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Journal of Bacteriology, January 1999, p. 434-443, Vol. 181, No. 2
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Discontinuous Occurrence of the hsp70
(dnaK) Gene among Archaea and Sequence Features
of HSP70 Suggest a Novel Outlook on Phylogenies Inferred from
This Protein
Simonetta
Gribaldo,1
Valentina
Lumia,1
Roberta
Creti,1
Everly
Conway
de Macario,2
Annamaria
Sanangelantoni,3 and
Piero
Cammarano1,*
Istituto Pasteur Fondazione Cenci-Bolognetti,
Dipartimento Biotecnologie Cellulari ed Ematologia, Università di
Roma I, Policlinico Umberto I°, 00161 Roma,1
and
Dipartimento di Genetica e Microbiologia "A. Buzzati
Traverso," Università di Pavia, 27100 Pavia,3 Italy, and
Wadsworth Center,
Division of Molecular Medicine, New York State Department of
Health, and Department of Biomedical Sciences, School of Public
Health, The University at Albany, Albany, New York
12201-05092
Received 6 July 1998/Accepted 19 October 1998
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ABSTRACT |
Occurrence of the hsp70 (dnaK) gene was
investigated in various members of the domain Archaea
comprising both euryarchaeotes and crenarchaeotes and in the
hyperthermophilic bacteria Aquifex pyrophilus and
Thermotoga maritima representing the deepest offshoots in
phylogenetic trees of bacterial 16S rRNA sequences. The gene was not
detected in 8 of 10 archaea examined but was found in A. pyrophilus and T. maritima, from which it was cloned
and sequenced. Comparative analyses of the HSP70 amino acid sequences
encoded in these genes, and others in the databases, showed that (i) in accordance with the vicinities seen in rRNA-based trees, the proteins from A. pyrophilus and T. maritima form a
thermophilic cluster with that from the green nonsulfur bacterium
Thermomicrobium roseum and are unrelated to their
counterparts from gram-positive bacteria, proteobacteria/mitochondria,
chlamydiae/spirochetes, deinococci, and cyanobacteria/chloroplasts;
(ii) the T. maritima HSP70 clusters with the homologues
from the archaea Methanobacterium thermoautotrophicum and
Thermoplasma acidophilum, in contrast to the postulated
unique kinship between archaea and gram-positive bacteria; and (iii) there are exceptions to the reported association between an insert in
HSP70 and gram negativity, or vice versa, absence of insert and gram
positivity. Notably, the HSP70 from T. maritima lacks the
insert, although T. maritima is phylogenetically unrelated to the gram-positive bacteria. These results, along with the absence of
hsp70 (dnaK) in various archaea and its
presence in others, suggest that (i) different taxa retained either one
or the other of two hsp70 (dnaK) versions (with
or without insert), regardless of phylogenetic position; and (ii)
archaea are aboriginally devoid of hsp70
(dnaK), and those that have it must have received it from
phylogenetically diverse bacteria via lateral gene transfer events that
did not involve replacement of an endogenous hsp70 (dnaK) gene.
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INTRODUCTION |
The 70-kDa heat-shock protein
(HSP70) is a member of a set of proteins (referred to as HSPs) which
undergo increased synthesis in response to a variety of physical and
chemical stresses (34). Originally identified as inducible
proteins, certain HSP70s are constitutively expressed and appear to be
essential for physiological cell growth (13, 26). HSP70 has
been found in all members of the domains Bacteria and
Eucarya investigated until now and in some members of the
domain Archaea (20, 22, 37). Certain bacteria
(Plantomycetales, Verrucomicrobiales, and
Synechococcus spp.; Escherichia coli) contain
more than one gene for HSP70 (39, 45, 52), and eucaryotic
genomes encode multiple HSP70 versions that are localized to the
various cell compartments (cytosol, endoplasmic reticulum,
mitochondria, and chloroplasts) (13). In accordance with the
proposed bacterial origins of mitochondria and chloroplasts, the
(nucleus-encoded) HSP70s of cell organelles are most similar in
sequence to homologues from members of the class
Proteobacteria and cyanobacteria, respectively
(6).
Intriguingly, HSP70-based phylogenies (20-25) contradict
both the three-domain division of living organisms inferred from
analysis of small-subunit rRNAs (53, 54) and the sisterhood
of Archaea and Eucarya evidenced by reciprocally
rooted trees of primordially duplicated genes (7, 17, 19, 31,
35). Unlike phylogenies of small-subunit rRNA sequences, the
HSP70-based phylogenies predict a close and specific relationship
between the archaea and gram-positive bacteria on the one hand and
between the eucarya and gram-negative bacteria on the other, rather
than between eucarya and archaea (22). These relationships
are supported, among other arguments, by the finding of a relatively
conserved insert occurring in the same position in all of the HSP70s
from gram-negative bacteria and eucarya but absent in all of the
homologues from archaea and gram-positive bacteria (21, 22).
The mutual affinities between eucarya, gram-negative bacteria, archaea,
and gram-positive bacteria have been taken as evidence that (i) archaea
and gram-positive bacteria constitute the two primary (albeit
paraphyletic) lines of cellular descent, (ii) gram-negative bacteria
are a late offshoot of the primitive gram-positive line, and (iii) the
eucaryotic genome arose by chimerism through a unique endosymbiotic
event involving the engulfment of an archaeon by a gram-negative
bacterium (22).
To study the evolution of the hsp70 (dnaK) gene
family with a sample of molecules more representative than that used in
earlier works, we examined the sequences currently available in the
databases and added two new hyperthermophilic bacteria representing the Aquificales and the Thermotogales. These are
considered to be the deepest divergences in the bacterial 16S rRNA
phylogenetic tree (3, 10). In addition, we sought the
occurrence of the hsp70 (dnaK) gene in various
archaea representing the euryarchaeotes and crenarchaeotes.
Here we report (i) the deduced amino acid sequences of the HSP70s from
Aquifex pyrophilus and Thermotoga maritima and
(ii) results of comparative analyses of these sequences and their
homologues in the databases. Based on these results, and on the
findings of the distribution of the hsp70 (dnaK)
gene among the archaea investigated, we propose an explanation for the
anomalous HSP70 phylogenies, which differs from others that are also
based on HSP70 sequence comparisons.
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MATERIALS AND METHODS |
Bacterial and archaeal strains and plasmids.
The bacteria
A. pyrophilus (DSM 6858) and T. maritima (DSM
3109) were gifts from K. O. Stetter (Lehrstuhl für
Mikrobiologie, Regensburg, Germany). The archaea Desulfurococcus
mobilis (DSM 2161), Pyrococcus woesei (DSM 3773),
Thermoplasma acidophilum (DSM 1728), and Thermoproteus
tenax (DSM 2078) were obtained from W. Zillig (Max Planck Institut
für Biochemie, Martinsried, Germany); Methanopyrus
kandleri (DSM 6324), Methanothermus fervidus (DSM 2088), and DNA from Archaeoglobus fulgidus (DSM 4139) were
gifts from R. Huber (Lehrstuhl für Mikrobiologie, Regensburg,
Germany); Methanococcus vannielii was a gift from A. Böck (Lehrstuhl für Mikrobiologie, Munich, Germany);
Halobacterium halobium DNA was a gift from F. Pfeiffer (Max
Planck Institut für Biochemie, Martinsried, Germany).
Sulfolobus solfataricus (formerly Caldariella
acidophila MT4) was grown in our laboratories. The
Methanosarcina mazei S-6 hsp70 (dnaK)
gene (37) was subcloned for this work in a pBluescript-based vector from another construct with a larger insert (2,195 bp) that also
contained the other stress genes in the locus (33). PCR
products were cloned in pMosBlue vector (Amersham) according to the
manufacturer's instructions. pBluescript SKM13 (Stratagene) was used
as the vector, and E. coli TB1 (New England Biolabs) was
used as the host. Plasmid-containing strains were grown in LB medium
supplemented with ampicillin (75 µg/ml).
DNA preparation, sequencing and digestion.
Genomic DNA was
prepared as previously described (4). Isolation and
purification of plasmid DNA, recovery of DNA fragments from
low-melting-point agarose gels, transformation experiments, and
Southern blottings were done according to standard protocols (43). Southern blottings and colony hybridizations using
homologous probes were performed at 65°C in the absence of formamide.
Southern blottings using heterologous DNA were performed at 37°C in
5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.5%
sodium dodecyl sulfate with 25 to 30% formamide, using 7 µg of
genomic DNA and 10 to 30 ng of a randomly labeled AccI
fragment (1,200 bp) of M. mazei dnaK encompassing codons 169 to 570. All DNA probes were labeled by using
[
-32P]dATP (specific activity, 6,000 Ci/mmol) and a
random priming labeling kit from Boehringer Mannheim. DNA sequences
were determined on both strands by the dideoxynucleotide chain
termination method (44) using [35S]dATP
(>1,000 Ci/mmol) and both universal and de novo-synthesized primers
according to the protocol for the Sequenase sequencing kit (U.S.
Biochemical Corp.).
The restriction enzymes used for optimal digestion of genomic DNAs from
the species investigated were EcoRI/HindIII
(T. maritima), XbaI (S. solfataricus),
SacI (H. halobium), EcoRI (M. fervidus), HindIII/XbaI (M. kandleri), and HindIII (D. mobilis,
P. woesei, A. pyrophilus, T. tenax,
A. fulgidus, and M. vannielii).
PCR-mediated DNA amplification.
dnaK was amplified by
PCR in a Perkin-Elmer apparatus according to standard procedures in 3.0 mM MgCl2. Two degenerate primers were used:
5'-CA(AG)GC(ACGT)AC(ACGT)AA(AG)GA(CT)GC(ACGT)GG-3' (hspI),
corresponding to the DNA segment encoding the conserved sequence
QATKDAG (E. coli HSP70 residues 152 to 158); and
5'-GC(ACGT)AC(ACGT)GC(CT)TC(AG)TC(ACGT)GG(AG)TT-3' (hspII),
complementary to the DNA segment encoding the conserved sequence
NPDEAVA (E. coli HSP70 residues 366 to 372).
Database searches and sequence alignments.
The Genetic
Computer Group program suite (15) of the UK MRC Human Genome
Mapping Project Resource Centre (Cambridge University, Cambridge,
United Kingdom) was used for retrieval of HSP70-related sequences. This
was done by probing the DNA and protein databases with the tBLASTn and
FASTAp options of the BLAST (1) and FASTA (41)
programs. To minimize alignment errors, a preliminary alignment of the
full-length HSP70 sequences was generated by CLUSTAL W (49),
using default gap penalties. The CLUSTAL W alignment was then locally
refined by using the segment-to-segment comparison method implemented
in the program DIALIGN (38) with the BLOSUM similarity
matrix (27).
Tree-making methods.
Phylogenetic trees were constructed by
using maximum-parsimony (MP), evolutionary distance (ED), and
maximum-likelihood methods. The MP analyses used the program PROTPARS
implemented in PHYLIP version 3.57c (16). The PHYLIP
programs SEQBOOT, PROTPARS, and CONSENSE were used sequentially to
generate an MP tree which was replicated in 100 bootstraps; on this
basis bootstrap confidence levels (BCL) were determined. Evolutionary
distances between all pairs of taxa were calculated with the Dayhoff
option of the PHYLIP program PROTDIST, which estimates the number of
expected amino acid replacements per position, using a substitution
model based on the PAM 120 matrix. The resultant pairwise distances
were then used to construct a least-squares tree with the program
FITCH. The programs SEQBOOT, PROTDIST, FITCH, and CONSENSE were used sequentially to construct a consensus tree based on 100 bootstrap replications of the original alignment. For maximum-likelihood analyses, we used the program PUZZLE version 4.0 (47) with
the Jones-Taylor-Thornton (JTT) substitution model and a
gamma-distributed model of site-to-site rate variation using eight rate
classes to approximate the continuous gamma distribution, as well as a gamma distribution parameter estimated from the data set.
Nucleotide sequence accession numbers and alignment
retrieval.
The A. pyrophilus and T. maritima
dnaK sequences are deposited at the EMBL GenBank database with
accession no. AJ005800 and AJ005129, respectively. The sequence
alignment used in this analysis (file name hsp70.aln) is available at
ftp.bce.med.uniroma1.it: dir/cammara.
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RESULTS |
Cloning of hsp70 (dnaK) homologues.
Fragments of A. pyrophilus and T. maritima dnaK
genes were successfully amplified by PCR with two degenerate primers
used in the past to clone dnaK genes from a number of
bacteria and archaea (20, 52). The amplified DNA fragments,
having the expected length of 650 nucleotides, were found to correspond
to the sequence encoding residues 152 through 372 of E. coli
HSP70 (see Materials and Methods). These fragments were used as probes for isolating genomic clones containing the A. pyrophilus
and T. maritima dnaK genes, and dnaK was
subcloned and sequenced. In contrast, when PCR was carried out with DNA
from a variety of archaea comprising both crenarchaeota (D. mobilis, S. solfataricus, and T. tenax) and
euryarchaeota (P. woesei, M. kandleri, M. fervidus, M. vannielii, A. fulgidus,
T. acidophilum, and H. halobium), successful amplification of dnaK-related sequences was observed only
with T. acidophilum and H. halobium DNA (results
not shown). Evidence for dnaK among the archaea listed above
was further sought by means of Southern blotting using a 1,200-bp
AccI fragment of M. mazei dnaK encompassing
codons 169 to 570 (see Materials and Methods). Again, the probe
hybridized only with restriction fragments of T. acidophilum
and H. halobium; these gave single hybridization bands upon
digestion with HindIII (T. acidophilum) and
SacI (H. halobium) (results not shown). Although
the absence of a gene cannot be asserted with confidence on the basis
of negative hybridization results, lack of dnaK in some of
the archaea listed above is demonstrated unequivocally by the recent
genome sequencing data (see Discussion).
Alignment and sequence comparisons of Aquifex and
Thermotoga HSP70s.
The deduced amino acid sequences of
the A. pyrophilus and T. maritima HSP70s were
aligned with most of the available homologues by using CLUSTAL W and
the DIALIGN segment-to-segment comparison method. In addition to
A. pyrophilus and T. maritima, the global alignment included 68 sequences. Of those, 17 were from gram-positive bacteria (of both the low- and high-G+C subdivisions), 5 were from
archaea; 29 covered the genera Deinococcus and
Thermus, the green nonsulfur bacteria, chlamydiae and
spirochetes, - 
-, and 
subdivisions of the class
Proteobacteria, mitochondria, cyanobacteria, and
chloroplasts; and 17 sequences were eucaryotic cytosolic HSP70s representing a broad sampling of eucaryal diversity. The deduced sequence of the Aquifex aeolicus HSP70 (14) that
became available after databank submission of the A. pyrophilus sequence was also used. The complete alignment of 70 HSP70 sequences is retrievable via anonymous ftp as described in
Materials and Methods. A subset of the global alignment highlighting
strongly conserved sequence motifs constraining the alignment topology
is shown in Fig. 1. An excerpt of the
alignment focusing on the insertion segment situated in the N-terminal
portion of many HSP70 sequences (21, 22) is shown in Fig.
2.
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FIG. 1.
Abridged alignment of the A. pyrophilus and
T. maritima HSP70 sequences with homologues from the low-G+C
gram-positive bacteria (Bsu), the high-G+C gram-positive bacteria
(Mtu), the archaea (Hma), the green nonsulfur bacteria (Tro), the
deinococci (Dpr), the proteobacteria (Eco), and the eucarya (Gin). Plus
signs indicate amino acid positions selected for construction of the
phylogenetic trees shown in Fig.s 3 to 5; highlighted blocks indicate
positions occupied by an identical amino acid in more than 90% of the
70 aligned sequences; shaded areas indicate positions occupied by
similar amino acids (ILVM, DEKRH, ST, GA, FYW, NQ) in 100% of the
seventy sequences. For reasons of space, the C-terminal portion of the
alignment (comprising residues not selected for tree reconstruction) is
not shown. Species abbreviations are listed in the legend to Fig. 2.
Vertical arrows delimit a sequence region that could not be confidently
aligned between the procaryotic and eucaryotic HSP70s. The boxed region
in the top block delimits the insertion segment found in many HSP70s.
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FIG. 2.
Excerpt of the HSP70 sequence alignment focusing on the
insertion segment which is found in many HSP70 sequences. Highlighting
and shading have the same meaning as in Fig. 1. Species abbreviations:
Aae (Aquifex aeolicus), Apy (Aquifex pyrophilus),
Atu (Agrobacter tumefaciens), Ava (Anabaena
variabilis), Bbu (Borrelia burgdorferi), Bja
(Bradyrhizobium japonicum), Bme (Bacillus
megaterium), Bov (Brucella ovis), Bsp
(Buchnera sp.), Bst (Bacillus
stearothermophilus), Bsu (Bacillus subtilis), Cac
(Clostridium acetobutylicum), Ccr (Caulobacter
crescentum), Cpa (Cryptosporidium parvum), Cpe
(Clostridium perfringens), Cpn (Chlamydia
pneumoniae), Ctr (Chlamydia trachomatis), Ddi
(Dictyostelium discoideum), Dpr (Deinococcus
proteolyticus), Eco (Escherichia coli), Eeu
(Euplotes eurystomus), Ehi (Entamoeba
histolytica), Erh (Erysipelothrix rhusiopathiae), Gin
(Giardia intestinalis), Hcu (Halobacterium
cutirubrum), Hma (Halobacterium marismortui), Lam
(Leishmania amazonensis), Lin (Leishmania
infantum), Lla (Lactococcus lactis), Lma
(Leishmania major), Lpn (Legionella pneumophila),
Mav (Mycobacterium avium), Mca (Mycoplasma
capricolum), Mge (Mycoplasma genitalium), Mle
(Mycobacterium leprae), Mma (Methanosarcina
mazei), Mpa (Mycobacterium paratuberculosis), Mpn
(Mycoplasma pneumoniae), Mth (Methanobacterium
thermoautotrophicum), Mtu (Mycobacterium
paratuberculosis), Neu (Nitrosomonas europaea), Ono
(Oxytricha nova), Pce (Pseudomonas cepacia), Pfa1
and Pfa2 (Plasmodium falciparum), Psa ch (Pisum
sativum, chloroplast), Rca (Rhodobacter capsulatus),
Reu (Ralstonia eutropha), Rme (Rhizobium
meliloti), Rsp (Rhodopseudomonas sp.), Sau
(Staphylococcus aureus), Sce (Saccharomyces
cerevisiae), Sco (Streptomyces coelicolor), Sgr
(Streptomyces griseus), Spn (Streptococcus
pneumoniae), Spy (Streptococcus pyogenes), Syc
(Synechococcus sp.), Syn (Synechocystis sp.), Tac
(Thermoplasma acidophilum), Tbr (Tripanosoma
brucei), Tcr mt (Tripanosoma cruzi, mitochondrion), Tcr
(Trypanosoma cruzi), Tma (Thermotoga maritima),
Tro (Thermomicrobium roseum), Tth (Thermus
thermophilus), Tva mt (Trichomonas vaginalis,
mitochondrion), Tva (Trichomonas vaginalis), Vne mt
(Vairimorpha necatrix, mitochondrion), Xla (Xenopus
laevis), Zma (Zea mays). Databank accession numbers are
shown to the right of species designations. NS, nonsulfur.
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Consistent with previous reports (
22,
37), the HSP70s from
the gram-positive bacteria and the five archaea (
T. acidophilum,
Methanobacterium thermoautotrophicum,
M. mazei, and two members
of
Halobacteriales)
lacked the insertion segment seen in the HSP70s
from the gram negative
organisms. Surprisingly, the
T. maritima HSP70, unlike that
of the other gram-negative bacteria, including
A. pyrophilus, lacked the distinctive
insertion.
Phylogenetic analysis of HSP70 sequences.
Figures
3 and 4
show MP and ED trees inferred from 492 amino acid positions that could
be confidently aligned among all sequences; a short stretch of 14 to 15 residues that was not unambiguously alignable between the procaryotic
and eucaryotic sequences was not included in the data set. The ED tree
was constructed by the least-squares method using a matrix of
evolutionary distances based on the Dayhoff PAM 120 amino acid
replacement model. The MP tree was one of two equally parsimonious
trees (6,438 steps) that differed in minor details of the branching
order.
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FIG. 3.
MP tree constructed with the program PROTPARS from the
492 amino acid positions marked by + in Fig. 1. Numbers on
internal branches are BCL based on 100 bootstrap replications of the
original alignment. Only BCL of >30% are shown. Branch lengths do not
represent number of steps. Abbreviations: NS, nonsulfur; mt,
mitochondria; chl, chloroplasts. Archaeal sequences are boxed;
asterisks indicate the two new sequences reported here. For full
organism names, see the legend to Fig. 2.
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FIG. 4.
ED tree constructed with the program FITCH from the 492 amino acid positions marked by + in Fig. 1. ED matrices were
calculated by the PAM 120 amino acid substitution matrix, using the
Dayhoff option of the program PROTDIST. Numbers on internal branches
are BCL based on 100 bootstrap replications. Only BCL of >50% are
shown. The scale bar represents 0.1 amino acid substitution per site.
For abbreviations and other details, see the legend to Fig. 3.
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In both trees, the deepest divide separated the eucarya from a
composite procaryotic cluster showing the archaea interspersed
among
(and associated with) different bacterial groupings. The
topologies of
the two trees were basically consistent in reproducing
(i) the same
clustering of the taxa (a monophyletic gram-positive
bacterium clade,
the proteobacteria, a chlamydia-spirochete grouping,
a strongly
supported cyanobacterium-
Deinococcus clade); (ii) a
similar
internal branching order within the clusters with few
minor
discrepancies; and (iii) the same specific associations
of mitochondria
with the proteobacteria and of chloroplasts with
the cyanobacteria. The
specific relationships seen in previous
reports (
22) between
M. mazei and the
Clostridium group of low-G+C
gram-positive bacteria, and between the
Halobacterium
cutirubrum/H. marismortui pair and the high G+C gram-positive
bacteria, were
also confirmed by the analyses. However, due to lack of
robustness
of the deepest nodes (<50% bootstrap support), the mutual
relationships
between the principal procaryotic clusters remain
statistically
indeterminate.
In both trees, the two novel (
A. pyrophilus and
T. maritima) sequences formed an independent, albeit tenuously
supported,
grouping of thermophilic organisms with the green nonsulfur
bacterium
Thermomicrobium roseum and the euryarchaeotes
M. thermoautotrophicum and
T. acidophilum. The
two archaea were strongly related to one
another (100% bootstrap
support) and appeared to share a last
common ancestor with
T. maritima. It is noteworthy that the three
thermophilic bacterial
taxa (
Aquifex,
Thermotoga, and
Thermomicrobium)
that clustered together in the HSP70-based
trees become resolved
into three independent but adjacent lineages in
phylogenetic trees
of small-subunit rRNA sequences (
3,
10).
These show
Aquificales,
Thermotogales, and green
nonsulfur bacteria, in that order, as
three consecutive offshoots of
the bacterial rRNA tree, situated
immediately below the
Deinococcus-cyanobacterium
radiations.
The relationships between the archaeal and bacterial HSP70 sequences
seen in Fig.
3 and
4 were further analyzed by quartet
puzzling (QP), a
maximum-likelihood algorithm that accounts for
site-to-site rate
variation and gives only fully resolved groupings.
As expected from the
lack of robustness of the ED and MP trees,
the topology of the QP tree
(Fig.
5) was largely star-like,
indicating
that the phylogenetic content of the HSP70 data set does not
allow
the resolution of the deepest relationships between the largest
procaryotic groupings (
48). Importantly, however, the
tree-like
component of the QP phylogeny recovered the association of
T. maritima with the
Methanobacterium-Thermoplasma pair (52% QP reliability)
and
confirmed the previously reported relationships between
Methanosarcina and the clostridia (87% QP reliability) and
between the halobacteria
and the high-G+C gram-positive bacteria (55%
QP reliability).
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FIG. 5.
Maximum-likelihood tree of the HSP70 alignment (the 492 positions marked in Fig. 1). The QP method was used with a
gamma-distributed model of site-to-site rate variation using eight rate
categories. The gamma distribution parameter estimated from the
data set was 0.71 ± 0.04, and the log-likelihood of the tree was
 19,510.8. Numbers above nodes are QP reliability values. The scale
bar represents 0.1 amino acid substitution per site. For other details,
see the legend to Fig. 3.
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Evolutionary distances between HSP70 sequences.
Average
maximum-likelihood distances between HSP70s from Archaea,
Eucarya, and gram-positive and gram-negative bacterial taxa were calculated by the JTT amino acid substitution model and are shown
in matrix form in Table 1. In accordance
with results based on the Dayhoff amino acid substitution model
(42; see Discussion), the average distances between
the eucaryal sequences and the procaryotic homologues are significantly
larger than those seen among procaryotes. This finding supports the
argument (42) that the most likely rooting of the HSP70 tree
lies between the procaryotic cluster and Eucarya, rather
than between gram-positive bacteria and Archaea (24), or between a gram-positive/archaeal clade and a
gram-negative/eucaryal clade (25).
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DISCUSSION |
Compared to previous studies of HSP70 sequences, the present data
set includes HSP70s from three deep-branching bacteria (A. pyrophilus and T. maritima [this report] and A. aeolicus [14]) and from the archaeon M. thermoautotrophicum (46). The results cast a new light
on the evolutionary history of dnaK genes by calling into
question such critical issues as (i) the orthology of the
dnaK sequences; (ii) the paraphily of Archaea
with respect to the gram-positive bacteria; and (iii) the ubiquity of
dnaK in the evolutionary spectrum.
Lack of phylogenetic specificity of the HSP70 insertion
segment.
According to Gupta and coworkers (18, 20-25),
the discrete insertion seen in the N-terminal region of many HSP70
sequences (Fig. 2) is an evolutionary landmark distinguishing
Gram-negative bacteria and eucarya (all of which possess the HSP70
insert) from gram-positive bacteria and archaea (which lack the HSP70
insert). Homologues lacking the insert (I
) were assumed
to represent the ancestral form of the protein from which all the
gram-negative bacterial homologues were derived (21), while
eucarya would have obtained the insert-containing form of HSP70
(I+) through the bacterial partner of a postulated
endosymbiotic event involving the fusion of a gram-negative bacterium
and an archaeon (18, 22-25).
These conclusions are challenged by the evidence in this report that
A. pyrophilus and
T. maritima, two gram-negative
bacteria
(
29,
30) representing adjacent offshoots in the 16S
rRNA tree
(
3,
10), differ from one another in that
A. pyrophilus possesses
and
T. maritima lacks the
insertion
segment.
The possibility that the genus
Thermotoga is
phylogenetically linked to the gram-positive bacteria (
8,
12), or that it
obtained its
dnaK gene from a
gram-positive bacterium, is unlikely.
First, the HSP70-based
phylogenies place
Thermotoga outside any
of the
gram-positive bacterial clusters and close to
Aquificales and green nonsulphur bacteria (
Thermomicrobium). Second, a
similar
placement of
Thermotoga is predicted by phylogenies
of small subunit
rRNA (
3,
10,
51), elongation factors (EF)
(
2,
5,
36), and aminoacyl-tRNA synthetases (
7).
These phylogenies
concur in placing the
Thermotogales as the
deepest or the second
deepest grouping in the bacterial tree
somewhat
deeper than the
green nonsulfur bacteria and the deinococci, and
definitely remote
from the later-branching gram-positive bacteria and
proteobacteria.
Such incongruity as exists between the placement of
Thermotoga in trees of molecular sequences, and its linkage
to the gram-positives
argued from lack of the HSP70 insert, can be
rationalized only
by positing that (i) different groupings retained
either one or
the other of two paralogous versions of the
dnaK gene or (ii)
the insert is an ancestral trait lost more
than once in bacterial
evolution. Indeed, by taking as a reference the
topologies of
the small-subunit rRNA and EF trees (
2,
3),
the distribution
of the two HSP70 forms throughout the bacterial phyla
would require
multiple insertion/deletion events as one moves from
Aquificales (I
+) to
Thermotogales
(I
), to green nonsulfur bacteria and deinococci
(I
+), to cyanobacteria (I
+), to gram-positives
bacteria (I
), and to proteobacteria (I
+).
Clustering of archaea with gram-positive bacteria.
An
additional question raised by the phylogenetic placement of the
Thermotogales concerns the postulated paraphily of
Archaea with respect to gram-positive bacteria
(20-25). In contrast to the tenet that archaea are
specifically related to (and arose polyphyletically within) the
gram-positive bacteria, our results indicate a specific relationship
between the archaea T. acidophilum and M. thermoautotrophicum and the bacterium T. maritima.
Although the robustness of the ((Thermoplasma,
Methanobacterium), Thermotoga) clade is not
impressive (52% QP reliability), the association of these three
thermophilic taxa is recovered by all the tree-making methods used.
Inasmuch as Thermotoga is not affiliated with any of the
gram-positive groupings, our observation renders less compelling the
general argument of Gupta and coworkers (18, 22-25) that gram-positive bacteria and archaea constitute the two primary (albeit
paraphyletic) lines of cellular descent, the gram-negative bacteria
being a later offshoot of the primitive gram-positive line.
Absence of dnaK among the archaea.
Several
considerations, based on the occurrence of hsp70
(dnaK) among the archaea (Table
2) and the topology of the prokaryotic cluster in the HSP70-based phylogenies (Fig. 3 to 5), support the
notion that archaea do not harbor dnaK genes except for taxa that recruited a dnaK sequence from sympatric bacteria.
First,
dnaK-related sequences are not detectable, by PCR
amplification or Southern blotting, in the euryarchaeotes
A. fulgidus,
M. vannielii,
P. woesei,
M. kandleri,
M. fervidus,
Methanococcus jannaschii,
Methanococcus voltae,
Methanospirillum hungatei, and
in the crenarchaeotes
T. tenax,
D. mobilis,
S. solfataricus,
and
a
Sulfolobus species. Secondly, in accordance with the
DNA hybridization
results, no
dnaK homologues have been
identified in the completely
sequenced genomes of
A. fulgidus,
M. jannaschii and
Pyrococcus horikoshi. Third, the HSP70 sequences from the archaea that
possess
a
dnaK gene (
T. acidophilum,
M. thermoautotrophicum,
M. mazei,
H. marismortui, and
H. cutirubrum) are distributed among,
and
clustered with, different bacterial groupings (Fig.
3 to
5) and
are
not distinguishable from the bacterial homologues by any unique
signature. Last, by taking as a reference the 16S rRNA-based phylogeny
(
40), the
dnaK distribution among the archaea is
nonsystematic
in character:
dnaK genes are missing in the
crenarcheotes and
are haphazardly scattered throughout the major
euryarchaeal phyla,
regardless of ranking order and phylogenetic
kinship. Also, this
gene is not shared by members of sister taxa such
as
M. hungatei (
Methanomicrobiales) and
M. mazei (
Methanosarcinales), or even
by members of the
same taxon such as
M. fervidus and
M. thermoautotrophicum (
Methanobacteriales).
Taken together, the above observations strongly suggest that archaeal
dnaK homologues, whenever they occur, were derived from
bacterial donors through lateral gene transfer events. Horizontal
transfer of
dnaK between the two prokaryotic domains is in
fact
suggested by the clustering of HSP70 sequences from organisms
(
Thermotoga,
M. thermoautotrophicum, and
Thermoplasma) possibly
thriving in similar (hot)
environments. Also, horizontal transfer
of protein-coding genes between
the two prokaryotic domains has
been reported repeatedly and is
evidenced by the clustering of
euryarchaeotes and gram-positive
bacteria in phylogenetic trees
of glutamine synthetase I sequences
(
8,
50).
A similar interpretation of the anomalous HSP70-based phylogenies had
been offered by Roger and Brown (
42) before the absence
of
hsp70 (
dnaK) in some archaea became obvious from
genome sequencing
data. They argued, based on a comparison of
evolutionary distances
between the HSP70 sequences, that (i) the HSP70
tree should be
rooted between eucarya and prokaryotes, rather than
between gram-positive
bacteria and archaea; and (ii) this rooting is
compatible with
the canonic Gogarten/Iwabe rooted tree of life
(
17,
31) if
the anomalous placement of archaea is the result
of a lateral
transfer and replacement of their endogenous
hsp70 (
dnaK) genes
by those from bacteria. The
recognition that archaea are aboriginally
devoid of
dnaK
simplifies the argument in that the transfer event
does not involve a
replacement of the homologous resident
gene.
Two explanations can account for the lack of
dnaK in
archaea; one is based on the Iwabe/Gogarten tree of life, and the other
relies on the fusion or chimeric model of cellular evolution advocated
by Gupta and coworkers (
18,
22-25) and Zillig et al.
(
55).
In the former case,
Archaea and
Eucarya constitute sister domains
sharing a last common
ancestor corresponding to the archaeal-eucaryal
branch of the universal
tree (
17,
31,
54). If an ancestral
dnaK existed
in the last common ancestor of extant organisms,
the lack of a
dnaK gene in
Archaea, and its persistence in
Eucarya,
could be (most parsimoniously) explained only by
positing a unique
gene extinction event occurring in the archaeal
branch of the
tree, provided
Archaea represents a
monophyletic grouping (see
references
2,
11, and
19 for a paraphyletic-
Archaea option).
The alternative possibilities that
dnaK either arose in the
bacterial
branch, or became extinct in the archaeal-eucaryal branch,
can
be ruled out, as in both cases the eucaryotic cytosolic HSP70s
could have been obtained only through duplication and divergence
of the
(nucleus-encoded) mitochondrial
dnaK, and there is no
evidence
from the HSP70-based trees (reference
6 and
this report) that
the cytosolic HSP70s arose from within the
proteobacterial/mitochondrial
cluster.
The absence of
dnaK in various archaea could also be simply
explained in the context of the fusion or chimeric model by assuming
that one of the two postulated primary lines either contained
(
Bacteria) or lacked (
Archaea) a dnaK sequence.
The HSP70 of the
hypothetical eukaryotic chimera was thus contributed
by the bacterial
parent of the fusion, regardless of whether the
bacterium (
18,
22-25) or the archaeon (
55)
provided the engulfing partner. However,
the fusion model of cellular
evolution predicts that reciprocally
rooted trees for primordially
duplicated genes that are contributed
by the bacterial parent of the
chimera will show
Bacteria and
Eucarya, rather
than
Archaea and
Eucarya, as the two sister
domains.
Given this premise, the fusion model is not a persuasive
alternative
until such a set of paralogous genes is identified and a
sisterhood
of
Eucarya and
Bacteria is
convincingly
demonstrated.
| |
ACKNOWLEDGMENTS |
We thank A. J. L. Macario for his input to this work
and for many helpful suggestions during preparation of the manuscript.
This work was supported by grants from Ministero Università e
Ricerca Scientifica e Tecnologica, Project Protein-Nucleic Acids
Interactions, and from CNR Progetto Finalizzato Biotecnologie.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Dipt.
Biotecnologie Cellulari, Sezione Genetica Molecolare, Policlinico
Umberto I°, Viale Regina Elena 324, 00161, Roma, Italy. Phone:
0039-0-6-4940609. Fax: 0039-0-6-4462891. E-mail:
Cammarano{at}bce.med.uniroma1.it.
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Abstract
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