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Journal of Bacteriology, December 2000, p. 6913-6920, Vol. 182, No. 24
Centre de Recherche en Infectiologie, Centre
Hospitalier Universitaire de Québec (Pavillon CHUL), Sainte-Foy,
Québec G1V 4G2,1 and Division de
Microbiologie, Faculté de Medicine,2 and
Département de Biochimie et de Microbiologie,
Faculté des Sciences et de Genie,3
Université Laval, Sainte-Foy, Québec G1K 7P4, Canada
Received 23 June 2000/Accepted 26 September 2000
The elongation factor Tu, encoded by tuf genes, is a
GTP binding protein that plays a central role in protein synthesis. One to three tuf genes per genome are present, depending on the
bacterial species. Most low-G+C-content gram-positive bacteria carry
only one tuf gene. We have designed degenerate PCR primers
derived from consensus sequences of the tuf gene to amplify
partial tuf sequences from 17 enterococcal species and
other phylogenetically related species. The amplified DNA fragments
were sequenced either by direct sequencing or by sequencing cloned
inserts containing putative amplicons. Two different tuf
genes (tufA and tufB) were found in 11 enterococcal species, including Enterococcus avium, Enterococcus casseliflavus, Enterococcus
dispar, Enterococcus durans, Enterococcus
faecium, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus
mundtii, Enterococcus pseudoavium, and
Enterococcus raffinosus. For the other six enterococcal species (Enterococcus cecorum, Enterococcus
columbae, Enterococcus faecalis, Enterococcus
sulfureus, Enterococcus saccharolyticus, and
Enterococcus solitarius), only the tufA gene
was present. Based on 16S rRNA gene sequence analysis, the 11 species
having two tuf genes all have a common ancestor, while the
six species having only one copy diverged from the enterococcal lineage
before that common ancestor. The presence of one or two copies of the tuf gene in enterococci was confirmed by Southern
hybridization. Phylogenetic analysis of tuf sequences
demonstrated that the enterococcal tufA gene branches with
the Bacillus, Listeria, and
Staphylococcus genera, while the enterococcal
tufB gene clusters with the genera Streptococcus and Lactococcus. Primary
structure analysis showed that four amino acid residues encoded within
the sequenced regions are conserved and unique to the enterococcal
tufB genes and the tuf genes of streptococci
and Lactococcus lactis. The data suggest that an ancestral
streptococcus or a streptococcus-related species may have horizontally
transferred a tuf gene to the common ancestor of the 11 enterococcal species which now carry two tuf genes.
The elongation factor Tu (EF-Tu) is
a GTP binding protein playing a central role in protein synthesis. It
mediates the recognition and transport of aminoacyl-tRNAs and their
positioning to the A site of the ribosome (20). The highly
conserved function and ubiquitous distribution render the elongation
factor a valuable phylogenetic marker among eubacteria and even
throughout the archaebacterial and eukaryotic kingdoms (3,
31). The tuf genes encoding EF-Tu are present in
various copy numbers per bacterial genome. Most gram-negative bacteria
contain two tuf genes (5, 15, 19, 39, 41, 43). As
found in Escherichia coli, the two genes, while being almost
identical in sequence, are located in different parts of the bacterial
chromosome (15, 20, 41). However, recently completed maps of
microbial genomes revealed that only one tuf gene is found
in Helicobacter pylori as well as in some obligate parasitic
bacteria, such as Borrelia burgdorferi, Rickettsia prowazekii, and Treponema pallidum, and in some
cyanobacteria (16, 18, 24, 32, 41, 44). In most
gram-positive bacteria studied so far, only one tuf gene was
found (8, 14, 17, 22, 28-30, 32, 35, 39). However, Southern
hybridization showed that there are two tuf genes in some
clostridia (39) as well as in Streptomyces
coelicolor and Streptomyces lividans (46,
47). Up to three tuf-like genes have been identified in Streptomyces ramocissimus (48).
Although massive prokaryotic gene transfer is suggested to be one of
the factors responsible for the evolution of bacterial genomes
(12, 27, 42), the genes encoding components of the translation machinery are thought to be highly conserved and difficult to transfer horizontally due to the complexity of their interactions (23). However, a few recent studies demonstrated evidence
that horizontal gene transfer has also occurred in the evolution of some genes coding for the translation apparatus, namely, 16S rRNA and
some aminoacyl-tRNA synthetases (6, 27, 45, 48, 49). No
further data suggest that such a mechanism is involved in the evolution
of the elongation factors. Previous studies concluded that the two
copies of tuf genes in the genomes of some bacteria resulted
from an ancient event of gene duplication (10, 39). Moreover, a study of the tuf gene in R. prowazekii suggested that intrachromosomal recombination has taken
place in the evolution of the genome of this organism (41).
To date, little is known about the tuf genes of enterococcal
species. In this study, we analyzed partial sequences of tuf genes in 17 enterococcal species, namely, Enterococcus
avium, E. casseliflavus, E. cecorum,
E. columbae, E. dispar, E. durans, E. faecalis, E. faecium, E. gallinarum, E. hirae, E. malodoratus, E. mundtii, E. pseudoavium, E. raffinosus, E. saccharolyticus, E. solitarius, and E. sulfureus. We report here the
presence of two divergent copies of tuf genes in 11 of these
enterococcal species. The six other species carried a single
tuf gene. The evolutionary implications are discussed.
(This study was presented in part at the 100th General Meeting of the
American Society for Microbiology, Los Angeles, Calif., 21 to 25 May
2000.)
Bacterial strains.
Seventeen enterococcal strains and other
gram-positive bacterial strains obtained from the American Type Culture
Collection (ATCC; Manassas, Va.) were used in this study (Table
1). All strains were grown on sheep blood
agar or in brain heart infusion broth prior to DNA isolation.
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Evidence for Horizontal Gene Transfer in Evolution
of Elongation Factor Tu in Enterococci
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
tuf gene sequences obtained in our laboratory
DNA isolation. Bacterial DNAs were prepared using the G NOME DNA extraction kit (Bio101, Vista, Calif.) as previously described (25).
Sequencing of putative tuf genes. In order to obtain the tuf gene sequences of enterococci and other gram-positive bacteria, two sequencing approaches were used: (i) sequencing of cloned PCR products and (ii) direct sequencing of PCR products. A pair of degenerate primers (U1, 5'-AAYATGATIACIGGIGCIGCICARATGGA-3', and U3, 5'-CCIACIGTICKICCRCCYTCRCG-3') were used to amplify an 886-bp portion of the tuf genes from enterococcal species and other gram-positive bacteria as previously described (25). For E. avium, E. casseliflavus, E. dispar, E. durans, E. faecium, E. gallinarum, E. hirae, E. mundtii, E. pseudoavium, and E. raffinosus, the amplicons were cloned using the Original TA cloning kit (Invitrogen, Carlsbad, Calif.) as previously described (25). Five clones for each species were selected for sequencing. For E. cecorum, E. faecalis, E. saccharolyticus, and E. solitarius as well as the other gram-positive bacteria, the sequences of the 886-bp amplicons were obtained by direct sequencing. Based on the results obtained from the earlier rounds of sequencing, two pairs of primers were designed for obtaining the partial tuf sequences from the other enterococcal species by direct sequencing. One pair of primers (EntA1, 5'-ATCTTAGTAGTTTCTGCTGCTGA-3', and EntA2, 5'-GTAGAATTCAGGACGGTAGTTAG-3') was used to amplify the enterococcal tuf gene fragments from E. columbae, E. malodoratus, and E. sulfureus. Another pair of primers (U1 and EntB, 5'-GTAGAAYTGTGGWCGATARTTRT-3') was used to amplify the second tuf gene fragments from E. avium, E. malodoratus, and E. pseudoavium.
Prior to direct sequencing, PCR products were electrophoresed on a 1% agarose gel at 120 V for 2 h. The gel was then stained with 0.02% methylene blue for 30 min and washed twice with autoclaved distilled water for 15 min. The gel slices containing PCR products of the expected sizes were cut out and purified with the QIAquick gel extraction kit (QIAgen Inc., Mississauga, Ontario, Canada) according to the manufacturer's instructions. PCR mixtures for sequencing were prepared as described previously (25). DNA sequencing was carried out with the Big Dye Terminator Ready Reaction cycle sequencing kit using a 377 DNA sequencer (PE Applied Biosystems, Foster City, Calif.). Both strands of the amplified DNA were sequenced. The sequence data were verified using the Sequencher 3.0 software (Gene Codes Corp., Ann Arbor, Mich.).Sequence analysis and phylogenetic study.
Nucleotide
sequences of the tuf genes and their respective flanking
regions in E. faecalis, Staphylococcus
aureus, and Streptococcus pneumoniae were retrieved
from The Institute for Genomic Research (http://www.tigr.org) microbial
genome database, and sequences of Streptococcus pyogenes
were obtained from the University of Oklahoma database
(http://www.genome.ou.edu/strep.html). DNA sequences and deduced
protein sequences obtained in this study were compared with those in
all publicly available databases by using the BLAST (2) and
FASTA programs. Unless specified, sequence analysis was conducted with
the programs from the GCG package (version 10; Genetics Computer Group,
Madison, Wis.). Sequence alignment of the tuf genes from 74 species representing all three kingdoms of life (Tables 1 and
2) was carried out by use of Pileup and was corrected upon visual analysis. The N- and C-terminus extremities of the sequences were trimmed to yield a common block of 201 amino acids, and equivocal residues were removed. Phylogenetic analysis was
performed with the aid of PAUP 4.0b4, written by David L. Swofford
(Sinauer Associates, Inc., Publishers, Sunderland, Mass.). The distance
matrix and maximum parsimony were used to generate phylogenetic trees,
and bootstrap resampling procedures were performed, using 500 and 100 replications in each analysis, respectively.
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Protein structure analysis. The crystal structures of (i) Thermus aquaticus EF-Tu in complex with Phe-tRNAPhe and a GTP analog (34) and (ii) E. coli EF-Tu in complex with GDP (40) served as templates for constructing the equivalent models for enterococcal EF-Tu. Homology modeling of protein structure was performed using the SWISS-MODEL server and inspected using the SWISS-PDB viewer version 3.1 (21).
Southern hybridization. In a previous study (25), we amplified and cloned an 803-bp PCR product of the tuf gene fragment from E. faecium. Two divergent sequences of the inserts, which we assumed to be tufA and tufB genes, were obtained. The recombinant plasmid carrying either tufA or tufB sequence was used to generate two probes labeled with digoxigenin (DIG)-11-dUTP by PCR incorporation following the instructions of the manufacturer (Boehringer Mannheim, Laval, Québec, Canada). Enterococcal genomic DNA samples (1 to 2 µg) were digested to completion with restriction endonucleases BglII and XbaI as recommended by the supplier (Amersham Pharmacia Biotech, Mississauga, Ontario, Canada). These restriction enzymes were chosen because no restriction sites were observed within the amplified tuf gene fragments of most enterococci. Southern blotting and filter hybridization were performed using positively charged nylon membranes (Boehringer Mannheim) and QuikHyb hybridization solution (Stratagene Cloning Systems, La Jolla, Calif.) according to the manufacturers' instructions, with modifications. Twenty microliters of each digest was electrophoresed for 2 h at 120 V on a 0.8% agarose gel. The DNA fragments were denatured with 0.5 M NaOH and transferred by Southern blotting onto a positively charged nylon membrane (Boehringer Mannheim). The filters were prehybridized for 15 min and then were hybridized for 2 h in the QuikHyb solution at 68°C with either DIG-labeled probe. Posthybridization washings were performed twice with 0.5× SSC-1% sodium dodecyl sulfate (SDS) at room temperature for 15 min and twice in the same solution at 60°C for 15 min (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Detection of bound probes was achieved using disodium 3-(4-methoxyspiro(1,2-dioxetane-3,2'-(5'-chloro)tricyclo(3,3.1.13.7)decan)-4-yl) phenyl phosphate (CSPD) (Boehringer Mannheim) as specified by the manufacturer.
Nucleotide sequence accession numbers. The GenBank accession numbers for partial tuf gene sequences generated in this study are given in Table 1. Sequences were assigned accession no. AF124220 to AF124224 and AF274715 to AF274747.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Sequencing and nucleotide sequence analysis. In this study, all gram-positive bacteria other than enterococci yielded a single tuf sequence of 886 bp using primers U1 and U3 (Table 1). Each of the four enterococcal species E. cecorum, E. faecalis, E. saccharolyticus, and E. solitarius also yielded one 886-bp tuf sequence. On the other hand, for E. avium, E. casseliflavus, E. dispar, E. durans, E. faecium, E. gallinarum, E. hirae, E. mundtii, E. pseudoavium, and E. raffinosus, direct sequencing of the 886-bp fragments revealed overlapping peaks according to their sequence chromatograms, suggesting the presence of additional copies of the tuf gene. Therefore, the tuf gene fragments of these 10 species were cloned first and then sequenced. Sequencing data revealed that two different types of tuf sequences (tufA and tufB) are found in eight of these species, namely, E. casseliflavus, E. dispar, E. durans, E. faecium, E. gallinarum, E. hirae, E. mundtii, and E. raffinosus. Five clones of both E. avium and E. pseudoavium yielded only a single tuf sequence. These new sequence data allowed the design of new primers specific for the enterococcal tufA or tufB sequences. Primers EntA1 and EntA2 were designed to amplify only enterococcal tufA sequences, and a 694-bp fragment was amplified from all 17 enterococcal species. The 694-bp sequences of tufA genes from E. columbae, E. malodoratus, and E. sulfureus were obtained by direct sequencing using these primers. Primers U1 and EntB were designed for the amplification of 730-bp portion of tufB genes and yielded the expected fragments from 11 enterococcal species, including E. malodoratus and the 10 enterococcal species in which heterogeneous tuf sequences were initially found. The sequences of the tufB fragments for E. avium, E. malodoratus, and E. pseudoavium were determined by direct sequencing using the primers U1 and EntB. Overall, tufA gene fragments were obtained from all 17 enterococcal species but tufB gene fragments were obtained from only 11 enterococcal species (Table 1).
The identities between tufA and tufB for each enterococcal species were 68 to 79% at the nucleotide level and 81 to 89% at the amino acid level. The tufA gene is highly conserved among all enterococcal species, with identities ranging from 87 to 99% for DNA and 93 to 99% for amino acid sequences, while the identities among tufB genes of enterococci ranged from 77 to 92% for DNA and from 91 to 99% for amino acid sequences, indicating their different origins and evolution (Table 3). Since E. solitarius has been transferred to the genus Tetragenococcus (13), which is also a low-G+C-content gram-positive bacterium, our sequence comparison did not include this species as an enterococcus. The G+C content of enterococcal tufA sequences ranged from 40.8 to 43.1%, while that of enterococcal tufB sequences ranged from 37.8 to 46.3%. Based on amino acid sequence comparison, the enterococcal tufA gene products shared higher identities with those of Abiotrophia adiacens, Bacillus subtilis, Listeria monocytogenes, S. aureus, and Staphylococcus epidermidis. On the other hand, the enterococcal tufB gene products shared higher percentages of amino acid identity with the tuf genes of S. pneumoniae, S. pyogenes, and Lactococcus lactis (Table 3).
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-helices and two 
-strands
of domain I, the entire domain II, and the N-terminal part of domain
III on the basis of the determined structures of E. coli
EF-Tu (40).
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-strand of
domain II. This region is partially responsible for the interaction
between the EF-Tu and aminoacyl-tRNA by the formation of a deep pocket
for any of the 20 naturally occurring amino acids (34, 40).
According to our three-dimensional model (data not illustrated), the
substitution Thr230
Ser in domain II of EF-Tu may have little
impact on the ability of the pocket to accommodate any amino acid.
However, the high conservation of Thr230 compared to the unique Ser
substitution found only in streptococci and 11 enterococci could
suggest a subtle functional role for this residue.
The tuf gene sequences obtained for
E. faecalis, S. aureus, S. pneumoniae, and S. pyogenes were compared with
their respective incomplete genome sequences
(http://www.tigr.org/tdb/mdb/mdbinprogress.html). Contigs with greater
than 99% identity were identified. Analysis of the E. faecalis genome data revealed that the single E. faecalis tuf gene is located within an str operon in which
tuf is preceded by fus, which encodes the
elongation factor G. This str operon is present in S. aureus and B. subtilis but not in the two streptococcal genomes examined. The 700-bp or so sequence upstream of the S. pneumoniae tuf gene has no homology with any known gene sequences. In S. pyogenes, the gene upstream of tuf is
similar to a cell division gene, ftsW, suggesting that the
tuf genes in streptococci are not arranged in an
str operon.
Phylogenetic analysis.
Phylogenetic analysis of the
tuf amino acid sequences with representatives of eubacteria,
archaebacteria, and eukaryotes using neighbor-joining and maximum
parsimony methods showed three major clusters representing the three
kingdoms of life. Both methods yielded similar topologies consistent
with the rRNA gene data (data not shown). Within the bacterial clade,
the tree is polyphyletic, but tufA genes from all
enterococcal species always clustered with those from other
low-G+C-content gram-positive bacteria (except for streptococci and
lactococci), while the tufB genes of the 11 enterococcal
species form a distinct cluster with streptococci and L. lactis (Fig. 2). Duplicated genes
from the same organism did not cluster together, thereby not suggesting
evolution by recent gene duplication.
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Southern hybridization.
Southern hybridization of
BglII-XbaI-digested genomic DNA from 12 enterococcal species tested with the tufA probe (DIG-labeled tufA fragment from E. faecium) yielded
two bands of different sizes in nine species, which also carried two
divergent tuf sequences according to their sequencing data.
For E. faecalis and E. solitarius, a single band
was observed, indicating that one tuf gene is present (Fig.
3). A single band was also found when
digested genomic DNAs from S. aureus, S. pneumoniae, and S. pyogenes were hybridized with the
tufA probe (data not shown). For E. faecium, the
presence of three bands can be explained by the existence of an
XbaI restriction site in the middle of the tufA
sequence, which was confirmed by sequencing data. Hybridization with
the tufB probe (DIG-labeled tufB fragment of
E. faecium) showed a banding profile similar to the one
obtained with the tufA probe (data not shown).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this study, we have shown that two divergent copies of genes encoding EF-Tu are present in some enterococcal species. Sequence data revealed that both genes are highly conserved at the amino acid level. One copy (tufA) is present in all enterococcal species, while the other (tufB) is present in only 11 of the 17 enterococcal species studied. Based on 16S rRNA sequence analysis, these 11 species are members of three different enterococcal subgroups (E. avium, E. faecium, and E. gallinarum species groups) and a distinct species (E. dispar). Moreover, 16S rDNA phylogeny suggests that the 11 species that possess two tuf genes all have a common ancestor from which they evolved further to become the current species (36). Since the six other species having only one copy diverged from the enterococcal lineage before that common ancestor, it appears that the presence of one tuf gene in these six species is not attributable to gene loss.
Two clusters of low-G+C-content gram-positive bacteria were observed in the phylogenetic tree of the tuf genes: one contained a majority of low-G+C-content gram-positive bacteria and the other contained lactococci and streptococci. This is similar to a previous finding based on phylogenetic analysis of the 16S rRNA gene and the hrcA gene coding for a unique heat shock regulatory protein (1). The enterococcal tufA genes branched with most of the low-G+C-content gram-positive bacteria, suggesting that they originated from a common ancestor. On the other hand, the enterococcal tufB genes branched with the genera Streptococcus and Lactococcus, which form a distinct lineage separated from other low-G+C-content gram-positive bacteria (Fig. 2). The finding that these EF-Tu proteins share some conserved amino acid residues unique to this branch also supports the idea that they may have a common ancestor. Although these conserved residues might result from convergent evolution upon a specialized function, such convergence at the sequence level, even for a few residues, seems to be rare, making it an unlikely event. Moreover, no currently known selective pressure, if any, would account for keeping one versus two tuf genes in bacteria. The G+C contents of enterococcal tufA and tufB sequences are similar, indicating that they both originated from low-G+C-content gram-positive bacteria, in accordance with the phylogenetic analysis.
The tuf genes are present in various copy numbers in different bacteria. Furthermore, the two tuf genes are normally associated with characteristic flanking genes (10). The two tuf gene copies commonly encountered within gram-negative bacteria are part of either the bacterial str operon or the tRNA-tufB operon (5, 10, 41). The arrangement of tufA in the str operon was also found in a variety of bacteria, including Thermotoga maritima, the earliest divergent bacterium sequenced so far (33), Aquifex aeolicus (11), cyanobacteria (7, 24), Bacillus spp. (28, 29), Micrococcus luteus (35), Mycobacterium tuberculosis (9), and Streptomyces spp. (46, 47). Furthermore, the tRNA-tufB operon has also been identified in A. aeolicus (11), Thermus thermophilus (38), and Chlamydia trachomatis (10). The two widespread tuf gene arrangements argue in favor of their ancient origins (10). It is noteworthy that most obligate intracellular parasites, such as Mycoplasma spp. (17, 22), R. prowazekii (41), B. burgdorferi (16), and T. pallidum (18), contain only one tuf gene. Their flanking sequences are distinct from the two conserved patterns as a result of selection for effective propagation by an extensive reduction in genome size by intragenomic recombination and rearrangement (10, 16, 18, 41).
Most gram-positive bacteria with low G+C content that have been sequenced to date contain only a single copy of the tuf gene as a part of the str operon. This is the case for B. subtilis, S. aureus, and E. faecalis. PCR amplification using a primer targeting a conserved region of the fus gene and the tufA-specific primer EntA2, but not the tufB-specific primer EntB, yielded the expected amplicons for all 17 enterococcal species tested, indicating the presence of the fus-tuf organization in all enterococci (data not shown). However, in the genomes of S. pneumoniae and S. pyogenes, the sequences flanking the tuf genes differ, although the tuf gene itself remains highly conserved. The enterococcal tufB genes are clustered with those of streptococci, but at present we do not have enough data to identify the genes flanking the enterococcal tufB genes. Furthermore, the functional role of the enterococcal tufB genes remains unknown. One can only postulate that the two divergent gene copies are expressed under different conditions.
The amino acid sequence identities between the enterococcal tufA and tufB genes are lower than either of (i) those between the enterococcal tufA and the tuf genes from other low-G+C-content gram-positive bacteria (streptococci and lactococci excluded) or (ii) those between the enterococcal tufB and streptococcal and lactococcal tuf genes. These findings suggest that the enterococcal tufA genes have a common ancestor with other low-G+C-content gram-positive bacteria via the simple scheme of vertical evolution, while the enterococcal tufB genes are more closely related to those of streptococci and lactococci. The facts that some enterococci possess an additional tuf gene and that the single streptococcal tuf gene is not clustered with those of other low-G+C-content gram-positive bacteria cannot be explained by the mechanism of gene duplication or intrachromosomal recombination. According to sequence and phylogenetic analysis, we propose that the presence of the additional copy of the tuf gene in 11 enterococcal species is due to horizontal gene transfer. The common ancestor of the 11 enterococcal species now carrying tufB genes acquired a tuf gene from an ancestral streptococcus or a streptococcus-related species through gene transfer during enterococcal evolution before the diversification of modern enterococci. Further study of the flanking regions of the gene may provide more clues to the origin and function of this gene in enterococci.
Recent studies of genes and genomes have demonstrated that considerable horizontal transfer occurred in the evolution of aminoacyl-tRNA synthetases in all three kingdoms of life (6, 26, 48). The heterogeneity of 16S rRNA is also attributable to horizontal gene transfer in some bacteria, such as Streptomyces, Thermomonospora chromogena, and Mycobacterium celatum (37, 45, 49). In this study, we provide the first example in support of a likely horizontal transfer of the tuf gene encoding EF-Tu. This may be an exception since stringent functional constraints do not allow for frequent horizontal transfer of the tuf gene as with other genes. However, enterococcal tuf genes should not be the only such exception as we have noticed that the phylogeny of Streptomyces tuf genes is at least as complex as that of enterococci. For example, the three tuf-like genes in one high-G+C-content gram-positive bacterium, S. ramocissimus, branched with the tuf genes of phylogenetically divergent groups of bacteria (Fig. 2). Another example may be the tuf genes in clostridia, which represent a phylogenetically very broad range of organisms and form a plethora of lines and groups of various complexities and depths. Four species belonging to three different clusters within the genus Clostridium have been shown by Southern hybridization to carry two copies of the tuf gene (39). Further sequence data and phylogenetic analysis may help in interpreting the evolution of EF-Tu in these gram-positive bacteria. Since the tuf genes and 16S rRNA genes are often used for phylogenetic study, the existence of duplicate genes originating from horizontal gene transfer may alter the phylogeny of microorganisms when the laterally acquired copy of the gene is used for such analyses. Hence, caution should be taken in interpreting phylogenetic data. In addition, the two tuf genes in enterococci have evolved separately and are distantly related to each other phylogenetically. The enterococcal tufB genes are less conserved and unique to the 11 enterococcal species. We previously demonstrated that the enterococcal tufA genes could serve as a target to develop a DNA-based assay for identification of enterococci (25). The enterococcal tufB genes would also be useful in the identification of these 11 enterococcal species.
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ACKNOWLEDGMENTS |
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We thank members of the Rapid Diagnostic group at the Centre de Recherche en Infectiologie of Laval University for their help in obtaining the tuf sequences. We thank Sonia Paradis and Pascal Lapierre for their help with phylogenetic analysis and Dominique Boudreau for his contribution to the three-dimensional structure analysis of EF-Tu and preparation of figures. Sequencing of E. faecalis, S. aureus, and S. pneumoniae genomes by the Institute for Genomic Research was accomplished with support from The National Institute of Allergy and Infectious Diseases, National Institutes of Health. We also thank the Streptococcal Genome Sequencing Project funded by USPHS/NIH grant no. AI38406 and B. A. Roe, S. P. Linn, L. Song, X. Yuan, S. Clifton, R. E. McLaughlin, M. McShan, and J. Ferretti from Department of Chemistry and Biochemistry, the University of Oklahoma, Norman, and the University of Oklahoma Health Science Center, Department of Microbiology and Immunology, Oklahoma City, for making available the S. pyogenes genomic sequence before publication.
This study was supported by grant PA-15586 from the Medical Research Council (MRC) of Canada and by Infectio Diagnostic (I.D.I.) Inc., Sainte-Foy, Québec, Canada. M. Ouellette is an MRC Scientist.
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FOOTNOTES |
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* Corresponding author. Mailing address: Centre de Recherche en Infectiologie, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, 2705 Boul. Laurier, Sainte-Foy, Québec G1V 4G2, Canada. Phone: (418) 654-2705. Fax: (418) 654-2715. E-mail: Michel.G.Bergeron{at}crchul.ulaval.ca.
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Top
Abstract
Introduction
Materials and Methods
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Discussion
References
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Journal of Bacteriology, December 2000, p. 6913-6920, Vol. 182, No. 24
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