Split dnaE Genes Encoding Multiple Novel Inteins in Trichodesmium erythraeum*
Xiang-Qin Liu
and
Jing Yang
From the
Department of Biochemistry & Molecular Biology, Dalhousie University,
Halifax, Nova Scotia B3H 4H7, Canada
Received for publication, May 13, 2003
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ABSTRACT
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Three inteins were found when analyzing a pair of split dnaE genes
encoding the catalytic subunit of DNA polymerase III in the oceanic
N2-fixing cyanobacterium Trichodesmium erythraeum. The
three inteins (DnaE-1, DnaE-2, and DnaE-3) were clustered in a 70-amino acid
(aa) region of the predicted DnaE protein. The DnaE-1 intein is 1258 aa long
and three times as large as a typical intein, due to the presence of large
tandem repeats in which a 57-aa sequence is repeated 17 times. The DnaE-2
intein has a more typical size of 428 aa with putative protein splicing and
endonuclease domains. The DnaE-3 intein is a split intein consisting of a
102-aa N-terminal part and a 36-aa C-terminal part encoded on the first and
second split dnaE genes, respectively. Synthesis of a mature DnaE
protein is predicted to involve expression of two split dnaE genes
followed by two protein cis-splicing reactions and one protein
trans-splicing reaction. Tandem repeats in the DnaE-1 intein
inhibited the protein splicing activity of this intein when tested in
Escherichia coli cells and may potentially regulate DnaE synthesis
in vivo.
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INTRODUCTION
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Inteins are protein sequences embedded in precursor proteins, and they
catalyze the protein-splicing reaction that excises the intein and joins the
flanking sequences (Nand C-exteins) with a peptide bond
(1). A typical protein splicing
reaction has 4 steps including N
S (or N
O) acyl shift at the N
terminus of intein, trans-esterification forming a branched
intermediate, cyclization of an Asn residue at the C terminus of intein, and
formation of a peptide bond between the flanking exteins
(24).
A typical intein is
400
aa1 long and
bi-functional, having both protein-splicing function and endonuclease
function. The two functions correspond to two separable structural domains of
the intein, in which the Nand C-terminal parts of intein make up the protein
splicing domain and the middle part of intein makes up the endonuclease domain
(5). The protein splicing
function ensures the self-removal of intein from the host protein, and the
endonuclease function promotes the maintenance and spread of the coding
sequence of intein via a gene conversion (intein homing) mechanism
(68).
Nearly 200 intein and intein-like sequences have been found, and most are
listed in the Intein Registry
(9,
10). These inteins are
distributed sporadically in many different proteins among bacteria, Archaea,
and eukaryotes, which is consistent with inteins being mobile genetic elements
capable of lateral gene transfers. Although inteins generally show low levels
of sequence conservation, they appear similar in overall structure, function,
and evolution origin
(1113).
Inteins were found most frequently in DNA-related proteins such as DNA
polymerase, although also found in many other types of proteins, and it is
possible that intein hotspots may exist in some proteins for yet unknown
reason (11,
13). Some inteins lack an
endonuclease domain and exist as mini-inteins with protein splicing function
only. A DnaE intein in some cyanobacterial species not only lacks an
endonuclease domain but also exists as a split intein with its Nand C-terminal
parts encoded on two separate genes. Synthesis of a mature Ssp DnaE
protein required the expression of the two split dnaE genes followed
by a protein trans-splicing reaction catalyzed by the split intein
(14). In this study we
analyzed the dnaE gene of the oceanic N2-fixing
filamentous cyanobacterium Trichodesmium erythraeum that forms
massive surface bloom (rapid cell proliferation) with important ecological
consequences (15,
16). Our analysis revealed
three inteins clustered in a small region of the DnaE protein, which included
a typical bi-functional intein, a split mini-intein, and an unusual intein
containing large tandem repeats that inhibited protein splicing.
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EXPERIMENTAL PROCEDURES
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Gene Cloning and AnalysisGenBankTM searches and
protein sequence alignments were performed using the BLAST search program
(17) and the Clustal W program
(18). Parts of the Ter
dnaE gene were amplified by doing polymerase chain reaction (PCR) from
total genomic DNA of T. erythraeum strain IMS101. The DnaE-1 intein
coding sequence was PCR-amplified using primers M144
(5'-ATGTCCTTCGTCGGTCYTCCATATC-3') and M145
(5'-ATCAATAAATCGCCTTCACATTGTAATC-3') and cloned in a pDrive
plasmid vector (Qiagen). To allow complete DNA sequence determination, nested
deletions were made in the cloned DNA by doing partial digestion at
XmnI restriction sites present once in each of the 171-bp repeat
sequences. In Southern blot analysis,
2 µgof T. erythraeum
genomic DNA was digested with restriction enzymes BfuAI and
BglII, resolved by electrophoresis in 1% agarose gel, and blotted
onto nylon membrane. The membrane was hybridized to a specified
32P-labeled DNA probe for 2024 h and washed under high
stringency (65 °C in 0.2x SSC and 1% SDS) before exposing x-ray
films.
Protein Splicing Analysis in Escherichia coli CellsTo
construct gene expression plasmids, Ter DnaE-1 intein coding sequence
(PCR-amplified using the above M144 and M145 primers) was inserted in the
previously made pMST plasmid
(19) between XhoI and
AgeI sites, replacing the Ssp DnaB intein coding sequence of
pMST'. Protein production in E. coli cells, gel electrophoresis
and Western blot analysis were performed as before
(19). Briefly, cells
containing the expression plasmid were grown in liquid Lurie Broth medium at
37 °C to late log phase (A600, 0.5).
isopropyl-1-thio-
-D-galactopyranoside was added to a final
concentration of 0.8 mM to induce production of the recombinant
protein, and the induction was continued for 3 h at 37 °C. Cells were then
harvested and lysed in SDSand dithiothreitol-containing gel loading buffer in
a boiling water bath before electrophoresis in SDS-polyacrylamide gel. Western
blots were carried out using anti-thioredoxin antibody (American Diagnostica)
and the enhanced chemiluminescence detection kit (ECL). Intensity of protein
band was estimated using a gel documentation system (Gel Doc 1000 coupled with
Molecular Analyst software, Bio-Rad).
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RESULTS
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Split dnaE Genes Contain Tandem RepeatsTo find new split
dnaE genes using our previous finding
(14), we searched the
GenBankTM containing the nearly complete genome sequence of T.
erythraeum strain IMS101 that had been determined at the Department of
Energy Joint Genome Institute
(www.jgi.doe.gov).
As illustrated in Fig. 1, parts
of putative split dnaE genes were found on three sequences with
accession numbers AAAU01000106, AAAU01000117, and AAAU01000050, respectively.
Our initial analysis indicated that the first split dnaE gene spans
an undetermined sequence gap between the first two GenBankTM sequences.
To determine this sequence gap, it was PCR-amplified from T.
erythraeum genomic DNA using primers specific to the two GenBankTM
sequences, and the resulting 4-kb DNA fragment was cloned
(Fig. 2). DNA sequence
determination of the cloned DNA produced the missing part of the first split
dnaE gene sequence and revealed large tandem repeats in which a
171-bp DNA sequence is repeated 17 times. To confirm the presence and size of
the tandem repeats in the T. erythraeum genome, Southern blot
analysis was performed after digesting the genomic DNA with BfuAI and
BglII restriction enzymes that cut outside the tandem repeats
(Fig. 2). A 0.3-kb DNA probe
next to the repetitive sequence hybridized to a BfuAI-BglII
DNA fragment that matched closely the 3216-bp size predicted from DNA
sequence. No DNA fragment of other sizes was detected, indicating a uniform
size of the tandem repeats in the cell population and their genomes. A 1.3-kb
DNA probe, which is specific to the tandem repeats, also hybridized to the
3.2-kb BfuAI-BglII DNA fragment as predicted. This DNA probe
did not hybridize to DNA fragment of other sizes, indicating that the 171-bp
repetitive sequence was not detectable in other parts of the genome under
these conditions. This is consistent with results of Blast searches of the
GenBankTM, in which we did not find the 171-bp repetitive sequence
outside the dnaE gene in the nearly complete genome sequence of
T. erythraeum.

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FIG. 1. Illustration of T. erythraeum split dnaE genes and
predicted protein products. Top, three solid lines (not drawn to
scale) represent three GenBankTM DNA sequences with accession numbers
AAAU01000106 (A-106), AAAU01000117 (A-117), and AAAU01000050
(A-050), respectively. The doted line connecting
A-106 and A-117 represents DNA sequence that was cloned and
determined in this study. Middle, the split dnaE genes
predicted two precursor proteins (drawn to scale) consisting of four exteins
(E1, E2, E3, and E4 (black boxes)) plus three inteins (open
boxes) marked as DanE-1 intein (including the tandem repeats), DnaE-2
intein, and DnaE-3 split intein. Bottom, predicted protein products
after two cis-splicing reactions and one trans-splicing
reaction, including a mature DnaE protein and the excised inteins.
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DnaE Protein Contains Multiple Novel InteinsThe above
findings revealed in T. erythraeum (Ter) a pair of split
dnaE genes that together encode a complete DnaE protein
(Fig. 1). The predicted
Ter DnaE protein contains three inteins named Ter DnaE-1,
Ter DnaE-2, and Ter DnaE-3 inteins, respectively. The three
inteins divide the DnaE protein sequence into four parts that are named E1,
E2, E3, and E4 exteins and are 719, 26, 44, and 481 aa long, respectively. The
predicted mature DnaE protein (1270 aa extein sequences, excluding the intein
sequences) is over 60% identical to DnaE proteins of two distantly related
cyanobacterial species, and this level of DnaE sequence identity is similar to
that between the other two cyanobacterial species. Blast searches of
GenBankTM sequences did not reveal additional dnaE-or
dnaE-like genes (complete or partial) in the nearly complete T.
erythraeum genome sequences. The three inteins in Ter DnaE
protein are clustered in a 70-aa region of the DnaE extein sequences, with the
DnaE-1, DnaE-2, and DnaE-3 inteins located after residues Leu719,
Arg745, and Tyr789, respectively, of the DnaE extein
sequences (Fig. 3A).
The extein-intein boundaries were readily defined, because the extein
sequences are very similar to DnaE proteins of other cyanobacteria and also
because of the conserved intein sequence features
(Fig. 3, B and
C). A Cys follows immediately the DnaE-1 intein in the
Ter DnaE protein, whereas Val is found at corresponding positions in
other DnaE proteins lacking the intein. Similarly, a Cys follows immediately
the DnaE-2 intein in the Ter DnaE protein, whereas Ala is found at
corresponding positions in other DnaE proteins lacking the intein. The DnaE-3
intein is followed by Cys in all three compared DnaE proteins containing the
intein. These are consistent with the requirement of a nucleophilic amino acid
(e.g. Cys) after an intein
(3) and may explain the absence
of DnaE-1 and DnaE-2 inteins in the other DnaE proteins.

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FIG. 3. Protein sequence comparisons. A, DnaE extein sequences of
T. erythraeum (Ter) are aligned with corresponding DnaE
sequences of Synechocystis sp. strain PCC6803 (Ssp) and
Nostoc punctiforme (Npu). Only extein sequences proximal to
the intein sequences are shown. Numbers mark amino acid positions in
the 1270-aa complete extein sequence (excluding the intein sequences) of
Ter DnaE protein. Names of the three inteins are shown in
parentheses, and they are positioned in the extein sequence after
amino acid residues Leu719, Arg745, and
Tyr789, respectively. B, sequence alignment of the T.
erythraeum DnaE-1 intein (Ter DnaE-1), the T.
erythraeum DnaE-2 intein (Ter DnaE-2), and the S. sp.
PCC6803 DnaB intein (Ssp DnaB). Tandem repeats
R1R17 in the Ter DnaE-1 sequence
represent the 17 57-aa repeating units. Consensus sequence of
R1R16 is shown in uppercase letters,
with sequence variations shown in lowercase letters. C, the T.
erythraeum DnaE-3 split intein sequence (Ter DnaE-3) is aligned
with the S. sp. PCC6803 DnaE split intein sequence (Ssp
DnaE), with the position of split indicated in parentheses. Putative
intein sequence motifs (A through H) are
underlined. Symbols: - represent gaps introduced to optimize the
alignment; * and. mark positions of identical and similar amino acids,
respectively.
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The Ter DnaE-1 intein has a predicted 1258-aa sequence consisting
of a 299-aa N-terminal sequence, a 969-aa repetitive sequence (17 tandem
repeats of a 57-aa sequence), and a 40-aa C-terminal sequence
(Fig. 3B). The
Ter DnaE-1 intein would be 339 aa long, after excluding the tandem
repeats, and has putative intein sequence motifs
(2022)
for a protein splicing domain and an endonuclease domain
(Fig. 3B), although it
showed less than 15% sequence identity to other known inteins. Presence of the
tandem repeats makes the Ter DnaE-1 intein three times as large as a
typical intein. The tandem repeats are located at or near the C-terminal
boundary between the putative endonuclease domain and the splicing domain.
Coding sequence of the tandem repeats is most likely translated, because its
2907-bp sequence is maintained as a continuous open reading frame with its
flanking coding sequences, while its non-coding frames contain numerous
termination codons as expected. Among the 17 tandem repeats, the first 16
(R1R16) are 91100% identical to each other
in DNA sequence and 88100% identical to each other in protein sequence,
and the last repeat (R17) differs from the others at the C-terminal
end (Fig. 3B).
The Ter DnaE-2 intein resembles typical bi-functional
self-splicing inteins, with a predicted 428-aa sequence that has putative
sequence motifs for both a protein splicing domain and an endonuclease domain
(Fig. 3B). The
Ter DnaE-2 intein is significantly similar to the Ssp DnaB
intein, showing 27% protein sequence identity and 46% protein sequence
similarity.
The Ter DnaE-3 intein is a split intein encoded by two split
dnaE genes found on two yet unlinked sequence contigs
(Fig. 1). These two split
dnaE genes could be on opposite DNA strands of the T.
erythraeum genome, as was the case in another cyanobacterium
(14), or they could be on the
same DNA strand and at least 56,449 bp apart, based on the available DNA
sequences. Predicted protein sequences of the DnaE-3 split intein consist of a
102-aa N-terminal part encoded on the first split dnaE gene and a
36-aa C-terminal part encoded on the second split dnaE gene (Figs.
1 and
3C). GTG was assumed
to be the start codon of the second split dnaE gene, based on the
absence of an upstream ATG codon or an appropriate down-stream ATG codon, and
also based on amino acid sequence comparisons with other DnaE split inteins
(Fig. 3C). The
Ter DnaE-3 split intein is 59% identical to the previously
characterized Ssp DnaE split intein, although the N-terminal part of
the Ter DnaE-3 split intein is 21 aa shorter than that of the Ssp
DnaE split intein (Fig.
3C).
Tandem Repeats in Ter DnaE-1 Intein Affect Protein
SplicingTo determine whether the Ter DnaE-1 intein is
functionally affected by the presence of the tandem repeats, we tested its
protein splicing activity in E. coli cells. A plasmid-borne fusion
gene was constructed to produce a fusion protein in which the Ter
DnaE-1 intein (plus its 5-aa native extein sequence on each side) was fused to
an N-terminal maltose-binding protein and a C-terminal thioredoxin
(Fig. 4). Similar fusion
protein containing other inteins has been used in previous studies
(19), so that the protein
splicing products are readily identified using SDS-polyacrylamide gel
electrophoresis and Western blotting. Precursor protein, spliced protein, and
excised intein were identified by their predicted sizes, and the first two
were further identified using anti-thioredoxin antibody. Variations of the
fusion gene were also made after deleting parts or all of the tandem repeats
coding sequence. When the complete tandem repeats (17 57-aa repeating units)
were present in the intein, only the precursor protein was observed at either
37 or 25 °C incubation temperature, indicating the absence of protein
splicing. When the number of the 57-aa repeating units was reduced to less
than 4, both spliced protein and excised intein were observed in addition to
the precursor protein, indicating that protein splicing had occurred. To
estimate the efficiency of protein splicing, intensity of individual protein
band on Western blot was used to measure the amount of that protein, and
protein splicing efficiency was calculated as the amount of spliced protein
divided by the sum of spliced protein and precursor protein. The efficiency of
protein splicing was estimated to be 73, 55, 50, and 45% for Ter
DnaE-1 intein containing 0, 1, 2, and 3 of the 57-aa repeating units,
respectively. Ter DnaE-1 intein containing 4 of the 57-aa repeating
units showed less than 5% protein splicing.

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FIG. 4. Protein splicing of Ter DnaE-1 intein. Top,
schematic illustration of the fusion protein construct consisting of
maltose-binding protein sequence (M), intein sequence (black
box), and thioredoxin sequence (T). Middle, predicted
sizes of protein products from different fusion protein constructs (numbered
as lanes 2 through 7) containing the specified number of the
57-aa repeating units (DnaE-1 intein, 0, 1, 2, 3, 4, and 17 repeats). Fusion
protein construct containing the Ssp DnaB mini-intein (numbered as
1) was included as a known standard for identifying the spliced
protein (19). Bottom,
observation of protein splicing. Total cellular proteins of E. coli
cells producing the specified fusion protein were resolved by SDS-PAGE and
visualized by Coomassie Blue staining (left panel) or Western blot
using anti-thioredoxin antibody (right panel). The lane numbers
correspond to the above fusion protein construct numbers. Position of the
precursor protein is marked by the letter P, the spliced protein by
the letter S, and the excised intein by a black dot.
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DISCUSSION
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Split dnaE Genes and Multiple InteinsWe have shown that two
split dnaE genes together encode a DnaE protein containing three
inteins in T. erythraeum, predicting that the synthesis of mature
DnaE protein involves expression of the two split dnaE genes followed
by two protein cis-splicing reactions and one protein
trans-splicing reaction. The predicted mature DnaE protein, after
excluding the inteins, is very similar to known DnaE proteins of related
organisms in size and sequence, and no other dnaE-like gene was found
in the nearly complete genome sequence of T. erythraeum. The three
inteins were readily identified in the Ter DnaE protein, because they
are insertion sequences showing clear boundaries, conserved intein sequence
motifs, and significant sequence similarities to known inteins. The DnaE-1 and
DnaE-2 inteins may be relatively recent acquisitions in the Ter DnaE
protein, because they were found only in T. erythraeum, and they have
putative endonuclease domain for intein homing. Sequence similarities (27%
identical and 46% similar) between Ter DnaE-2 intein and the
cyanobacterial Ssp DnaB intein are significantly higher than those
seen among non-homologous inteins
(12,
23), which may suggest a
common ancestor for these two inteins, although their host proteins or
insertion sites have no apparent similarity. The Ter DnaE-1 intein is
of unknown origin, having very low sequence similarity to other known inteins
even after excluding the tandem repeats. The Ter DnaE-3 split intein,
present in at least two other cyanobacterial species
(9), likely has an ancient
origin preceding the divergence of the distantly related cyanobacterial
species. This split intein is least likely to spread through intein homing or
lateral gene transfer, because it lacks an endonuclease domain and is encoded
on split genes. The DnaE-1, DnaE-2, and DnaE-3 inteins are clustered in a
small (70 aa) region of the large (1270 aa, excluding the inteins) DnaE
protein, suggesting that this small region may be a hotspot for intein
insertions. Intein hotspots may also exist in other proteins, such as a DNA
replication factor protein in which three inteins (Mja RFC-1, RFC-2, and RFC-3
inteins) were found in an 87-aa region
(9). The Ter DnaE
protein is unique in that its three inteins include both cis-splicing
inteins and trans-splicing split intein.
Tandem Repeats Inside InteinThe Ter DnaE-1 intein
is most unusual with the presence of large tandem repeats, which was revealed
in sequence determination of the cloned DNA and confirmed in Southern blot
analysis of the genome. This is the first repetitive sequence found inside an
intein. The tandem repeats are strikingly large (57 aa repeated 17 times),
unlike the single amino acid repeats associated with Huntington's disease
(24). Also unlike typical
repetitive sequences that are dispersed in many parts of the genome and often
not translated, the tandem repeats of Ter DnaE-1 intein were
translated and not detected in other parts of the T. erythraeum
genome in GenBankTM searches and in Southern blot analysis. The tandem
repeats may have originated from duplications of a pre-existing piece of the
intein sequence, although they did not show similarity to known intein
sequences and were not required for the protein splicing function of the
intein. Alternatively, the tandem repeats may have originated from exogenous
sequences that entered the Ter DnaE-1 intein. Self-splicing inteins
have been thought as "safe havens" for homing endonucleases
present in most inteins, because the ability of self-removal through protein
splicing of the intein makes them tolerated by the host protein. The
Ter DnaE-1 intein can be such a safe haven to the tandem repeats, if
the latter were selfish genetic elements of unknown function. The Ter
DnaE-1 intein could also be a "waste bin," if the tandem repeats
had no function at all.
Possible Function of the Tandem RepeatsPerhaps more likely,
the tandem repeats may play a biological role in T. erythraeum, which
would provide a reason for this organism to maintain the tandem repeats.
Tandem repeats are prone to changes in length, because of polymerase slippage
in DNA replication and unequal crossing over in DNA recombination. Tandem
repeats of Ter DnaE-1 intein exhibited uniform size in PCR and
Southern blot analysis, which may suggest unknown mechanism or reason for
maintaining the number of the 57-aa repeating units at 17. Individual
repeating units of the tandem repeats showed less than 10% DNA sequence
heterogeneity and less than 12% protein sequence heterogeneity, which may also
suggest unknown mechanism or reason for maintaining the tandem repeats.
Different tandem repeats in a number of other proteins have been known to play
structural or ligand/substrate-binding roles, which include examples like
mucin, cell surface proteins, and proteins containing spectrin repeats
(2527).
Our results suggested that the tandem repeats of Ter DnaE-1 intein
may regulate the protein splicing activity of this intein, because protein
splicing in E. coli cells was inhibited when 4 or more of the 57-aa
repeating units were present in the intein. This may predict a regulatory
mechanism in the native T. erythraeum cell that overcomes the
inhibition of protein splicing by the tandem repeats or reduces the number of
the 57-aa repeating units to less than 4, to produce a mature DnaE protein
(catalytic subunit of DNA polymerase III). The predicted regulatory mechanism
may regulate DNA polymerase III synthesis and in turn DNA replication and cell
proliferation. Interestingly, the N2-fixing T. erythraeum
is known to form massive ocean surface bloom (rapid cell proliferation) with
important ecological consequences
(15,
16), although it is not known
what controls the bloom, and rapid DNA replication must accompany the rapid
cell proliferation.
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FOOTNOTES
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* This work was supported by research grants from the National Science and
Engineering Research Council of Canada and from the Canadian Institute of
Health Research. The costs of publication of this article were defrayed in
part by the payment of page charges. This article must therefore be hereby
marked "advertisement" in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact. 
To whom correspondence should be addressed: Dept. of Biochemistry &
Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada.
Tel.: 902-494-1208; Fax: 902-494-1355; E-mail:
pxqliu{at}dal.ca.
1 The abbreviation used is: aa, amino acid(s). 
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ACKNOWLEDGMENTS
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We thank Dr. J. B. Waterbury for the gift of T. erythraeum cells
used in this study.
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