by PCR amplification with universal and reverse primers
(Takara) which corresponded to both sides of the cloning site on the
vector. The amplified product was digested with either HhaI
or RsaI, examined for RFLP, and then sorted. Plasmid DNA was
prepared from a clone representative of each RFLP group by means of the
Wizard mini prep DNA purification system (Promega) and used as a
template for sequencing performed by using the Dye Terminator Cycle
Sequencing Kit and an automatic sequence analyzer (Applied Biosystems
model 377). Sequences showing no relatedness to nifH and the
corresponding RFLP groups were excluded and not counted.
Quantitative PCR.
For the quantification of mRNA, a
competitive quantitative PCR approach (15, 33, 37) was
applied. Two primers, NKN-QQV (5'-CAACAGGTGTTCATACAC-3') and
NKN-THG (5'-CGTATAGGCTCCGTGCGT-3'), were used for specific
amplification of the nifH gene of the termite alternative
nif methanogen (anf-methano) cluster I. This
primer pair was tested for its specificity by using plasmid DNA from various nifH clones of termite gut origin as templates. For
the quantitative PCR, a competitor plasmid (pNK-N) was constructed as
follows. A 310-bp DNA region of 
phage DNA (nucleotide positions 5205 to 5514; database accession no. X00906) was PCR amplified with
primers QQVFIH-LAM
(5'-CAACAGGTGTTCATACACATACCGAGGCTGACGT-3') and
THGAYT-LAM
(5'-CGTATAGGCTCCGTGCGTGTTGAGGATCCCCATAA-3'),
which contain the sequences of the primers NK-QQV and NK-THG
(underlined). The PCR product was cloned into the vector pGEM-T
(Promega) according to the manufacturer's instructions.
A set of standard samples containing a certain amount of competitor DNA
(from 1 to 100 pg) was prepared. To each reaction mixture, a constant
amount of the cDNA was added. The reaction conditions were 35 cycles of
94°C for 30 s, 50°C for 45 s, and 72°C for 2 min. The
PCR products were separated on a 2% agarose gel, the gel was stained
with ethidium bromide, and the stained products were visualized by
means of a fluorescence imaging analyzer, FMBIO II Multi-view
(Hitachi). Quantification of the PCR products was performed by using
FMBIO Analysis, version 6.0 (Hitachi). To adjust for the difference in
fluorescence based on the size of the fragment, the intensity of the
internal control was multiplied by the ratio of the target size to the
competitor size (256/346). Standard curves for the competitive PCR were
made by using data from a series of 10-fold dilutions of a known amount
of the competitor pNK-N with 10 pg of plasmid DNA of the clone NKN4
used as the target. In ordinary experiments, cDNA corresponding to 2.6 µg of total RNA gave a linear curve and this curve was used for the quantification. When linearity was not obtained, the cDNA was diluted
and the cDNA corresponding to 0.5 µg of total RNA gave the linearity
and was used. The mRNA concentrations were expressed as femtomol per
milligram of total RNA, assuming that the RTase reaction was complete.
For the quantification of the genomic nifH gene, 50 pg of
total DNA of the microbial community was used.
Cloning, sequencing, and phylogenetic analysis of the nitrogenase
structural gene cluster.
The DNA region downstream of the
nifH gene in termite anf-methano cluster I was
isolated and characterized. Entire termite guts were squeezed in 0.4%
NaCl solution and used as a template for PCR. The primers used for the
amplification were NKN-QQV and ANFK (5'-GGYTGRCAXGTRAADATXGG-3').
The latter primer corresponded to a conserved amino acid sequence
in anfK (the Azotobacter vinelandii anfK amino
acid positions 16 to 22 [database accession no. M23528]). The
reaction conditions were 35 cycles of 94°C for 30 s, 50°C for
45 s, and 72°C for 2 min. The PCR products were purified on an
agarose gel and cloned into the vector pGEM-T. Deletion derivatives of
the clones were constructed by means of a kilosequence deletion kit
(Takara). The plasmid DNA was prepared and used as templates in
sequencing as described above. Open reading frames (ORFs) in the
determined nucleotide sequence were analyzed by means of GENETYX software (Software Development), and the amino acid sequences were
aligned by using the CLUSTAL W package (32). The PHYLIP version 3.5c phylogeny inference software package (13) was
used to infer the protein phylogeny. PROTDIST with the Dayhoff PAM matrix option was used to calculate evolutionary distances.
Phylogenetic trees were constructed from the evolutionary distance data
by the neighbor-joining method, implemented through the program
NEIGHBOR. A total of 100 bootstrapped replicate resampling data sets
for PROTDIST were generated with the program SEQBOOT to provide
confidence estimates for tree topologies.
Nucleotide sequence accession numbers.
The sequences
determined in this study will appear in the nucleotide sequence
databases under accession no. AB027742 to AB027751.
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RESULTS |
Comparison of the transcripts and genomic DNA of nifH
genes by FLT-RFLP.
In order to investigate the contribution of
each nifH gene in the gut of N. koshunensis to
nitrogen fixation in the termite, expression of the nifH
gene was investigated. The nifH mRNA was amplified from
total RNA extracted from the gut microflora by RT-PCR to obtain
products of the anticipated size. In the absence of the RTase reaction
step, no amplification was detected. This result indicated that the
RT-PCR could specifically detect nifH mRNA, and the products
were not derived from the genomic DNA.
The composition of the amplified nifH sequences was examined
by FLT-RFLP analysis comparing those derived from the nifH
mRNA and those derived from nifH genes in genomic DNA. As
shown in Fig. 1, the diversity of the
nifH genes in the DNA extracted from the gut mixed
population was confirmed. In contrast, only limited heterogeneity was
detected in the mRNA from the gut. The terminal restriction fragment
(T-RF) of 161 bases, especially, gave a strong fluorescent signal (more
than 60% of the total fluorescence intensity detected). However, the
fluorescence intensity of the 161-base-sized T-RF was less than 10% of
the total in the FLT-RFLP profile of the PCR products amplified from
the genomic DNA. Regardless of the length of time the termites were
kept in the laboratory, the feeding conditions, or the termite colonies
examined, the heterogeneity of the T-RFs detected in the RT-PCR
products was less than that in the case of the PCR products amplified
from genomic DNA. The T-RF of 161 bases was always a major component in
the FLT-RFLP profiles of the RT-PCR products. In the case of some
colonies, a T-RF of 54 bases also showed strong fluorescence intensity, up to 40% of the total (Fig.
2D). These results
suggested that among the diverse nifH genes in the termite
gut only a few represented by the T-RFs of 54 and 161 bases were
preferentially expressed.
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FIG. 1.
A comparison of the nifH constituents of the
RT-PCR and PCR products derived from the gut microflora of the termite
N. koshunensis. Electropherograms of the
HhaI-digested nifH genes amplified from the
genomic DNA by PCR (A) and from the mRNA by RT-PCR (B) are shown. The
termites were fed filter paper for 5 days before nucleic acid
extraction. The nifH genes were amplified with the
5'-fluorescently labeled primer IGK and the primer VCG. Numbers below
the electropherograms show base lengths.
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FIG. 2.
Effects of termite feeding conditions on nitrogen
fixation activity, the levels of nifH mRNA, and the amount
of DNA of the termite anf gene. The termites were shifted
from a diet of infested wood to filter paper moistened with distilled
water or filter paper moistened with an aqueous solution of either 1 mM sodium molybdate (+Mo column) or 2% ammonium sulfate
[+(NH4)2SO4 column]. (A)
C2H2 reduction activity of live termites. (B)
Quantification of the amount of mRNA of the termite anf
gene. The RT-PCR product was not detected (ND), and thus the amount of
mRNA was below the detection limit. (C) Quantification of the termite
anf gene in the genomic DNA of the gut microflora. Results
in panels A to C are means of two to six determinations, and the error
bars indicate standard deviations. (D) FLT-RFLP analysis of the
nifH RT-PCR products amplified from the mRNA of the gut
microflora. Since no amplification by RT-PCR was observed, the profile
for the termites fed filter paper with ammonium sulfate is not shown.
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Identification of the preferentially expressed genes.
Based on
the sequence information on nifH genes previously isolated
from the gut microbial community of N. koshunensis
(26), it is possible to predict the sequence identity for
most profiles obtained in the FLT-RFLP analysis. Among the isolated
nifH clones, only the sequences of termite
anf-methano cluster I were predicted to correspond to the
T-RF of 161 bases. The T-RF of 54 bases corresponds to the sequence of
either termite anf-methano cluster I or termite pseudo
nif cluster I.
In order to determine their exact identity, the RT-PCR products were
clonally isolated and analyzed. A termite colony showing a significant
amount of both the 54- and 161-base-sized T-RFs in the FLT-RFLP profile
of the RT-PCR products was used. The 21 nifH clones isolated
were sorted into nine RFLP groups, and representatives of these groups
were analyzed by nucleotide sequencing, to obtain five different amino
acid sequences. Table 1 shows the results of analysis of the RT-PCR clones. A large proportion of the clones (18 of the 21 clones) consisted of clones sharing close similarity to each
other, and these were affiliated with termite anf-methano cluster I. The predicted T-RF size of these sequences was either 54 or
161 bases. This finding was congruent with the results of FLT-RFLP
analysis of the RT-PCR products, although more of the 54-base-sized
T-RFs were isolated than in the case of the 161-base-sized T-RFs,
probably reflecting a difference in cloning efficiency. The results
indicated that the gene of termite anf-methano cluster I was
preferentially expressed and thus might be critical for nitrogen
fixation in N. koshunensis.
Quantification of mRNA and effect of termite feeding
conditions.
Since a gene belonging to the termite
anf-methano cluster I was suggested to be responsible for
the most abundant type of nifH transcripts in the gut
microbial community, its expression was investigated in a quantitative
manner. For quantification, competitive PCR targeting the product of RT
was applied. In order to detect the sequences specifically, specific
primers for the termite anf gene were designed. An internal
DNA standard (competitor) for the competitive PCR which contained the
same primer binding sites as the target and from which PCR products of
a different size were produced was constructed. The relative
amplification efficiency of the target compared to that of the
competitor was determined from a plot of the log ratio of target
intensity to competitor intensity against the log ratio of the
concentrations of target to input competitor. Coamplification of the
sequence from a clone of termite anf-methano cluster I with
a series of dilutions of the competitor resulted in a line with a slope
of 0.595 and a regression value of 0.983, indicating that the standard curve prepared could be used for quantification of samples within this range.
To investigate the relationship between nitrogen-fixing activity and
the amount of mRNA, the termite N. koshunensis was fed under
different conditions (Fig. 2). The termites were shifted from a diet of
infested wood to filter paper moistened with water with or without the
inclusion of a nitrogen source. The termites fed filter paper without
any added nitrogen source showed more than fivefold greater levels of
C2H2 reduction activity than the termites fed
wood (Fig. 2A). The filter paper probably contained a lower amount of
combined nitrogen than the wood. The termites fed the diet with a
nitrogen source added showed diminished activity, to the extent of
0.1-fold, as compared to those fed wood. A similar fluctuation of
nitrogen fixation activity has been demonstrated in the case of the
termite Coptotermes formosanus (8).
Quantitative analysis of the levels of mRNA expressed from the termite
anf gene showed that the total RNA extracted from termites fed filter paper contained a 2.2-fold higher concentration of this mRNA
than that from termites fed wood (Fig. 2B). In the case of feeding the
diet with a nitrogen source added, the level of mRNA expressed was
below the detection limit. The nitrogen-fixing activity and the amount
of mRNA from the termite anf gene were well correlated. Such
a correlation was also observed with another termite colony with a
different feeding regiment. Under these feeding conditions, mRNAs
derived from the termite anf gene were the major mRNA
species detected by RT-PCR and the following FLT-RFLP analysis (Fig.
2D). Thus, the amount of nitrogen fixation activity in N. koshunensis appears to be determined by the levels of mRNA expressed from the termite anf gene. As shown in Fig. 2D, a
minor signal corresponding to a T-RF of 43 bases, which was predicted to correspond to a nifH gene of termite pseudo
nif cluster I, was observed when the diet was wood. However,
in spite of the increased activity, the signal disappeared when the
termites were fed filter paper.
It is known that expression of nitrogenases of the anf group
is repressed at the transcriptional level in the presence of a trace
amount of Mo (4, 19). The effect of Mo on the levels of
nitrogen fixation activity and expression of the nifH gene was investigated. The termites were fed filter paper moistened with a
Mo-containing solution without any added nitrogen source. The
C2H2 reduction activity was slightly increased
(around 1.4-fold) compared with that of the termites fed filter paper
without Mo (Fig. 2A). Interestingly, the Mo in the diet had no effect
on the amount of mRNA detected (Fig. 2B). This finding indicates that
expression of the termite anf gene is independent of the presence of Mo, as opposed to the ordinary anf genes.
FLT-RFLP analysis of the mRNA produced in the presence of Mo revealed a significant amount (up to 50% of the total fluorescence intensity) of
expression of mRNA derived from a non-anf group of the
nifH sequences represented by T-RFs which were 266 and 307 bases in size (Fig. 2D). The corresponding nifH sequences
for both were predicted to be those affiliated with termite anaerobe
cluster III (26). This increased expression was probably
responsible for the slight increase in nitrogen-fixing activity observed.
Whether the population of microorganisms possessing the termite
anf gene had changed or not was examined by means of the
quantitative competitive PCR targeting these genes in genomic DNA
extracted from the gut microbial community. In contrast to the
C2H2 reduction activity and the amount of mRNA
detected, only a slight fluctuation of the nifH copy number
per unit amount of genomic DNA was observed (Fig. 2C). This result
indicates that the nitrogen fixation activity observed was not
influenced by a change in the population of microorganisms. Rather, the
activity observed reflected the expression level per cell in the
population of microorganisms.
Cloning and phylogenetic analysis of the anf gene
cluster.
The structural features of the nitrogenase gene cluster
which is responsible for nitrogen fixation in N. koshunensis
were further characterized. Since nifH, nifD, and
nifK usually occur in this order in nitrogenase operons, the
entire region encoded by nifD was amplified by PCR. One of
the primers used was specific for the nifH genes of termite
anf-methano cluster I, and the other was a degenerate primer
corresponding to a conserved region of nifK. A single band,
3.6 kb in size, was detected upon electrophoresis of the PCR products
and was then cloned. Nucleotide sequences corresponding to the
nifH region were determined for several clones. They showed
high sequence similarity, sharing more than 97% amino acid identity
and showing around 98% amino acid identity with the RT-PCR clone
NKN-RT11. The 3.6-kb DNA region of a clone designated anfK33 was
completely sequenced, and putative genes for the alternative nitrogenase were identified: anfH, ORF105, ORF122,
anfD, anfG, and anfK, in this order
(Fig. 3A).
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FIG. 3.
(A) Schematic representation of the genes identified in
the DNA region downstream of the termite anf gene. A 3.6-kb
DNA region of the clone anfK33 was completely sequenced, and
presumptive coding regions were identified by comparison with
nif operons in the DNA sequence databases. (B) Unrooted
phylogenetic tree for nifD amino acid sequences. The tree
was inferred from 201 unambiguously aligned amino acid positions by the
neighbor-joining method. Bootstrap values above 50 from 100 resamplings
are shown for each node. The scale bar denotes 0.1 substitutions per
site. Abbreviations, with accession numbers in parentheses: Ansp,
Anabaena sp. strain PCC7120 (V01482-2); Anvar,
Anabaena variabilis (L20472); Azchr-VNF, A. chroococcum (X51756); Azvin, A. vinelandii (X06886);
Azvin-VNF, A. vinelandii vnf (M32371); Azvin-ANF, A. vinelandii anf (M23528); Clhun-ANF, C. hungatei
(U59415); Clpas, C. pasteurianum (M21537); Clpas-ANF,
C. pasteurianum (L09762); Frsp, Frankia alni ArI3
(L41344); Heser, Herbaspirillum seropedicae (Z54207); Klpne,
K. pneumoniae (X07748); Mcmar, Methanococcus
maripaludis (U75887); Mbthe, Methanobacterium
thermoautotrophicum (X87971); Msbar, M. barkeri nifH2
(U11291); Rhcap, R. capsulatus (M15270); Rhcap-ANF, R. capsulatus anfD (X70033); Rhjap, Rhizobium japonicum
(X01045). (C) Unrooted tree for glnB, ORF105, and ORF12X (X
represents 2 to 8). The tree was inferred from 133 unambiguously
aligned amino acid positions by the neighbor-joining method. Bootstrap
values above 50 from 100 resamplings are shown for each node. The scale
bar denotes 0.1 substitutions per site. Abbreviations: Klpne, K. pneumoniae (X14012); Azvin, A. vinelandii (U91902);
Heser, H. seropedicae glnB (Z54207); Azbra,
Azospirillum brasilense (X51499); Brjap,
Bradyrhizobium japonicum BJ110d (M26753); Mbjan,
Methanobacterium jannassu (U67464); Clcel, C. cellobioparum (U59414); Mcthe, Methanococcus
thermolithotrophicus (X13830); Mbthe delta H, M. thermoautotrophicum delta H (X87971); Mbthe Marburg, M. thermoautotrophicum Marburg (AE00916); Msbar1, M. barkeri (X56072); Msbar2, M. barkeri (X56073); Mcmar,
Methanococcus maripaludis (U75887); Mbiva,
Methanobacterium ivanobii (X56071).
|
|
Phylogenetic analysis based on the sequence of nifD has
shown that Mo-independent nitrogenases form monophyletic groupings separate from the primary Mo nitrogenase (10, 11, 16). When the anfD sequence isolated here was included in the analysis
(Fig. 3B), it was clearly in a cluster together with the members of the
alternative nitrogenase group (Fe nitrogenases). A bootstrap value of
100% at the node supported this clustering. anfD in the isolated clone shared 84% amino acid identity with the anfD
genes of Clostridium pasteurianum and A. vinelandii, respectively. More than 78% amino acid identity of
the anfD gene in the isolated clone to other anfD
genes was evident, whereas it showed less than 62% and less than 44%
amino acid identity to the vnfD and nifD genes,
respectively. The genes for the third subunit of dinitrogenases, anfG and vnfG, are only known to be present in
the gene clusters encoding Mo-independent nitrogenases. The
anfG sequence in the isolated clone was the most similar to
anfG of C. pasteurianum (48% amino acid
identity), and it showed more than 38% amino acid sequence identity to
anfG from other organisms but less than 27% identity to the
product encoded by vnfG. Although the anfK
sequence was too short to characterize, the phylogenetic positions and sequence comparisons of anfD and anfG suggested
that the gene cluster representing termite anf-methano
cluster I may encode an alternative nitrogenase having neither Mo nor V
as a cofactor.
The isolated gene cluster was also found to contain two ORFs, ORF105
and ORF122, inserted between anfH and anfD that
showed significant amino acid similarity to the glnB-like
PII proteins. In enteric bacteria, glnB encodes the PII
protein which participates in the nitrogen regulatory cascade
(21). The amino acid sequences encoded by these ORFs were
grouped together with those of ORF105 and ORF12X (X represents 2 to 8)
reported in the nif gene cluster of the Archaea
domain. The products of ORF105 and ORF122, especially, showed the
highest amino acid identities, 61 and 55%, respectively, with those
located downstream of nifH1 of Methanosarcina
barkeri 227. The tyrosyl residue (amino acid position 49), the
site of uridylylation of the PII protein in enterobacteria
(21), was present in ORF105 but not in ORF122.
| |
DISCUSSION |
In the symbiotic microbial community in the gut of N. koshunensis, the anf gene affiliated with termite
anf-methano cluster I has been shown to be the most critical
gene responsible for nitrogen fixation in the termite. Transcripts of
the anf gene were the major mRNA species derived from
nifH detected in the gut community by RT-PCR, and the amount
of anf mRNA was well correlated with the level of nitrogen
fixation activity in the termite. The results of feeding experiments
performed under several conditions suggested that the level of
expression of the anf gene determines the level of nitrogen
fixation activity in the termite. Generally, in order to investigate a
certain biological activity in natural environments, a survey using
genes for key functional enzymes may be beneficial. However, as
described in this study, monitoring the expression of the genes
detected is necessary to evaluate their real contributions.
The results indicating that only the anf gene was
preferentially expressed are surprising because there are diverse
nifH sequences in the DNA extracted from the gut microbial
community of N. koshunensis. Other than the anf
gene, nifH genes affiliated with the anaerobe group and the
proteobacteria-cyanobacteria group were isolated (26).
However, only small amounts of the transcripts of these non-anf genes were detected by RT-PCR, and thus they seem to
be nonfunctional under the standard experimental conditions employed here. However, the transcription of some of these genes was induced when an excess amount of Mo was present (Fig. 2D). Simultaneously, nitrogen fixation activity also increased slightly but significantly, suggesting that these genes are functionable when they are expressed. Thus, the non-anf genes appear to encode potentially active
nitrogenases. These results imply that the metal supply may not be
enough to induce the expression of these non-anf genes under
natural states of the termite. On the other hand, not all the
non-anf genes were found to be induced in the presence of
Mo. There may be some reason, other than Mo availability, for the
repression of their expression.
The presence of diverse nifH sequences in the gut community
has been reported for various termites, and the nifH genes
of the anf group were not always present in all of the
termites investigated (26, 27). The existence of
anf genes can be explained simply as being due to inadequate
amounts of Mo in their diets, as the ordinary Mo-dependent nitrogenases
need Mo as a cofactor for expression of nitrogen-fixing activity. The
termite species possessing no anf genes in the gut community
can probably obtain sufficient Mo from their food.
Transcription of the anf gene in the gut community of the
termite was found to be repressed upon the addition of fixed nitrogen to the termite diet. The repression of transcription by fixed nitrogen
is a common feature of the regulation of nitrogenase expression. In
general, expression of anf genes is known to be repressed in
the presence of Mo. Depending on the availability of this metal as a
cofactor, expression of the Mo-dependent primary nitrogenase and
expression of the alternative nitrogenase are switched on and off at
the transcriptional level and are exchanged with each other, to express
the activity (4, 19). However, the regulation of expression
of the termite anf gene was found to be independent of the
presence of Mo (Fig. 2B). Since the amount of Mo added was excessive
(around 100-fold compared to the concentrations repressing ordinary
anf genes [4, 19]) and since the induction of some non-anf genes was observed, the Mo concentration in
the gut is believed to be sufficient to regulate gene expression. The
Mo-independent regulatory feature is unique to the anf gene in the termite symbiotic system. One possible explanation for the
Mo-independent expression is that the Mo availability may be limited in
the termite gut, and due to the long-term symbiotic relationship within
the termite gut, the gene may have lost the regulatory mechanism
sensing Mo. Alternatively, the symbiont possessing the anf
gene may have lost the gene encoding the Mo-dependent primary
nitrogenase, and thus, under diazotrophic conditions, the organisms
evolved to constitutively express the Mo-independent anf
nitrogenase regardless of the presence of Mo. Further analyses are
necessary to clarify these possibilities.
Some methanogenic archaea possess nifH genes affiliated with
the anf-methano group of nifH. However, these
nitrogenases contain Mo as a cofactor, and their nifD genes
are phylogenetically grouped with those of the Mo-dependent
nitrogenases (16) (database accession no. X87971). The
Mo-independent regulation of gene expression in the case of the termite
anf gene might suggest a Mo-dependent ordinary enzyme
encoded by it as in methanogens. However, this is probably not the case
for the anf gene cluster of the termite. The results of
sequencing and phylogenetic analysis indicated that anfH,
anfD, and anfG in the anf gene cluster
identified here encode an alternative nitrogenase that is both Mo and V independent.
The gene organization and sequence features of the termite
anf gene were found to be distantly related to those of
well-characterized organisms. Thus, information about the taxonomy of
organisms possessing the anf gene is limited. Except for the
presence of two ORFs homologous to glnB, the phylogenetic
character of anfD and anfG described here and
also that of anfH reported previously (26)
suggests that particular species in the domain Bacteria
possess the anf gene cluster. In the Archaea
domain, the presence of a Mo-independent alternative nitrogenase and
the existence of anfD and anfG orthologous genes
have never been reported. However, the anfH gene was
distantly related to any known bacterial anfH gene. In the
case of anfD analysis (Fig. 3B), taxonomy of the organism
possessing the termite anf gene could not be predicted
because anfD sequences of phylogenetically related organisms
did not form clusters within the Fe nitrogenase group (C. pasteurianum-Clostridium hungatei and A. vinelandii-Rhodobacter capsulatus). The presence of the two ORFs
between the nifH and nifD genes seems to be a
common feature within the Archaea domain (11,
30). All diazotrophic methanogenic archaea share this feature of
gene organization. However, the sequence homologous to ORF105 has
recently been reported for Clostridium cellobioparum (sequence database accession no. U59414). Because the nucleotide sequence of the DNA region corresponding to ORF122 has not yet been
reported, the existence of ORF122-like genes is now not certain. Nevertheless, the presence of ORF105 in the genome of an organism in
the Bacteria domain indicates that its presence is no longer characteristic of the Archaea domain. Still, the two ORFs in
the termite anf gene cluster are most closely related to
those of a methanogen, M. barkeri. An organism of
yet-uncharacterized diazotroph probably possesses the termite
anf gene. Further investigations are necessary to
taxonomically identify the organism that possesses the anf
gene and thus the organism most responsible for nitrogen fixation in
the termite.
The molecular approaches described in this study have been shown to be
powerful tools to investigate and characterize the expression of a
certain gene within a mixed microbial community. Of course, for more
detailed taxonomic, physiological, and genetic analyses, isolation and
cultivation of the responsible microorganisms are important. Our
preliminary attempts to isolate microorganisms harboring the
anf gene by enrichment, however, have failed, though a
variety of the enrichment conditions have been used (24). Although diazotrophic bacterial growth has been observed under some
conditions, it has never been shown whether the bacteria possess the
anf gene. Isolation of such bacteria is thought to be
difficult under ordinary conditions. An in situ hybridization technique
with a specific probe may be useful for identification of the cell
types expressing this gene (1, 22, 28). Even if isolation of
these cells is successful, the cultivation conditions may not represent
the natural state of the organisms, since complicated symbiotic
relationships with the termite and with other members of the community
may be involved. Mimicry of the environment within the termite gut in
vitro cannot be expected to be achieved. For these reasons,
culture-independent approaches are believed to be beneficial and
advantageous in order to understand the real nature of the microbial
community consisting of a mixed population.
| |
ACKNOWLEDGMENTS |
This work was partially supported by grants from the Biodesign
Research Program, the Genome Research Program, and the Eco Molecular
Science Research Program from RIKEN and by a grant from the
International Cooperative Research Project (Bio-Recycle Project) from
Japan Science and Technology Corporation. S.N. was supported by a grant
from the Junior Research Associate Program from RIKEN.
We thank I. Yasuda for advice on termite collection.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Microbiology
Laboratory, RIKEN (The Institute of Physical and Chemical Research),
Hirosawa 2-1, Wako, Saitama 351-0198, Japan. Phone: 81-48-462-1111, ext. 5724. Fax: 81-48-462-4672. E-mail:
mohkuma{at}mailman.riken.go.jp.
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Applied and Environmental Microbiology, November 1999, p. 4935-4942, Vol. 65, No. 11
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Abstract
Introduction
