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Colloquium
A genomic perspective on nutrient provisioning by
bacterial symbionts of insects
Nancy A. Moran*~t, Gordon R. Plague*t, Jonas P. Sandstrom§, and Jennifer L. Wilcox*
*Department of Ecology and Evolutionary Biology and tCenter for Insect Science, University of Arizona, Tucson, AZ 85721; and Department of Entomology,
Swedish University of Agricultural Sciences, SE-75598 Uppsala, Sweden
Many animals show intimate interactions with bacterial symbionts
that provision hosts with limiting nutrients. The best studied such
association is that between aphids and Buchnera aphidicola, which
produces essential amino acids that are rare in the phloem sap diet
Genomic studies of Buchnera have provided a new means for
inferring metabolic capabilities of the symbionts and their likely
contributions to hosts. Despite evolutionary reduction of genome
size, involving loss of most ancestral genes, Buchnera retains
capabilities for biosynthesis of all essential amino acids. In contrast,
most genes duplicating amino acid biosynthetic capabilities of
hosts have been eliminated. In Buchnera of many aphids, genes for
biosynthesis of leucine and tryptophan have been transferred from
the chromosome to distinctive plasmids, a feature interpreted as a
mechanism for overproducing these amino acids through gene
amplification However, the extent of plasmid-associated amplifi-
cation varies between and within species, and plasmid-borne
genes are sometimes fewer in number than single copy genes on
the (polyploid) main chromosome. This supports the broader in-
terpretation of the plasmid location as a means of achieving
regulatory control of gene copy number and/or transcription.
Buchnera genomes have eliminated most regulatory sequences,
raising the question of the extent to which gene expression is
moderated in response to changing demands imposed by host
nutrition or other factors. Microarray analyses of the Buchnera
transcriptome reveal only slight changes in expression of nutrition-
related genes in response to shifts in host diet, with responses less
dramatic than those observed for the related nonsymbiotic species,
Escherichia coli.
Symbioses between bacteria and eukaryotes, i.e., chronic
infections that are part of the normal life history of the host
and are often beneficial, are ubiquitous in nature but have
historically received little attention from experimental biologists
or ecologists. This situation has been reversed in the last few
years, which have seen a surge of interest and progress in this
field (14. One useful view of these symbioses is as persistent
intimate associations in which partners interact through the
transfer of molecules, particularly small molecules that are
essential to the growth requirements of each organism. However,
the interdependence of partners that makes symbiosis so intrigu-
ing also has made it difficult to study. In particular, the inability
to culture most symbionts has thwarted characterization of their
chemical interactions with hosts. Recently, however, the acqui-
sition of DNA sequences, including whole genomic sequences, of
symbionts has enabled major progress in defining the biosyn-
thetic capabilities that underlie contributions to hosts.
Invertebrates generally, and insects especially, show a partic-
ularly striking variety of symbioses with bacteria. Many of these
associations are conspicuous because of the presence of a
"bacteriome" (or "mycetome"), a specialized structure in the
host body that houses the symbionts and that can occupy a
substantial proportion of the host biomass. The symbionts are
often intracellular but frequently enclosed within the cytoplasm
www.pnas.org/cgi/doi/10. 1 073/pnas.21 35345 1 00
by a host-derived membrane. Bacteriome-associated symbioses
of animals, with emphasis on arthropods, were studied exten-
sively by light microscopy during the first half of the 20th century
and summarized in a book by the prominent symbiosis re-
searcher Paul Buchner (2~. The volume describes a kind of
fairyland of intimate and highly specialized biological interac-
tions, many of which have not been further studied. Despite the
anatomical and histological diversity of these symbiotic arrange-
ments, Buchner proposed a unified functional role for bacteri-
ome occupants, hypothesizing that their raison d'etre is the
provisioning of nutrients that animals are unable to synthesize
themselves and that are absent or limiting in the specialized diets
exploited by particular animal groups. Extensive sections of his
book are devoted to insects with highly restricted diets, such as
plant sap or vertebrate blood. Although nutritional provisioning
has been better documented for some associations than others,
the overall evidence supports the view that symbiosis has often
enabled the expansion of animal niches and has contributed
substantially to evolutionary diversification. The macroevolu-
tionary role of nutrition-based symbiosis can be appreciated by
recogn izing that insects feeding on phloem or xylem sap, includ-
ing aphids, psyllids, whiteflies, scale insects, planthoppers, cica-
das, spittlebugs, and most leafhoppers, are dependent for their
way of life on obligate bacterial endosymbionts.
Recently, molecular approaches have enabled substantial
progress in understanding the evolution and functioning of the
obligate symbionts of several insect hosts. Based on DNA
sequence data, we now know that intracellular, bacteriome-
associated symbiotic associations of insects, involving vertical
transmission through eggs, typically descend from ancient infec-
tions of ancestors dating back 100 million years or more (1~.
Phylogenetic studies have also revealed that many of the intra-
cellular symbionts within insects are closely related to the well
studied experimental organism, Escherichia coli, for which func-
tional information is available for many gene products. This fact
has provided an ideal basis for using gene sequences to infer
symbiont metabolic capabilities, including their contributions to
host nutrition.
Here we consider the question of how a bacterial genome has
been modified in the context of the new demands of symbiosis,
especially nutrient provisioning to host tissues. We focus on the
best-studied insect symbiont, Buchnera aphidicola, which has
convolved with its aphid hosts for >100 million years (3~. We
have divided the question into two main aspects: (i) modifica-
t~ons of genome contents and organization and (ii) modification
of controls on gene expression. Because different hosts encoun-
This paper results from the Arthur M. Sackier Colloquium of the National Academy of
Sciences, "Chemical Communication in a Post-GenomicWorici," heic! January 17-19, 2003,
at the Arnold and Mabel Beckman Center of the National Academies of Science and
Engineering in Irvine, CA.
tTo whom correspondence should be acIdressed. E-maii: nmoran~u.arizona.edu.
@) 2003 by The National Academy of Sciences of the USA
PNAS 1 November 25, 2003 1 vol. too 1 suppl. 2 1 14543-14548
OCR for page 32
ter varying nutritional demands and varying dietary conditions, A2s
we expect some heritable modifications manifested as differ-
ences among species or populations. In addition, individual host a. 20
species must deal with a variety of nutritional limitations during ',,
the course of a season or while using different resources or O
habitats; thus, we might expect that symbiont productivity can be ~ 15
modified as a result of environmental factors. E
5 10
Nutritional Provisioning as a Primary Task of Symbionts in
Sap-Feeders
Animals generally are lacking in pathways underlying amino acid 5
production, typically requiring 10 "essential" amino acids in the
diet; this limitation must stem from ancient gene loss in an
ancestor of Metazoa. Animals also have a large number of
dietary requirements for enzymatic cofactors (or "vitamins"~.
Studies of phloem sap composition, based on collections from
the severed mouthparts of aphids, show that nitrogen is almost
exclusively present as amino acids, and that nonessential amino
acids predominate (44. In general, the essential amino acids are
present in relative concentrations considerably lower than those
found in aphid proteins or in dietary requirements determined
for insects. This imbalance implies that insect growth potential
can be enhanced by symbionts, which absorb abundant amino
acids and sugars from the host and use these to generate limiting
amino acids through biosynthetic capabilities lacking in insects.
However, both the total concentrations of pooled amino acids as
well as the relative concentrations of individual essential amino
acids can differ dramatically among plant species and pheno-
logical stages (e.g., refs. 5 and 6~. Amino acid levels and profile
also fluctuate, depending on the plant response to aphid feeding
(7~. Experimental studies have provided some evidence that the
nonessential amino acid glutamate is the major nitrogenous
compound exported from the aphid and used by Buchnera for
production of the essential, limiting amino acids (8-10~.
Reading the Genome Sequences of Insect Symbionts: Old Tools
for New Tasks
Complete genome sequences are available for Buchnera of three
distantly related aphid species fAcyrthosiphon pisum (11), Schiza-
phis graminum (12), and Baizongia pistaciae (13~], for W-ggles-
worthia brevipalpis, the obligate symbiont of tsetse fly (14), and
for Blochmannia camponotii, the obligate symbiont of carpenter
ants (15~. The genome sequences of several other insect symbi-
onts are expected to be available soon. Genomic sequences allow
us to identify which pathways symbionts possess, and, even more
uniquely, which pathways they lack. This information has greatly
extended our knowledge of the nutritional role of symbionts
within hosts and provides a foundation for further studies of
chemical exchange between host and symbiont.
Based on the systems studied so far, bactenome associates in
insects show extensive genomic modifications, including massive
loss of ancestral genes and many rearrangements affecting order
and orientation of genes on the chromosome. For Buchnera,
~gglesworthia, and Baumannia, genomes are reduced to only
~20% of the genes present in ancestors and modern relatives,
and each contains many unique gene arrangements, yielding only
small regions of synteny with related bacteria (15-17~. Further-
more, in contrast to most other bacterial genomes ~ncluding
those of its closest relatives, these symbiont genomes show little
evidence of gene uptake. Indeed, Buchnera lineages appear to
lack any novel genes not present in related free-living bacteria,
and comparisons of Buchnera genomes, that diverged with their
ancestors >100 million years ago, show lower levels of gene
acquisition or genomic rearrangements than do any other fully
sequenced bacteria (11~.
The combination of genome reduction and absence of gene
acquisition implies that, in these insect symbioses, the basis for
the symbiotic contributions lies in ancestral genes and pathways,
14544 1 www.pnas.org/cgi/doi/10.1073/pnas.2135345100
OBuchnera(Sg): 541 Genes
· E. cold MG1655: 3587 Genes
· _
· 1. ]! Is 1~' ~ 1
a,~,n,n,n,8.~. ~ ~
B25
| 1.~ ·Buchnera Favorable Plant
~20
15
-
e,. 10
~ 51 ;
u,
~ Buchnera Resistant Plant
· E. col~ Rich Medium
~ E. cold Minimal Medium
~ ~ = ~Q .~~n ~ ~ ~ ~ - =~ ~ ~ ~8Q ~Q ~ C~ =~
O O ~ ~ tO ~ ~ o o o ~ o U, ~ ,~ o ~
° E z ,~ ~ Z E e~ O ~ ,,, t;
Fig. 1. (A) Proportional representation of genes grouped by functional
categories within the genomes of Buchnera (S. graminum) and E. coli, based
on functional categorizations established for the Clusters of Orthologous
Groups (COGs) (52). (B) Representation of COG functional categories in the
transcriptomes of Buchnera (S. graminum) and E. co/i. Buchnera were from
mixed-age host aphids grown at 20°C on plants differing in phloem concen-
trations of total and essential amino acids (barley versus resistant wheat
cultivar; plants as in ref. 7). E. colidata are from the public ASAP database (53)
and depict transcriptomes of early log-phase cultures grown in amino acid
"Rich" LB medium versus "Minimal" Mops medium plus 0.2% glucose (J.
Glasner, personal communication). Microarray attributes, RNA extraction
procedures, and data normalization steps were similar for both bacterial
species (42, 49). Bar height is the sum of gene signals in a given COG divided
by the number of genes in that COG; bars are standard deviations based on
experimental replicates (five replicates per Buchnera treatment; two and six
replicates for E. cold LB and Minimal Media treatments).
present in free-living relatives, that were coopted for the purpose
of provisioning hosts after the initiation of symbiosis. For both
Buchnera and ~gglesworthza, the maintenance of ancestral genes
for nutrient biosynthesis provides a clear indication of their
mutualistic relationship to hosts and sharply contrasts with the
loss of these same pathways from genomes of obligately parasitic
bacteria, which acquire metabolites from host tissues (1~. Buch-
nera of A. pisum has genes for all of the essential amino acid
pathways (total of 54 loci), plus genes for fixation of inorganic
sulfur and for synthesis of riboflavin (13~. Because unneeded
genes are quickly eliminated from these genomes, the retention
of these biosynthetic pathways gives a clear indication of a
continued contribution of the corresponding nutrients to hosts.
The prominence of essential amino acid production as an
activity of Buchnera is evident from the set of genes retained
(Fig. lA). The E. cold genome encodes ~60 genes for the core
biosynthetic pathways underlying production of essential amino
acids, ~20 for the core pathways for nonessential amino acids
Moran et a/.
OCR for page 33
Table 1. The ratios of copies of plasmid-borne amino acid biosynthetic genes (leuABCD, trpEG) to chromosomal
gene copies for Buchnera of different aphid species and strains
Aphid host Aphid clone/population leuABCD trpEG
A. pisum 12 United Kingdom clones - 2.4~16.2*
N. A. Moran lab clone 5A (Madison, Wl) 0.6 4.8
Diuraphisnoxia P. Baumann late clone (Lincoln, NE) 0.9t 1.8$
South Africa population 0.3 0.4
Rhopalosiphum maidis N. A. Moran lab clone (Tucson) - 0.3
S. graminum Biotype B (K. A. Shuiran lab clone) - 0.5
Biotype E (T. Mittler lab clone) 23.5t 14.5§
Biotype E (P. Baumann lab clone) 1.4 2.1
Biotype E (N. A. Moran lab clone) 1.9 1.S
Biotype E (K. A. Shufran lab clone) 1.6 2.6
Biotype G (K. A. Shuiran lab clone) 0.5 2.4
Biotype SC (K. A. Shufran lab clone) - 0.5
Uroleucon ambrosias 86 individuals, 15 U.S. populations 0.5-2.8~ 0.3-~.9~
Unless otherwise noted, values are previously unpublished and were estimated by using real-time quantitative PCR as in ref. 25 (primer
sequences are available upon request). The reason for the discrepancy among Buchnera (S. graminum) estimates is uncertain, although
the concordance of the quantitative PCR estimates across biotypes provides support for their accuracy.
*Ref. 27; calculated by using quantitative DNA hybridization.
tRef. 26; calculated by using quantitative DNA hybridization.
tRef. 30; calculated by using quantitative DNA hybridization.
§Ref. 18; calculated by using quantitative DNA hybridization.
~Ref. 25; calculated by using real-time quantitative PCR.
plus >50 genes involved in transport of amino acids. Of these,
Buchnera retains almost all of the genes for production of
essential amino acids and only two (or four, depending on how
amino acids are categorized) for synthesis of nonessential amino
acids or amino acid transport (13~. Overall, the core genes
for biosynthesis of essential amino acids comprise a larger
proportion (~10~o) of the Buchnera genome than of the E. cold
genome (<2%~.
How Are 6enomes of Obligate Symbionts That Provision Hosts
Modified Relative to Those of Fre+Living Relatives?
The large majority of genes underlying amino acid biosynthetic
pathways are confined to the main Buchnera chromosome.
However, one of the early striking discoveries about the Buch-
nera genome, seemingly linked to the nutrient-provisioning role,
was the finding that rate-limiting genes for biosynthesis of
tryptophan (trpEG, ref. 18) and the genes for biosynthesis of
leucine (leuABCD, ref. 19) are encoded on two distinct types of
multicopy plasmids. Each of these plasmids has apparently
evolved only one or very few times (20-22), and some early-
branching Buchnera lineages retain these genes on the main
chromosome in an ancestral position (e.g., refs. 12 and 23~.
The initial interpretation of the plasmid position of trpEG and
leuABCD was that it enabled overproduction of the end products
for host nutrition, because the plasmid location allowed ampli-
fication of gene copy number relative to single copy chromo-
somal genes. The subsequent discovery that each Buchnera cell
contains multiple chromosomal copies (24) raised the more
general interpretation of the plasmid location of trpEG and
leuABCD as an arrangement allowing either increase or decrease
in copy number relative to chromosomal genes (25), and perhaps
allowing regulation of the level of amplification in response to
environmental conditions. Ratios of plasmid-borne trpEG and
leuABCD copies to chromosomal gene copies do vary among
species, possibly in a direction that is related to host needs (26~.
Table 1 summarizes previously published and newly estimated
ratios of plasmid-borne amino acid biosynthetic genes to single
copy chromosomal genes in Buchnera of different aphid strains
and species. The ratios of plasmid-borne genes to single-copy
chromosomal genes range from much less than 1-fold (implying
fewer plasmid copies than chromosome copies within a cell) to
Moran et a/.
>10-fold, with rather similar ranges for relative amplification of
both leu and trp genes (ref. 26 and Table 1~. Values vary both
within and between species, with at least part of the variation
heritable (e.g., ref. 27~. Thus, the ratios of copies of trpE to trpB
(a single copy chromosomal gene) appear stable among different
aphids of the same maternal line (27~. Also, the amplification
level may be quite constant for some species and more variable
for others, based on analyses of geographically dispersed isolates
of several species (refs. 25, 27, and 28 and Table 1~.
In the case of t~pEG, the variation in ratio of plasmid-borne
to chromosomal genes is due both to differences in numbers of
repeats per plasmid (21) and to differences in relative plasmid
copy number. But another variable affecting the number of
functional trpEG copies is the frequent inactivation of some gene
copies. Early stop codons, large deletions, and frameshift mu-
tations have been found in at least some of the plasmid-borne
t7pEG copies of several aphid species (13, 28-31) and can be
geographically widespread within a species (28~.
The rarity of origins of plasmid amplification, the apparent
heritability and stability of amplification levels, and the frequent
inactivation of trpEG all suggest that levels of plasmid amplifi-
cation do not provide a route for quick responses to changes in
demands for leucine or tryptophan production. Thus, although
the plasmids are likely to be involved in adjusting production of
amino acids, the exact mechanism is not clear. This picture is
supported by data suggesting that tryptophan production is not
correlated with the ratio of trpEG to single copy chromosomal
gene copies among different A. pisum strains (27), although
these ratios could have included inactivated trpEG copies (see
32~. Observations so far do not exclude the possibility of some
feedback control of plasmid copy number by amino acid con-
centrations, as a major heritable component does not rule out
plasticity in the ratio of plasmid-borne to chromosomal genes.
Degradation of Symbiont Provisioning Through Gene
Inactivation and Loss
In contrast to the situation in A. pisum in which all of the essential
amino acids pathways are conserved, several lines of evidence
indicate that individual pathways are sometimes inactivated or
lost in particular Buchnera strains. The absence of a gene can
only be definitively concluded when whole genome sequences
PNAS I November 25, 2003 1 vol. 100 1 suppl. 2 1 14545
OCR for page 34
are determined. Among the three Buchnera genomes now avail-
able for distantly related aphid species with very different
patterns of host plant use, loss of the pathway for fixation of
inorganic sulfur occurred independently in two lineages; this loss
precludes synthesis of cysteine and methonine by using inorganic
sulfur sources (11, 12~. In addition, Buchnera of Baizongza
pistaciae has lost capacity for arginine biosynthesis, with the
relevant genes deleted from positions at several locations in the
genome (12~. The fact that two of the three sequenced Buchnera
genomes have lost functionality of some nutrition-related path-
ways strongly suggests that such losses are widespread evolu-
tionary events. This hypothesis is reinforced by nutritional
studies suggesting that certain amino acids are dietary require-
ments for some aphid/Buchnera isolates. Results of nutritional
studies on A. pisum and several other aphid species have been
interpreted as indicating species-specific or strain-specific re-
quirements for most of the 10 essential amino acids (including
arginine, histidine, isoleucine, leucine, lysine, phenylalanine,
threonine, tryptophan, and valine) (e.g., refs. 33 and 34~. Nu-
tritional studies also have shown variation among species in
whether inorganic sulfate could replace either or both of the
sulfur-containing amino acids (e.g., ref. 35~.
Genomic sequence data show that the essential amino acid
pathways are generally present in Buchnera but sometimes inacti-
vated or lost (11, 12), providing striking support for the following
interpretation of nutritional results. First, the usual absence of
dietary requirement for essential amino acids, which contrasts with
animals generally, reflects provisioning by Buchnera. Second, in-
dividual pathways are repeatedly lost in ~ndividualBuchnera strains,
resulting in reversion to host dependence on dietary sources.
Because the Buchnera of most aphids appear to have retained a
large complement of amino acid biosynthetic pathways, a corollary
is that the lineages undergoing these inactivation events may be
evolutionary dead ends. The report of substantially smaller ge-
nomes in the Buchnera of some aphid species (36) raises the
possibility that even more erosion of biosynthetic capabilities has
occurred In some lineages, which may encounter enriched diets or
rely on additional, novel symbionts (or "secondary" symbionts)
(e.g., refs. 37-40) for some amino acids.
The emerging picture is that provisioning services of obligate
symbionts can be eroded by losses of gene function. The lack of
recombination or gene uptake by Buchnera implies that such
losses are irreversible. Inactivation of genes underlying biosyn-
thetic abilities may occur under circumstances in which dietary
supplies are sufficient, and data on phloem sap contents indi-
cates that particular amino acids may be adequate in some plants
(4, 41~. Nonetheless, these losses are expected to impose per-
manent limitations on future abilities of aphid lineages to
colonize novel foodplants. Possibly, associations with secondary
symbionts may be driven by the need to replace functions
formerly provided by Buchnera.
Integration of Symbiont Gene Expression with the Nutritional
Economy of Hosts
Experiments based on full genome microarrays provide a general
overview of patterns of gene expression in a symbiont genome,
allowing us to ask questions of how expression diverges from that
of related free-living bacteria and to address the issue of whether
symbionts are able to alter gene expression to fit host needs.
Laboratory and statistical methods underlying the microarray
results presented here have been described (42), and we limit
ourselves to a generalized overview of symbiont gene expression
patterns. Fig. 1B contrasts transcript abundances, grouped by
gene functional categories, between Buchnera of Schizophis
graminum and E. coli. Buchnera is represented as samples
derived from aphids feeding on nutritionally favorable and
unfavorable plants (7~; E. cold is represented as cultures from
growth media with and without preformed amino acids (J. D.
14546 1 www.pnas.org/cgi/cioi/10. 1 073/pnas.21353451 00
16.0
14.0
o
12.0
l
~ 10.0 ,
:~ 8.0 ,
,
6.0 l
_
.u ;
' 4.0
2.0
16 3
S
CL
12 C
="
8 ~
In
8
4 91
en
o
SO
lo
lo:
I ·Buchnera Favorable Plant
~BlJchnera Resistant Plant
·E. cold Rich Medium
~ E. cold Minimal Medium
' Essential Amino Acids Ess-nt'8
Fig. 2. Transcript abundances of genes in essential and nonessential amino
acid biosynthetic pathways. Colored bars and leftyaxis are average transcript
signals summed for a given pathway and averaged over experimental repli-
cates. Gray bars and rightyaxis show numbers of genes per pathway grouping
and represent expected signal intensity based on numbers of genes. All data
are for orthologs shared between Buchnera(Sg) and E. colt. ARG, argA, argB,
argC, argD, argE, cars, care, argF, argG, and argH; ILE/LEU/VAL, ilvH, ilvl, ilvC,
and ilvD; LEU, leuA, leuC, leuD, and leuB; TRP/PHE, aroH, aroB, aroD, aroE,
aroK, aroA, and aroC; TRP, trpE, trpD, trpC, trpA, and trpB; PHE, pheA;
PHE/HIS, hiss; HIS, hisG, hisl, hiss, hisH, hisF, hisB, and hiss; LYS, dapA, dapB,
dapD, dapF, and IysA; LYS/THR, thrA, asd, thrB, and thrC; MET, mete, and
metF; GLY, g/yA; CYS, cysE, and cysK. Block shading below the graph groups
amino acid pathways as branched, aromatic, etc.
Glasner, personal communication). For both organisms, the
profiles across the functional categories are strikingly similar for
genome content (Fig. 1A) versus transcriptome content (Fig.
1B). The fraction of genes in a particular clusters of orthologous
groups (COG) category shows a highly significant linear rela-
tionship to the pooled expression level for that category, both for
Buchnera (for both treatments, R2 = 0.98, P < 0.0001) and E. cold
(for both treatments, R2 = 0.93, P < 0.0001~. As noted above, the
Buchnera genome has differentially retained genes for essential
amino acid biosynthesis (a subset of the amino acid metabolism
category found in E. cold as shown in Fig. 1), and this greater
representation extends to the transcriptome, suggesting greater
investment in functions that underlie host provisioning. Another
notable feature is that chaperoning are relatively highly ex-
pressed in Buchnera, even under nonstress conditions, as previ-
ously noted (Fig. 1B and ref. 42~.
The parallel between number of genes and transcript abun-
dance extends to the level of individual amino acids pathways for
both Buchnera and E. coli, although, as expected, the growth
media has a major influence on the expression levels in E. cold
(Fig. 2~. The main amino acids that are deficient in the S.
graminum diet (the phloem sap of grasses) are arginine and
lysine, which are two of the most common amino acids in the
insect proteins (4~. Transcript abundances for genes in these
pathways tend to be high (Fig. 2~. In contrast, genes underlying
production of methionine and cysteine show relatively low
transcript levels, possibly reflecting the fact that these pathways
are of no use for amino acid provisioning in this Buchnera
species, because the upstream pathway for sulfur fixation has
been inactivated (11~.
As noted above, aphids feed on heterogeneous populations of
plants that impose varying demands on Buchnera's biosynthetic
capabilities, raising the question of how the symbiont genome
responds. In free-living bacteria, availability of a particular
amino acid has a dramatic effect on its rate of production, and
this feedback control arises from a myriad of mechanisms,
involving transcript production as well as inhibition of enzymes.
For the relatively long and energetically expensive pathways
Moran et a/.
OCR for page 35
underlying production of the essential amino acids, transcript
production is typically governed by multiple regulatory mecha-
nisms in E. cold and other bacteria. These same pathways
underlie the central contribution of Buchnera to host nutrition.
In view of the varied diets of hosts and the apparent cost of excess
production of biosynthetic enzymes and end products, we might
expect mechanisms for transcriptional regulation of these genes
in Buchnera. This expectation is reinforced by the fact that amino
acid provisioning comprises a relatively large proportion of the
genome and transcriptome of Buchnera.
But, if transcriptional control mechanisms are present at all, they
are altered dramatically in Buchnera. The first evidence for loss of
the usual regulatory mechanisms came from examination of regions
upstream to structural genes for the biosynthetic enzymes. These
lack leader peptides and show altered sequences that indicate
degraded or altered binding properties, as noted, for example, for
aromatic amino acid pathways (43, 44~. The first complete genome
of Buchnera revealed the lack of recognizable orthologs of any of
the regulatory proteins that bind with free amino acids to block or
activate transcriptional promoter sites (13), although metR was
identified as an intact ORE in the second genome (11~.
The loss of recognizable gene regulatory features in sequence
analyses constrasts with findings of physiological studies. Several
of these have provided evidence that aphids with an intact
symbiosis can down-regulate their production of specific amino
acids when abundant. This suggests that Buchnera has one or
more negative feedback mechanisms governing amino acid
production (45-47~. For example, in studies on Aphis fabae
provided with radioactively labeled glut amic acid, a greater
proportion of label was incorporated into the essential amino
acid isoleucine on diets lacking isoleucine than on diets con-
taining it (48~. Similarly, inA. pisum, neosynthesis of all essential
amino acids was depressed on a diet with plentiful essential
amino acids relative to diets with limiting amounts, with synthesis
of histidine and arginine completely suppressed (45~. Although
it is plausible that the apparent mechanisms for regulating amino
acid production involve only feedback inhibition of the enzymes
rather than transcriptional regulation, this would provide a stark
contrast to the situation in other bacteria, in which regulation of
transcription is a prominent means of control.
As reported in Wei et al. (49), the fraction of the E. cold
transcriptome devoted to amino acid biosynthetic genes in-
creases markedly for colonies grown on minimal media (no
exogenous amino acids provided). For Buchnera, in contrast,
pooled transcript abundance for genes underlying amino acid
biosynthesis differs little from aphids confined to plants with
dramatically different nutritional qualities (Fig. 1B). This rela-
tive constancy of transcript abundances extends to individual
1. Moran, N. A. (2002) Cell 108, 583-586.
2. Buchner, P. (1965) Endosymbcoscs of Animals with Plant Microorganisms (Wiley,
New York).
;3. Munson, M. A., Baumann, P., Clark, M. A., Baumann, L., Moran, N. A.,
Voeptlin, D. J. & Campbell, B. C. (1991) J. Bacteriol. 173, 6321-6324.
4. Sandstrom, J. P. & Moran, N. A. (2001) Physiol. Entomol. 26, 202-211.
5. Bernays, E. A. & Klein, B. A. (2002) Physiol. Entomol. 27, 275-284.
6. Sandstrom, J. & Pettersson, J. (1994) J. Insect Physiol. 40, 947-955.
7. Telang, A., Sandstrom, J., Dyreson, E. & Moran, N. A. (1999) Entomol. Exp.
Appl. 91, 402-412.
8. Febvay, G., Liadouze, I., Guillaud, J. & Bonnot, G. (1995)Arch. Insect Biochem.
Physiol. 29, 45-69.
9. Sasaki, T. & Ishikawa, H. (1995) J. Insect Physiol. 41, 41-46.
10. Whitehead, L. F. & Douglas, A. E. (1993) J. Gen. Microbiol. 139, 821-826.
11. Tamas, I., Klasson, L., Canback, B., Naslund, A. K., Eriksson, A. S.,
Wernegreen, J. J., Sandstrom, J. P., Moran, N. A. & Andersson, S. G. (2002)
Science 296, 2376-2379.
12. van Ham, R. C., Kamerbeek, J., Palacios, C., Rausell, C., Abascal, F., Bastolla,
U., Fernandez, J. M., Jimenez, L., Postigo, M., Silva, F. J., et al. (2003) Proc.
Natl. Acad. Sci. USA 100, 581-586.
Moran et a/.
biosynthetic pathways (Fig. 2~. The low response to changes in
plant quality could reflect a lack of symbiont ability to adaptively
alter transcription rates. However, other explanations are pos-
sible, including experimental limitations preventing detection of
fine-tuned feedback mechanisms; for example, the pooling of
RNA from maternal and embryonic symbionts~may obscure
biologically meaningful changes. Regulation of amino acid bio-
synthesis could be achieved, in part, through regulation of amino
acid transport from host to symbiont. The Buchnera genome
lacks apparent importers, but each Buchnera cell is enclosed
within a host membrane that is capable of active transport of
glutamate and aspartate, which are substrates for biosynthesis of
the other amino acids (10~. Such host control could govem the
overall levels of amino acid biosynthesis within Buchnera cells
but would not provide a way to fine-tune the production of
individual amino acids according to host needs. The aphids seem
not to alter their assimilation of essential amino acids in response
to different dietary concentrations of these compounds (50),
indicating that their regulation within aphids must involve
differential symbiont productivity.
Conclusions
The genomes of the obligate symbionts of insects provide a clear
picture of modification of an ancestral genome for a particular
role in exchanging nutrients with the host. The picture emerging
so far tells us that the essential genetic capabilities are ancient
and consist of pathways that are widely distributed among
nonsymbiotic bacteria. It appears that the maternally inherited,
obligate bacteriome-associates of insects are derived through
reduction and minor modifications of ancestral genomes rather
than through acquisition or invention of novel "symbiosis"
genes. Nonetheless, some of the critical elements for mainte-
nance of symbiosis remain unidentified. We do not yet know why
the ~y3 Proteobacteria have given rise to a large proportion of the
symbiotic lineages living in insects. Perhaps this group possesses
some yet-to-be-discovered key genes that enable symbiosis. One
possibility is the frequent presence of a type III secretion
apparatus that might serve as the initiator of the intracellular
association, as in the secondary symbionts of tsetse flies and the
related symbionts of grain weevils (51~. In addition, the control
of gene expression may involve novel mechanisms, a possibility
that is supported by the apparent plasticity of Buchnera contri-
butions depending on host diet.
We thank H. McLaughlin and H. Dunbar for laboratory assistance, K. A.
Shufran for providing aphid clones, J. Glasner for access to unpublished
data, and H. Ochman for comments on the manuscript. Funding was
from National Science Foundation Grant 9978518 (to N.A.M.), a
National Science Foundation postdoctoral fellowship (to J.L.W.), and a
National Institutes of Health postdoctoral fellowship (to G.R.P.~.
13. Shigenobu, S., Watanabe, H., Hattori, M., Sakaki, Y. & Ishikawa, H. (2000)
Nature 407, 81-86.
14. Akman, L., Yamashita, A., Watanabe, H., Oshima, K., Shiba, T., Hattori, M.
& Aksoy, S. (2002) Nat. Genet. 32, 402-407.
15. Gil, R., Silva, F. J., Zientz, E., Delmotte, F., Gonzalez-Candelas, F., Latorre,
A., Rausell, C., Kamerbeek, J., Gadau, J., Holldobler, B., et al. (2003) Proc.
Natl. Acad. Sci. USA 100, 9388-9393.
16. Moran, N. A. & Mira, A. (2001) Genome Biol. 2, RESEARCH0054.
17. Silva, F. J., Latorre, A. & Moya, A. (2001) Trends Genet. 17, 615-618.
18. Lai, C. Y., Baumann, L. & Baumann, P. (1994) Proc. Natl. Acad. Sci. USA 91,
3819-3823.
19. Bracho, A. M., Martinez-Torres, D., Moya, A. & Latorre, A. (1995) J. Mol.
Evol. 41, 67-73.
20. Baumann, L., Baumann, P., Moran, N. A., Sandstrom, J. & Thao, M. L. (1999)
J. Mol. Evol. 48, 77-85.
21. Rouhbakhsh, D., Clark, M. A., Baumann, L., Moran, N. A. & Baumann, P.
(1997) Mol. Phylogenet. Evol. 8, 167-176.
22. van Ham, R. C., Gonzalez-Candelas, F., Silva, F. J., Sabater, B., Moya, A. &
Latorre, A. (2000) Proc Natl. Acad. Sci. USA 97, 10855-10860.
23. Lai, C. Y., Baumann, P. & Moran, N. A. (1995) Insect Mol. Biol. 4, 47-59.
24. Komaki, K. & Ishikawa, H. (1999) J. Mol. Evol. 48, 717-722.
PNAS 1 November 25, 2003 ~ vol. 100 1 suppl. 2 1 14547
OCR for page 36
25. Plague, G. R., Dale, C. & Moran, N. A. (2003) Mol. Ecol. 12, 1095-1100.
26. Thao, M. L., Baumann, L., Baumann, P. & Moran, N. A. (1998) Curr. Microbiol.
36, 238-240.
27. Birkle, L. M., Minto, L. B. & Douglas, A. E. (2002) Physiol. Entomol, 27, 302-306.
28. Wernegreen, J. J. & Moran, N. A. (2000) Proc. R. Soc. London Ser. B 267,
1423-1431.
29. Baumann, L., Clark, M. A., Rouhbakhsh, D., Baumann, P., Moran, N. A. &
Voegtlin, D. J. (1997) Curr. Microbiol. 35, 18-21.
30. Lai, C. Y., Baumann, P. & Moran, N. A. (1996) Appl. Environ. Microbiol. 62,
332-339.
31. Wernegreen, J. J., Richardson, A. O. & Moran, N. A. (2001) Mol. Phylogenet.
Evol. 19, 479-485.
32. Birkle, L. M. & Douglas, A. E. (1999) Heredity 82, 605-612.
33. Srivastava, P. N. & Auclair, J. L. (1975) J. Insect Physiol. 21, 1865-1871.
34. Srivastava, P. N. (1987) in World Crop Pests, eds. Mink, A. K. & Harrewijn, P. 49.
(Elsevier, Amsterdam), Vol. 2A, pp. 99-122.
35. Turner, R. B. (1971) J. Insect Physiol. 17, 2451-2456.
36. Gil, R., Sabater-Munoz, B., Latorre, A., Silva, F. J. & Moya, A. (2002) Proc.
Natl. Acad. Sci. USA 99, 4454-4458.
37. Fukatsu, T. (2001) Appl. Environ. Microbiol. 67, 5315-5320.
38. Montllor, C. B., Madmen, A. & Purcell, A. H. (2002) Ecol. Entomol. 27, 189-195.
39. Sandstrom, J. P., Russell, J. A., White, J. P. & Moran, N. A. (2001) Mol. Ecol.
10, 217-228.
40. Tsuchida, T., Koga, R., Shibao, H., Matsumoto, T. & Fukatsu, T. (2002) Mol.
Ecol. 11, 2123-2135.
14548 1 www.pnas.org/cgi/doi/ 10.1 073/pnas.2135345 100
41. Sandstrom, J. & Moran, N. A. (1999) Ent. Exp. Appl. 91, 203-210.
42. Wilcox, J. L., Dunbar, H. E., Wolfinger, R. D. & Moran, N. A. (2003) Mol.
Microbiol. 48, 1491-1500.
43. Jimenez, N., Gonzalez-Candelas, F. & Silva, F. J. (2000) J. Bacteriol. 182,
2967-2969.
44. Kolibachuk, D., Rouhbakhsh, D. & Baumann, P. (1995) Curr. Microbiol. 30,
313-316.
45. Febvay, G., Rahbe, Y., Rynkiewiecz, M., Guillard, J. & Bonnot, G. (1999) J.
E;xp. Biol. 202, 2639-2652.
46. Leckstein, P. M. & Llewellyn, M. (1973) J. Insect Physiol. 19, 973-980.
47. Liadouze, I., Febvay, G., Guillaud, J. & Bonnot, G. (1995) J. Insect Physiol. 41,
33-40.
48. Douglas, A. E., Minto, L. B. & Wilkinson, T. L. (2001) J. E;xp. Biol. 204,
349-358.
Wei, Y., Lee, Y. M., Richmond, C., Blattner, F. R., Rafalski, J. A. & LaRossa,
R. A. (2001) J. Bacteriol. 183, 545-556.
50. Wilkinson, T. L. & Ishikawa, H. (1999) Entomol. Exp. Appl. 91, 195-201.
51. Dale, C., Plague, G. R., Wang, B., Ochman, H. & Moran, N. A. (2002) Proc.
Natl. Acad. Scr. USA 99, 12397-12402.
52. Tatusov, R. L., Natale, D. A., Garkavtsev, I. V., Tatusova, T. A., Shankavaram,
U. T., Rao, B. S., Kiryutin, B., Galperin, M. Y., Fedorova, N. D. & Koonin,
E. V. (2001) Nucleic Acids Res. 29, 22-28.
53. Glasner, J. D., Liss, P., Plunkett, G., III, Darling, A., Prasad, T., Rusch, M.,
Byrnes, A., Gilson, M., Biehl, B., Blattner, F. R. & Perna, N. T. (2003) Nucleic
Acids Res. 31, 147-151.
Moran et al.
Representative terms from entire chapter:
amino acid
