The Evolution and Genetics of Aphid Endosymbionts
Author(s): Paul Baumann, Nancy A. Moran, Linda Baumann
Reviewed work(s):
Source: BioScience, Vol. 47, No. 1 (Jan., 1997), pp. 12-20
Published by: University of California Press on behalf of the American Institute of Biological Sciences
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The
Evolution
and
Genetics of
Aphid Endosymbi
Molecularbiology has provided new insights into the genetics,
physiology,and evolutionarybiology of this intimateassociation
Paul Baumann, Nancy A. Moran, and Linda Baumann
One
of the strikingattributes
of prokaryotes (both Bacteria and Archaea) is the diversity of their catabolic pathways
and their biosynthetic capabilities
(Brock et al. 1994). Many prokaryotes are able to use unusual substrates for growth and synthesize all
of the constituents of cells from relatively simple compounds. In contrast, many eukaryotes lack such
capabilities and have developed close
associations in which they take advantage of the metabolic versatility
of prokaryotes. In some of these
associations, the organisms live in
close contact but remain separate.
In other cases, called endosymbioses, the prokaryote is sequestered
within the eukaryotic cell. The classical compilation of endosymbiotic
associations
is that of Buchner
(1965).
Insects are particularly prone to
endosymbiotic associations. Such
associations are widespread among
members of the orders Homoptera
(aphids, whiteflies, mealybugs, psyllids, and cicadas), Blattaria (cock-
Paul Baumann is a professor in the
Microbiology Section, University of
California, Davis, CA 9561.6-8665.
Nancy A. Moran is a professor in the
Department of Ecology and Evolutionary Biology, University of Arizona,
Tucson, AZ 85721. Linda Baumannis a
research associate in the Microbiology
Section, Universityof California, Davis,
CA 95616-8665. Reprint requests and
correspondence should be addressed to
P. Baumann. ? 1997 American Institute of Biological Sciences.
12
Aphids coevolve with a
prokaryoticendosymbiont, which resemblesa
free-livingbacterium
modified for the
overproductionof
essentialnutrients
roaches), and Coleoptera (beetles;
Buchner 1965, Douglas 1989). Many
of these insects are restricted to diets
deficient in one or more required
nutrients.
The endosymbionts,
through their biosynthetic activities,
are believed to provide the host with
the missing nutrients (Dadd 1985).
Generally, endosymbionts cannot
be cultured outside their hosts, perhaps reflecting their dependence on
the association. As a consequence of
this inability to cultivate endosymbionts on laboratory media, investigations of these organisms have been
limited. The advent of the methods
of molecular biology, primarily the
characterization of nucleic acids, has
expanded greatly the potential for
obtaining new information on organisms that cannot readily be cultured. Such advances are especially
evident in studies of intracellular
human pathogens, such as chlamydia and rickettsia, which cannot be
grown outside cells. In the last seven
years, a similar approach has been
used for studying the endosymbionts
of aphids (Baumann et al. 1995),
and in this article we present an
overview of this work.
Aphids and
their endosymbionts
Aphids are major pests of plants and
can be thought of as living syringes
that insert their needlelike stylets
into the plant phloem tissue and
suck up plant sap (Dixon 1973). In
addition to causing damage directly,
their mode of feeding makes aphids
vectors of plant viruses, which cause
major losses to agricultural crops
(Blackman and Eastop 1984). The
life cycle of many aphids is complex,
involving alternation of sexual and
asexual reproduction. Their most
rapidly reproducing generations consist of parthenogenetic
females,
which typically give birth to 50 or
more progeny per month. The mother
contains developing daughters that
may, in turn, contain developing
granddaughters. This telescoping of
generations permits rapid maturation and a high rate of increase in
numbers within an aphid colony.
Inside the body cavity of most
aphids is a bilobed structure called
the bacteriome, which consists of
60-90 polyploid cells called bacteriocytes (Baumann et al. 1995).
Within these cells are host-derived
vesicles containing gram-negative
spherical or oval bacteria (Figure 1).
These bacteria have been assigned to
the genus Buchnera. A fully mature
aphid (Schizaphis graminum) weighs
approximately 500 gg and contains
5.6 million cells of Buchnera. AsBioScience Vol. 47 No. 1
suming that the aphid has the same
density as water, this figure corresponds to approximately 1.1 x 1010
endosymbionts per ml (Baumann and
Baumann 1994).
The association between the aphid
and Buchnera is an example of obligate mutualism: each partner is dependent on the other for survival.
Buchnera does not live outside the
aphid host and has not been cultured
on laboratory media. Transmission
of Buchnera is maternal, with mother
aphids infecting either eggs or developing embryos before birth. Treatment of aphids with antibiotics
results in the elimination of endosymbionts, with a concomitant reduction in the rate of aphid growth
and eventual sterility of the aphid.
Most aphids contain Buchnera, although there are several interesting
exceptions in which endosymbionts
are absent. These exceptions include
aphids in the family Pemphigidae, in
which reproductive females contain
Buchnera but nonreproductive soldier morphs or dwarf males lack
endosymbionts as a result of not
being inoculated within the mother
(Buchner 1966, Fukatsu and Ishikawa 1992b). In addition, certain
aphid species of the tribe Cerataphidini lack both bacteriocytes and
Buchnera and instead possess extracellular, yeastlike organisms (Fukatsu and Ishikawa 1992a, 1996).
Surrounding the bacteriome is a
sheath consisting of a thin layer of
flattened cells. In some aphids, these
cells may harbor a gram-negative,
rod-shaped bacterium that is usually
called the secondary (S-) endosymbiont (Baumann et al. 1995). S-endosymbionts, which are also maternally transmitted, are generally fewer
in number than Buchnera. Many
species of aphid lack S-endosymbionts, and some species show variation among strains in whether or not
S-endosymbionts are present, suggesting that they are not essential for
host survival (Chen and Purcell in
press). It is not known whether Sendosymbionts have any beneficial
effects on their hosts.
Coevolution of Buchnera and
the aphid hosts
Phylogenetic analyses based on small
subunit (16S or 18S) ribosomal
January 1997
Figure 1. Electron micrographof Buchnerawithin bacteriocytes. Arrow indicates
the vesicle membrane. Bar = 1 gim. Photo courtesy of Mary Kinsey and Don
McLean.
DNAs (rDNA) are routinely used to
elucidate evolutionary relationships
within both prokaryotes and eukaryotes. Applying this approach to studies of Buchnera and their aphid
hosts has produced a remarkably
clear-cut picture of the evolutionary
history of this endosymbiotic association, as summarized in Figure 2.
Analyses of bacterial 16S rDNA show
that Buchnera from a diverse assemblage of aphids form a single monophyletic group. In other words, all
Buchnera are more related to one
another than to any other bacteria.
Furthermore, the relationships obtained within Buchnera are in agreement with the established host classification and with reconstructions
of host phylogeny based on morphology and on 18S rDNA (Baumann et al. 1995, Moran and Baumann 1993, Moran et al. 1995, von
Dohlen and Moran 1995).
This "matching" of phylogenetic
trees between aphids and Buchnera
strongly supports the view that a
single ancient infection of a com-
mon ancestor of all aphids has been
transmitted vertically through the
various lineages of aphids as they
diversified. Because the common ancestor of all aphids is estimated,
from fossil evidence, to be 150-250
million years old, the original infection must date back at least this far.
Following this initial infection, endosymbionts and aphid hosts appear
to have diversified in parallel, resulting in the present strains of
Buchnera that are associated with
the present species of aphid. The
congruence between the phylogenies
of host and endosymbiont indicates
that the vertical transmission of
Buchnera has been maintained since
the time of the original infection,
with no transfer of Buchnera between different aphid lineages.
Using the fossil record to estimate
times of divergence for aphid hosts
and extending the same dates to the
corresponding endosymbionts, it has
been possible to calculate the rate of
nucleotide base substitution within
the 16S rDNA gene of Buchnera.
13
which are imprecise due to the poor
fossil record. The analyses indicate
that the bacteria evolve much faster
S. graminum
than their hosts, with substitution
rates 36 times greater in Buchnera
originof
than in their hosts (Moran et al.
R.padi
R.maidis
association 1995). Furthermore, this difference
150-250 MY in rate appears to apply generally to
A.pisum
Buchnera
comparisons between bacteria and
origin
of
D.
insects, because both Buchnera and
noxia
association
aphids evolve at rates similar to reU. sonchif
150-250 MY I
lated bacteria and insects, respecU. rurale
tively. Among the plausible explanations for this large difference in
M.persicae
rates is the faster generation time of
bacteria relative to insects or posC. viminalis
sible differences in mutation rates
Mi.kinseyi
that could arise from different DNA
repair mechanisms.
P. betae
Mealybugs and whiteflies are related to aphids. Like aphids, these
Me.rhois
insect groups feed on plant sap as
Sl. chinensis
their sole diet and harbor endosymbionts. Phylogenetic studies based
on 16S rDNA have indicated that
species
the endosymbionts of each of these
groups descended from infections
Escherichia
coli
separate from that leading to Buch(A.pisum)
S-endosymbiont
nera. Similar investigations on the
Proteusvulgaris
endosymbionts of cockroaches, carpenter ants, and tsetse flies have
Ruminobacter
amylophilus
shown that these also result from
Figure2. Congruenceof the evolutionary relationships of Buchneraand the aphid additional, independent infections
hosts. The endosymbiont tree is based on 16S rDNA; the aphid tree is based on by free-living bacteria (Aksoy et al.
18S rDNA and the fossil record. Full generic and specific names are used for free- 1995, Bandi et al. 1995, Moran and
living bacteria. Aphid generic abbreviations and species names are used for Baumann 1993, Schr6der et al.
Buchnera and the aphid host. A = Acyrthosiphon; C = Chaitophorus; D = 1996). Together, these molecular
Diuraphis; M = Myzus; Me = Melaphis; Mi = Mindarus; P = Pemphigus; R = phylogenetic studies of endosymRhopalosiphum; S = Schizaphis; Si = Schlechtendalia; U = Uroleucon; MY bionts of diverse insect groups sugmillion years; S-endosymbiont = secondary endosymbiont.
gest that infections that led to endosymbiotic associations have occurred
repeatedly in different groups of hosts
The rate of 1%-2% per site per 50 events). This interpretation is con- and have arisen from a variety of
million years is roughly twice as fast sistent with the observation that the free-living bacterial groups. These
as previous approximate estimates acceleration in evolutionary rate is results also suggest that endosymbiof evolutionary rates in 16S rDNA concentrated at sites in the DNA otic associations within insects can
of free-living prokaryotes (Moran sequences that are subject to natural be evolutionarily stable for long
selection. The latter inference is timespans and through periods of
and Baumann 1993). Comparisons
codiversification of hosts and bacteof 16S rDNA of Buchnera and other based on the finding that nucleotide
bacteria likewise have indicated that sites within amino acid codons that ria.
In contrast to Buchnera, which
evolutionary rates within Buchnera result in a change in the amino acid
are somewhat higher than those of sequence are more accelerated than forms a clade rather distant from
sites not effecting a change (Moran any other bacteria in the same divifree-living bacteria.
One hypothesis for the cause of 1996).
sion, the S-endosymbionts of AcyrThe concordance between phy- thosiphon pisum (Figure 2) and
this accelerated evolution is that
endosymbionts are subject to bottle- logenies of aphids and Buchnera Macrosiphum rosae fall within the
allow direct comparison of evolu- monophyletic bacterial group Ennecks in population size, particuwhich includes
tionary rates of prokaryotes and terobacteriaceae,
larly during their transmission
from mother aphid to progeny,
eukaryotes over the same timespan. such well-studied organisms as EsThis approach allows comparisons
cherichia coli and Proteus vulgaris
resulting in more frequent fixation
of rates while circumventing reli- (Baumann et al. 1995, Chen and
of slightly deleterious mutations
through genetic drift (i.e., by chance ance on estimates of ancestral dates, Purcell 1996).
Bacteria
Aphid
species
Aphids
Bacterial
14
BioScience Vol. 47 No. 1
Buchnera genes resemble those
of free-living bacteria
The nucleotide sequence of more
than 65 kilobases (kb) of DNA has
been determined for Buchnera from
the aphid S. graminum. The results
show thatBuchnera has many of the
genes present in free-living bacteria
(see box this page). The detected
genes include ones coding for proteins that are involved in DNA replication, messenger RNA synthesis,
protein synthesis, amino acid biosynthesis, glycolysis, ATP generation, secretion, and protein folding.
The presence of these genes with
diverse functions is consistent with
previous studies indicating that in
isolated cells of Buchnera, the synthesis of more than 210 proteins can
be detected. In addition, Buchnera
can incorporate radioactive precursors into DNA and rRNA (Ishikawa
Buchnera
Aromaticaminoacid family
aroA,aroE,aroH (genesof the commonportionof the
pathway)
trpA,trpB,trpC(F),trpE,trpG(genesof the tryptophan
branchof the pathway)
Cysteinebiosysthesis
cysE (firstenzymein cysteinebiosynthesis)
Chaperones
groEL, groES, hscA
Protein secretion
secB
ATP-proton motive force interconversion
atpBEFHAGDC(F1andF0 componentsof ATPsynthase)
Glycolysis
gapA (glyceraldehyde-3-phosphate
dehydrogenase),tpiA
(triosephosphateisomerase)
IDNA replication
dnaA(initiationof DNAreplication),dnaG
(primase),dnaN
1989).
During growth of the aphid, the
increase in number of Buchnera parallels the increase in aphid weight
(Baumann and Baumann 1994). This
observation implies a strict regulation of endosymbiont number and
also suggests that the orderly events
characteristic of bacterial growth,
involving the expression of many
genes and the synthesis of several
proteins, occur during Buchnera
growth. The doubling time of
Buchnera in the aphid is approximately 1.5-2.0 days, a timespan
much longer than doubling times
attainable by many free-living bacteria. Because bacteria growing at a
slow rate have a reduced demand for
ribosomes (needed for protein synthesis), many bacteria with long
doubling times have only one or two
copies of the ribosomal RNA operon
(Baumann et al. 1995). This is also
the case with Buchnera, which contains a single copy of the genes for
ribosomal RNAs. The organization
of the ribosomal genes in Buchnera
is also distinctive. In most bacteria
they form a single transcription unit
(16S-23S-5S
rRNA), whereas in
Buchnera they are arranged as two
transcription units (16S rRNA and
23S-5S rRNA; Baumann et al. 1995).
The significance of this arrangement
is not known.
Endosymbionts are sometimes
postulated to represent a transitional
January 1997
genes
Genesdetectedin Buchnerafrom the aphidSchizaphisgraminum.
(Forreferencesand a moredetaileddescriptionof the genes correspondingto the abbreviationssee Berlynet al. 1996.)
and dnaQ (DNA polymerase III subunits),
(gyrase)
DNA-dependent RNA polymerase
gidA, gyrB
rpoA, rpoB, rpoC, rpoD (sigma-70)
Degradation of RNA
rnh, rnpA
Ribosomal RNA
rrf (SS), rrl (23S), rrs (16S)
Amino acid acyl tRNA synthetases
argS, cysS, thrS
Ribosomal proteins
Other
rplL (L7/L12), rplT (L20), rpmH (L34), rpml (L35), rpsA
(Sl), rpsD (S4), rpsK (S11)
fdx (ferredoxin), himD (DNA bending, regulation), infC
(initiation factor-3), trmE (tRNA methyltransferase)
stage between free-living bacteria
and organelles, such as mitochondria or chloroplasts, that originated
from prokaryotes. A characteristic
of mitochondria and chloroplasts is
a major reduction of the genome
size, with a concomitant decrease in
gene number, and the transfer of
many essential genes to the host cell
nucleus. Buchnera differs from organelles in its large genome size and
in the retention of many genes that
are present in free-living bacteria
but absent from organelles (Baumann
et al. 1995). The basis for this difference may reflect the characteristics
of the host in which the endosymbiotic association first originated. The
ancestors of mitochondria and chlo-
roplasts infected a unicellular eukaryote; in contrast, the ancestor of
Buchnera infected a multicellular
animal host in which it was sequestered within somatic cells separate
from those of the germ line. This
separation may have limited the opportunities for the permanent transfer of genes from the prokaryotic
chromosome to the nuclear genome
of the host, because such transfer
would persist only if it occurred in
the germ line.
Buchnera and the synthesis of
essential amino acids
Plant phloem sap, the diet of aphids,
is rich in carbohydrates but deficient
15
Chorismate
trpG
trpG
tipE
od?.
S. graminum
Plasmid
(
16
14.3
kb
trpE
trpG
ftrpG
,_ trpE
ori?
Anthranilate
> Tryptophan
trpD trpC(F) trpB trpA s. graminum
S. chinensis
kbChromos
kb
4.8
Chromosome
3.9 kb
trpE trpG
Si. chinensis
Figure 3. Genetics of the tryptophan biosynthetic pathway in Buchnera from the
aphids S. graminumand SI. chinensis. Thin line = DNA; thick line = protein coding
regions. In Buchnerafrom S. graminum, trpEG is amplified by being on a plasmid
consisting of four tandem duplications of a 3.6-kb unit (top). In Si. chinensis,
trpEG is located on the endosymbiont chromosome (bottom). In Buchnera from
both aphid species, the remaining genes of the tryptophan biosynthetic pathway
are on the chromosome (middle). trpEG encodes the two subunits of anthranilate
synthase; trpD encodes phosphoribosyl anthranilatetransferase;trpC(F) encodes
a fusion protein consisting of indolglycerol phosphate synthetase and
phosphoribosyl anthranilate isomerase; trpBA encodes the two subunits of
tryptophan synthase. Each enzymatic reaction is designated by an arrow. Lines
link the enzymes (arrows) to their corresponding genes. Stippled bar on the
plasmid designates a conserved 0.5-kb region (ori?) having properties of a DNA
origin of replication. Arrowheads on plasmid indicate direction of transcription;
chromosomal genes are transcribed left to right.
in certain nitrogenous compounds,
including some of the ten essential
amino acids required by insects and
other animals (Dadd 1985). The biosyntheticactivitiesof endosymbionts
have been proposed as the source of
these amino acids for aphids (Dadd
1985). Evidence for this interpretation has come from a variety of
nutritional studies of aphid growth
on synthetic media. The most unequivocal support for this role of
endosymbiontswould be experimental resultsindicatingthat aphidswith
intact endosymbiontsgrow independently of essential amino acids and
that aphids treated with antibiotics
that eliminateendosymbiontsrequire
these amino acids for growth. In
practice, such clear-cut results are
not obtained. Many nutritionalstud16
ies are difficult to interpret because
aphids grow poorly on synthetic
media and frequently cannot be cultured beyond a few generations. In
addition, effects of antibiotics can
be only partially overcome by supplying essential amino acids. Furthermore, sustained growth of aphids
on synthetic media containing antibiotics is not possible regardless of
its composition. Despite these obstacles, recent investigations combining nutritional studies, radiolabeling experiments, and enzymatic
assays have produced direct evidence
for the synthesis by Buchnera of the
essential amino acids tryptophan,
cysteine, and methionine (Douglas
1990, Douglas and Prosser 1992).
Other studies suggest that essential amino acids are derived in part
from glutamine, which comprises a
major fraction of the amino acids
present in plant sap. In A. pisum
growing on plants, glutamine is also
a major fraction of the amino acids
in aphid tissue and aphid hemolymph
(Sasaki and Ishikawa 1995). Isolated
bacteriocytes are able to take up
glutamine and hydrolyze this compound to glutamate, whereas isolated Buchnera are able to take up
glutamate but not glutamine (Sasaki
and Ishikawa 1995, Whitehead and
Douglas 1993). Buchnera has been
shown to incorporate the nitrogen
of glutamate into seven amino acids,
which are excreted by the endosymbionts. Four of these amino acids are
essential for insects, and these experiments are consistent with their synthesis by Buchnera for the aphid host.
Gene amplification as a means of
tryptophan overproduction. Currently, the best available evidence
indicates that Buchnera overproduces tryptophan for the aphid host
(Baumann et al. 1995, Douglas and
Prosser 1992). This is an essential
amino acid for the aphid and is found
in low quantities in plant phloem.
The tryptophan biosynthetic pathway, from chorismate to tryptophan,
consists of seven enzymatic reactions. The genetics and biochemistry of this pathway have been elucidated in many different prokaryotes
(Crawford 1989). In almost all cases,
anthranilate synthase, the first enzyme of the pathway, is rate limiting
and is feedback inhibited by tryptophan. In free-living bacteria, this
mode of regulation assures that tryptophan synthesis is reduced when
the amino acid is available, thereby
conserving carbon and energy for
other cellular processes.
All of the genes for the tryptophan
biosynthetic pathway are present in
Buchnera from the aphid S. graminum (Figure 3). The endosymbionts
from this aphid show an unusual
modification that permits the increased synthesis of tryptophan for
use by the aphid host. The genes for
anthranilate synthase (trpEG) are
located on a plasmid, that is, a small
circular segment of DNA separate
from the main bacterial chromosome
(Lai et al. 1994). The remaining
genes [trpDC(F)BA] are on the
Buchnera chromosome. The trpEG
BioScience Vol. 47 No. 1
plasmid consists of four tandem duplications of a 3.6-kb unit that contains trpEG. Approximately four
such plasmids are present per
Buchnera chromosome, resulting in
an approximately 16-fold amplification of trpEG (Figure 3). Gene
amplification would increase the
amount of anthranilate synthase protein, thereby resulting in overproduction of tryptophan by Buchnera
for the aphid host. Even if there was
accumulation of tryptophan, resulting in some feedback inhibition of
anthranilate synthase activity, the
increased level of anthranilate synthase protein would still increase
In both
tryptophan
synthesis.
prokaryotes and eukaryotes, gene
amplification is a common mechanism for increasing the amount of an
enzyme activity that is limiting for
growth (Anderson and Roth 1977,
Lai et al. 1994).
The amplification of trpEG on
plasmids is an adaptation on the
part of Buchnera for being a better
mutualistic partner to its aphid host.
The natural selection underlying the
establishment of this inherited trait
would have occurred at the level
of whole aphids, with the betterprovisioned aphids outcompeting
others in the same population that
lack Buchnera with these plasmids.
Thus, the trpEG plasmids illustrate
the extent to which the aphidBuchnera association functions and
evolves as a single, fused organism.
Gene amplification of trpEG has
been detected in Buchnera from the
other members of the Aphididae that
have been examined, including the
aphids Rhopalosiphum padi, Rhopalosiphum maidis, and A. pisum
(Rouhbakhsh et al. 1996). R. maidis
has a plasmid consisting of a single
3.6-kb unit; the other aphid species
have plasmids consisting of four to
ten tandem duplications. All of these
aphids develop rapidly, reaching
maturity less than two weeks after
birth. By contrast, Schlechtendalia
chinensis has a development time of
more than six weeks. In Buchnera
from SI. chinensis, trpEG is not
amplified and is present in one copy
on the endosymbiont chromosome
(Baumann et al. 1995). The difference in the development times between SI. chinensis and the other
aphids may reflect a difference in the
January 1997
o ri?
trpE
trpG
40
0o
.
3.2 kb
40
30
? 20
20
itrpE
1111111
'"I"I"I"I"II"II
"1"1""11111
ytrpG
1
1.
3 .2 kb
Figure4. Gene silencing of trpEG in Buchnerafrom the aphid D. noxia. Bargraph
indicates the number of changes between the 3.2-kb units containing trpEG and
xtrpEG (pseudogenes). Each bar covers 100 successive nucleotides of the DNA
sequence, and the bar height shows the numberof differences.ori? = region having
properties of a DNA origin of replication.
demand for tryptophan. Aphids that
develop rapidly would require a
higher rate of tryptophan provision,
hence amplification of trpEG. In
contrast, in aphids that develop
slowly the demand for tryptophan is
reduced and gene amplification may
be unnecessary.
Phylogenetic analyses based on
trpEG sequences result in trees with
the same order of branching as trees
based on chromosomal trpB and 16S
rDNA as well as host mitochondrial
genes (Rouhbakhsh et al. 1996).
These results add support for the
long-term vertical transmission of
these genes and indicate a lack of
genetic transfer between plasmids
and endosymbionts of different aphid
lineages. In addition, the congruence of trees based on trpEG and on
other genes indicate that the amplified trpEG originated from a chromosomal trpEG and not from an
exogenous source. In all of the amplified 3.6-kb units, only an approximately 500 base pair segment upstream of trpEG is highly conserved.
The beginning segment of this region has some of the properties of an
origin of DNA replication (ori?), a
DNA site that binds the proteins and
enzymatic machinery that initiates
DNA replication (Rouhbakhsh et al.
1996). Directly upstream of trpE is
a sequence resembling a promoter
that may be used for trpEG expression. Note that neither of these functional attributes has been established
experimentally.
trpEG amplification may be a stable
attribute. In free-living bacteria, gene
amplification by tandem duplications may occur with a frequency as
high as 10-4-10-5 per cell per generation. Once the selective conditions
that favor gene amplification are
removed, the number of gene copies
is rapidly reduced by homologous
recombination, a process that removes DNA fragments that have
sequence homology (Anderson and
Roth 1977). During the life of the
aphid there may be periods in which
the tryptophan demand is reduced.
If this were to lead to a reduction in
trpEG to one copy, then a return to
a high tryptophan demand would
again necessitate a mutation resulting intrpEG amplification. Because
such a mutation would occur in only
a small fraction of the population it
would perhaps be advantageous for
Buchnera to stabilize gene amplification by removing the mechanism
that results in the rapid decrease in
gene number. These speculations
may provide an explanation for the
somewhat peculiar situation that is
found in the trpEG-containing plasmid of Diuraphis noxia, the Russian
wheat aphid.
Buchnera from this aphid contains a plasmid consisting of eight
3.2-kb units, slightly smaller than
those of the other Buchnera plasmids. There are approximately two
plasmids for each D. noxia endosymbiont chromosome (Lai et al.
1996). One of these units contains
an intact copy of trpEG, whereas
the remaining seven units contain
trpEG pseudogenes, that is, trpEG
sequences that cannot code for a
functional full-length polypeptide
17
becausetheyareriddledwith muta-
which are so-called chaperones, proteins whose major role is the prevention of misfolding, which might
occur during protein synthesis, transleuA
7.8 kb
location across membranes, and releuD
covery from stress (Zeilstra-Ryalls
et al. 1991). Many chaperones are
leuB
at high levels within cells
present
Leucine
lieuc
under laboratory conditions of cultivation. Because a variety of deletea-Keto
a-Keto
rious conditions, including an inisovalerate
isocaproate
crease in temperature, results in their
Figure 5. Plasmid containing genes of
the leucine biosynthetic pathway from increase, these proteins have also
Buchnera of the aphid R. padi. Each been called "stress" or "heat shock"
enzymatic reaction is designated by an proteins (Zeilstra-Ryalls et al. 1991).
arrow. leuA = isopropylmalate syn- In E. coli grown at 30?C, GroEL
thase; leuB = P-isopropylmalate dehy- constitutes 1% of the total protein,
drogenase; leuCD = two subunits of whereas at 46?C the concentration
isopropylmalate isomerase. Lines link is increased to 12% (Zeilstra-Ryalls
the enzymes (arrows) to their corre- et al. 1991). In this and most other
sponding genes. Arrowheads on plas- bacteria the
for GroES and
mids indicate direction of transcrip- GroEL are genes as a
arranged
single trantion. Redrawn from the nucleotide
in
the
unit
order
groELscription
sequence of Bracho et al. (1995).
groES. Their expression is regulated
primarily by •32, a heat shock refactor. This
reactions (Umbarger 1996). Some- sponse transcription
what analogous to the tryptophan protein recognizes a set of promotbiosynthetic pathway, the first en- ers different from that recognized by
zyme of the leucine pathway, iso- the principal transcription factor
propyl malate synthase (LeuA; Fig- used for the initiation of transcripure 5) is feedback inhibited by the tion of most genes during vegetative
end product, leucine (Umbarger growth.
Buchnera from the aphids A.
1996). The Buchnera plasmid contains the genes coding for the first, pisum and S. graminum have groESpresumably feedback-inhibited en- groEL arranged in the same order as
zyme, as well as the two subsequent in E. coli. These genes are also preenzymes (Figure 5). The fourth reac- ceded by a putative promoter contion, which is a transamination, can taining nucleotide sequences similar
in E. coli be catalyzed by several to those recognized by032 (Baumann
enzymes; the genes for these enzymes et al. 1995, Ohtaka et al. 1992).
are not included on the Buchnera When subjected to a variety of treatplasmid. A similar plasmid has also ments, isolated Buchnera have an
been found in Buchnera from R. increased incorporation of radioacmaidis, S. graminum, and two other tive amino acids into GroEL as is
species in the same family of aphids characteristic of the stress or heat
(Bracho et al. 1995). The number of shock response (Morioka and Ishcopies of the plasmid containing the ikawa 1992). In Buchnera from S.
Plasmid location of the genes under- leucine biosynthetic genes relative graminum, GroEL constitutes aplying leucine biosynthesis. Because to the Buchnera chromosome has proximately 10% of the total pronot been determined. Current re- tein (Baumann et al. 1996). This
Buchnera is thought to synthesize
many or all of the essential amino sults suggest that it is present in level is only slightly less than that in
acids for the aphid, it would be ex- multiple copies, which is consistent E. coli at 46?C, a temperature near
pected that genes for other amino with the amplification of the leucine the maximum at which growth ocacid biosynthetic pathways may also biosynthetic genes and the overpro- curs and one at which many of the
be found amplified on plasmids. This duction of this amino acid for the E. coli stress proteins are present in
high amounts. Many intracellular
has recently been found to be the aphid host.
pathogenic bacteria as well as sevcase with genes of the leucine patheral endosymbionts also have inet
al.
Bracho
is
GroEL
1995).
5;
way (Figure
overproduced
creased or high levels of GroEL, a
In E. coli and other organisms, this in Buchnera
finding that has been attributed to
pathway is initiated from a-ketoisothe "stress" of the "hostile" intraessenthe
All
to
leuis
converted
which
produce
prokaryotes
valerate,
tial proteins GroEL and GroES, cellular environment (Morioka and
cine by means of four enzymatic
tions resulting in stop codons and
reading frame shifts. A comparison
of the nucleotide differences between
trpEG and a trpEG pseudogene indicates that most of the differences
occur in the region corresponding to
the putative promoter and the Nterminal portion of trpE (Figure 4).
These changes would result in the
silencing of trpEG expression.
D. noxia inflicts major damage to
cereals. It differs from other cereal
aphids in causing much more severe
lesions, involving histolysis of the
plant cells and disintegration of chloroplasts and mitochondria. The damage may release some free tryptophan
into nearby phloem tissue. Availability of this tryptophan to the aphid
would reduce the demand for trpEG
amplification, and the energy and
nutritional costs of making the anthranilate synthase protein would
favor a reduction in its production
when it is not needed for host nutrition. As indicated above, in Buchnera of D. noxia, a reduction in the
energy wasteful synthesis of anthranilate synthase is accomplished by
gene silencing. Gene silencing and
the retention of pseudogenes may be
a consequence of stabilization of gene
amplification by the elimination of
the recombination mechanism that
leads to the rapid decrease of gene
copies. The persistence of one functional trpEG unit is consistent with
the fact that D. noxia sometimes
inhabits other grass species, on which
extensive lesions are not induced.
On these plants, tryptophan is probably not released into phloem sap,
and survival would require the synthesis of tryptophan on the part of
Buchnera.
18
BioScience Vol. 47 No. 1
Ishikawa 1992). However, to consider the high level of GroEL as
always indicative of stress is an unwarranted extension of a useful concept developed from studies of responses of organisms subjected to
potentially deleterious conditions.
Because the association between
Buchnera and aphids is 150-250
million years old, the intracellular
environment must be considered the
normal habitat for these bacteria and
can no longer be described as stressful. For Buchnera, the high level of
GroEL is the norm.
Conclusions
The aphid is a composite organism
intimately associated with and dependent on a prokaryotic endosymbiont (Buchnera). Application of the
methods of molecular biology has
given us new information on fundamental aspects of this association.
Molecular phylogenetic evidence
strongly supports a single origin of
the endosymbiotic association at
least 150-250 million years ago, in a
common ancestor of all modern
aphids. Subsequently there was
cospeciation of Buchnera and aphids,
with no transfer of endosymbionts
or endosymbiont genes between
aphid lineages. Relative to free-living bacteria, Buchnera and other
endosymbionts exhibit elevated rates
of sequence evolution, perhaps as a
result of decreased effectiveness of
selection under population structures
that increase genetic drift.
Despite its long history as an endosymbiont,Buchnera retains many
of the genetic features of free-living
bacteria. Some of these properties
have been modified and exploited
for the benefit of the host. Among
these are the plasmid association
and amplification of the genes coding for the limiting enzyme of tryptophan biosynthesis and the three
enzymes of leucine biosynthesis.
These essential amino acids are lacking in the phloem sap diet of aphids.
Amplification of the genes underlying their biosynthesis in Buchnera
appears to be a route for the overproduction of these nutrients, which
are supplied to the aphid host. Thus,
these plasmids represent adaptation
on the part of Buchnera to be a
better mutualist, and they illustrate
January 1997
the degree to which the aphid-Buchnera association functions as a single
composite organism. Another possible adaptation to an endosymbiotic existence in Buchnera is the
presence of only a single copy of
genes coding for rRNA. One or few
copies of rRNA genes is characteristic of bacteria that do not achieve
rapid rates of growth. Finally, as is
the case with many intracellular organisms,Buchnera has increased levels of GroEL, a chaperone involved
in the proper folding of proteins.
The role of this increase is unclear,
but it probably functions as an adaptation to the endosymbiotic lifestyle.
Although many questions remain
unanswered, the association between
Buchnera and aphids is currently the
best understood of any endosymbiosis in animals. Preliminary studies of
other endosymbionts suggest that
some of the features seen in Buchnera may be usual at least in insect
endosymbionts. For example, recent
molecular phylogenetic research on
endosymbioses of tsetse flies, carpenter ants, and cockroaches suggest that they result from ancient
infections followed by cospeciation
(Aksoy et al. 1995, Bandi et al. 1995,
Schr6der et al. 1996), and elevated
levels of GroEL protein recently have
been noted for tsetse fly endosymbionts (Aksoy 1995). Thus, the association between aphids and Buzchnera serves as a paradigm for studies
of associations between prokaryotic
endosymbionts and other animals.
Acknowledgments
Research from the authors' laboratories was supported by National
Science Foundation grants IBN9201285 and MCB-9402813 to P.
Baumann, DEB-9306495 to N. A.
Moran, DEB-9527635 to Moran and
Baumann, by Entotech Inc. (Novo
Nordisk) to Baumann, and by the
University of California Experiment
Station to Baumann. Due to space
limitations many references could
not be cited; most are included in
Baumann et al. 1995 and Moran et
al. 1995.
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