Differences between parasitism and symbiosis as seen in gene

Differences between parasitism and symbiosis as seen in gene repertoire H Ishikawa The University of the Air, 2-11 Wakaba, Mihama-ku, Chiba 261-8586, Japan

Abstract Aphids (Homoptera; Insecta) harbor intracellular bacterial symbionts, Buchnera, in their

specialized cells called bacteriocytes. Buchnera are exceptional bacteria in that they

possess many, usually more than 100, genomic copies per cell. Whereas Buchnera are

phylogenetically close to Escherichia coli, their genome size has been reduced to a

seventh that of E. coli, and in proportion to this the number of genes the bacteria contain

has decreased. The Buchnera genome retains genes necessary for biosynthesis of

essential amino acids that the host insect cannot synthesize. Meantime, these symbiotic

bacteria have lost most of the genes for biosynthesis of non-essential amino acids that

the host is able to synthesize. Thus, evidently Buchnera and their host are

complementary to each other with respect to the biosynthesis of amino acids. Such

mutual dependence is unique to the symbiosis between Buchnera and aphids, and has

never been observed in the associations between parasitic bacteria, such as Mycoplasma

and Rickettsia, and their hosts. Buchnera resemble parasitic bacteria in that many

essential genes are missing from their genome, most of whose products may be

synthesized and supplied by the host. It is also possible that multifunctional proteins

unique to Buchnera make up for part of these missing gene products.

Introduction So far, some 4,400 species of aphids (Homoptera, Insecta) have been described. Most, if

not all, of them harbor intracellular bacterial symbionts, called Buchnera, in the

cytoplasm of bacteriocytes, huge cells in the abdomen specialized for this purpose

(Buchner, 1965). Buchnera are Gram-negative bacteria 1-2 µm in diameter and belong

to the γ-3 subdivision of the Proteobacteria, and are phylogenetically close relatives of Escherichia coli (Munson et al., 1991). They are maternally transmitted to eggs and

parthenogentic embryos of aphids through host’s generations (Baumann et al., 1995).

Phylogenetic analysis of 16S rDNA sequences of Buchnera from numerous aphid

Endocytobiosis Cell Res. (2004) 15, 1-6

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species has suggested that the symbiotic association between Buchnera and aphids was

established 200-250 million years ago and led to cospeciation of the two associates

(Moran et al., 1993). In the course of symbiosis over 200 million years, the two have

developed intimate genetic and physiological relationships, and the association has

become so obligate that neither of the associates can any longer reproduce

independently (Houk and Griffiths, 1980; Ishikawa, 1989).

Aphids feed exclusively on phloem sap of plants, which is rich in carbohydrates, but

poor in nitrogenous and other compounds. It had long been a riddle what enables aphids,

feeding on nutritionally poor diet, to maintain their extraordinarily high fecundity. The

answer was that Buchnera supplement the aphids’ poor diet. There is now convincing

evidence that Buchnera provide essential amino acids (Sasaki and Ishikawa, 1995;

Douglas, 1998) and riboflavin (Nakabachi and Ishikawa, 1999) to the host insects. The

fact that Buchnera cannot reproduce independently is most likely due to evolutionary

changes of their physiology because of the prolonged symbiotic association with aphids.

In an effort to look into the evolutionary changes from free-living bacteria to

intracellular symbionts, we recently examined the Buchnera genome with respect to its

size and copy number per cell. As a result, it was unveiled that the Buchnera genome is

only a seventh that of E. coli (Charles and Ishikawa, 1999) though the bacteria are close

relatives of the latter. It was also demonstrated that Buchnera are very exceptional

bacteria in that they possess many, usually more than 100, genomic copies per cell

(Komaki and Ishikawa, 1999). With these results in mind, we set about the sequence

analysis of the Buchnera genome, the result of which highlighted the differences

between the mutualistic symbiont and parasitic bacteria (Shigenobu et al., 2000; 2001).

Materials and Methods For the genome analysis of Buchnera, we dissected about 2,000 individuals of the pea

aphid, Acyrthosiphon pisum, to isolate bacteriocytes manually. The isolated

bacteriocytes were crushed by pipetting and subjected to filtration through a 5 µm filter to obtain Buchnera cells without contaminations. Genomic DNA was prepared from the

cells by a standard phenol/chloroform protocol. Sequencing the Buchnera genome was

performed by the whole genome random shotgun sequencing method (Fleischmann et

al., 1995). We used two strategies for identifying open reading frames (ORFs). An

initial set of ORFs was identified by the Gene-Hacker program, a system for gene

structure prediction using a hidden Markov model (Yada and Hirosawa, 1996).

Predicted ORFs were compared against a non-redundant protein database using the


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BLAST programs. Combining these results, we identified and annotated the Buchnera

ORFs (Shigenobu et al., 2000).

General Features of the Buchnera Genome The result demonstrated that the genome of Buchnera from A. pisum, which we call

Buchnera sp. APS, is composed of one circular chromosome and two circular plasmids,

pLeu and pTrp. The chromosome is 640,681 bp, the smallest of the completely

sequenced genomes, except for that of Mycoplasma genitalium (Fraser et al., 1995).

Buchnera sp. APS contains a single copy of each of the three types of ribosomal RNA

and 32 transfer RNA genes. We identified 583 ORFs, whose average size is 988 bp,

accounting for about 88% of the entire genomic sequence. This gene density in the

genome is much the same as in the E. coli genome. The predicted isoelectric points (pIs)

of these gene products are much more basic than those of the corresponding

polypeptides of E. coli, as suggested earlier (Sato and Ishikawa, 1997; Matsumoto et al.,

1999). This is mostly due to increased occurrence of the codons for the amino acid

lysine in Buchnera genes, which probably results from the evolutionary A/T pressure

imposed upon the Buchnera genome (Ishikawa, 1987; Sueoka, 1988).

Similarity search permitted the functional assignment of 500 of the 583 ORFs, and other

80 ORFs are homologous to hypothetical proteins deposited for other bacteria, leaving

behind only three ORFs that do not find their matches. This is an exceptionally small

number, because in all the genomes, including those of parasitic bacteria, sequenced to

date, at least 20% of ORFs are so-called orphan genes. Recently, it was evidenced that

the three orphan genes found in Buchnera were actively expressed (Shimomura et al.,

2002). Among the 583 ORFs, as many as 569 have orthologs in the E. coli genome.

Moreover, the most similar counterparts of these genes are, in general, those of E. coli.

In addition, the operons of E. coli that are retained by Buchnera, their gene orders are

well conserved. These findings strongly suggest that the Buchnera genome is a subset of

the E. coli genome, reflecting the evolutionary relationship between the two bacteria.

Buchnera sp. APS resembles endocellular and epicellular parasites, such as Rickettsia

prowazekii (Andersson et al., 1998) and M. genitalium (Fraser et al., 1995), in that their

genome size has been extremely reduced. However, the genes left behind in the

Buchnera genome are remarkably different from those in the parasitic bacteria, which

depend on their hosts for most nutrients, and the reduction of the genome size of these

parasites is, in greater part, due to the loss of genes for biosynthesis of those nutrients.

By contrast, in the association between Buchnera and aphids, Buchnera are more of a


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provider than a recipient of biosynthetic products such as essential amino acids and

vitamins (Sasaki and Ishikawa, 1995; Douglas, 1998; Nakabachi and Ishikawa, 1999).

In accordance with such the role, Buchnera sp. APS retains many genes that are missing

from the genomes of parasites.

Mutualism as seen in gene repertoire The difference between symbionts and parasites is typically reflected on their gene

repertoire. Whereas the parasitic bacteria contain only a few, if any, genes responsible

for biosynthesis of amino acids, Buchnera sp. APS contains 54 genes in this category,

which account for about 10% of its total genes. Among the genes for biosynthesis of

amino acids that E. coli contains, Buchnera sp. APS has selectively lost those for

biosyntheis of the non-essential amino acids that animals can synthesize, suggesting that

the symbiotic bacterium depends on the host for these amino acids. Instead, unlike

parasitic bacteria, Buchnera sp. APS retains virtually all the genes for biosynthesis of

the essential amino acids that animals themselves cannot synthesize. Evidence suggests

that these genes are expressed (Nakabachi and Ishikawa, 1997). This complementarity

and mutual dependence seen in the gene repertoire typifies how successfully the

symbiosis is operating, in that the symbiont provides the host with what the host cannot

synthesize, and vice versa.

A similar mutual dependence is seen in biosynthesis of coenzyme A (CoA). In view of

the gene repertoire, Buchnera can synthesize pantothenate from pyruvate, whereas there

are no genes for the pathway from pantothenate to CoA. Meantime, it is generally

known that animals are able to produce CoA from pantothenate, but not pantothenate

from pyruvate. Despite a dramatic reduction of the gene number, Buchnera sp. APS

possesses complete gene sets for the sulfur reduction pathway and biosynthesis of

cysteine. Aphids must benefit from these abilities of Buchnera, because insects, in

general, cannot reduce sulfate to sulfide. Unlike parasitic bacteria whose genomes have

been sequenced to date, Buchnera retains almost complete gene sets for biosyntheses of

nucleotides. It remains to be known whether or not the pathways are only for

Buchnera’s own use.

Genes Missing from Buchnera sp. APS In contrast to relative richness in the genes involved in biosynthesis of nutrients, many

genes, including those that seem essential to maintain the identity as a cell, are missing

from Buchnera. First, Buchnera sp. APS lacks many transporter genes that are shared in


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common by all bacterial species whose genomes have been sequenced to date. This is

an unexpected finding because any intracellular symbiosis should necessarily be based

on frequent exchanges of various substances between the two associates. Second,

Buchnera lacks all the genes for the citric acid cycle except for those for the

2-oxoglutarate dehydrogenase complex. As convincing evidence suggests that Buchnera

respires aerobically, it is a riddle how this bacterium secures electron donors for the

electron transfer system to produce ATP. Third, according to its gene repertoire,

Buchnera has only a limited capacity for DNA repair and recombination. Fourth,

Buchnera has only a few genes for cell-surface components, suggesting that this

bacterium is not able to produce lipopolysaccharides (LPSs). There are only a few genes

for lipoproteins and outer membrane proteins. Scarcity of genes for these components

indicates that the cell surface of Buchnera is structurally vulnerable. This is in contrast

to other bacteria, including parasitic and free-living ones, which generally have complex

and flexible surface structures to evade attack by the host immune system or to survive

harsh environments. Fifth, genes responsible for phospholipid biosynthesis are almost

completely missing from the Buchnera genome, although phospholipids are

indispensable components in the formation of the membrane lipid bilayer. It is

interesting to know how Buchnera makes up for the lack of these genes to maintain its

identity as a cell.

Finally, genes for various regulatory systems are almost completely missing from the

Buchnera genome. Among these are those for two-component regulatory systems that

generally control gene expression in response to environmental changes. This can be a

reflection of the stable milieu where Buchnera lives. Of particular interest is that no

transcriptional regulator of amino acid biosynthesis is present despite the conservation

of many genes for amino acid biosynthesis, as mentioned above. In addition, genes of

Buchnera do not have leader sequences, and thus the bacterium is not equipped with a

transcription attenuation system. This suggests that, so far as appropriate substrates are

available, Buchnera will produce amino acids without limit. This situation is quite

favorable to the host aphids that require an enormous amount of amino acids to sustain

their high fecundity. However, a recent report suggests that it is not for the host’s

prosperity, but a means to protect its own genome against deleterious mutations

accumulating that Buchnera produces very much amount of amino acids (Fares et al.,

2002).


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Differences between parasitism and symbiosis as seen in gene

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