Previous Article | Next Article 
Journal of Bacteriology, March 2001, p. 1853-1861, Vol. 183, No. 6
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.6.1853-1861.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Degenerative Minimalism in the Genome of a
Psyllid Endosymbiont
Marta A.
Clark,1
Linda
Baumann,1
MyLo Ly
Thao,1
Nancy A.
Moran,2 and
Paul
Baumann1,*
Microbiology Section, University of
California, Davis, California 95616-8665,1 and
Department of Ecology and Evolutionary Biology, University
of Arizona, Tucson, Arizona 857212
Received 25 September 2000/Accepted 14 December 2000
 |
ABSTRACT |
Psyllids, like aphids, feed on plant phloem sap and are obligately
associated with prokaryotic endosymbionts acquired through vertical
transmission from an ancestral infection. We have sequenced 37 kb of
DNA of the genome of Carsonella ruddii, the endosymbiont of
psyllids, and found that it has a number of unusual properties revealing a more extreme case of degeneration than was previously reported from studies of eubacterial genomes, including that of the
aphid endosymbiont Buchnera aphidicola. Among the unusual properties are an exceptionally low guanine-plus-cytosine content (19.9%), almost complete absence of intergenic spaces, operon fusion,
and lack of the usual promoter sequences upstream of 16S rDNA. These
features suggest the synthesis of long mRNAs and translational coupling. The most extreme instances of base compositional bias occur
in the genes encoding proteins that have less highly conserved amino
acid sequences; the guanine-plus-cytosine content of some protein-coding sequences is as low as 10%. The shift in base
composition has a large effect on proteins: in polypeptides of C. ruddii, half of the residues consist of five amino acids with
codons low in guanine plus cytosine. Furthermore, the proteins of
C. ruddii are reduced in size, with an average of about 9%
fewer amino acids than in homologous proteins of related bacteria.
These observations suggest that the C. ruddii genome is not
subject to constraints that limit the evolution of other known eubacteria.
| |
INTRODUCTION |
The insect suborder Sternorrhyncha
(Hemiptera), which includes psyllids, also contains aphids, mealybugs,
and whiteflies, all of which feed primarily or exclusively on plant
phloem sap (5). All of these insects contain primary
endosymbionts corresponding to different bacterial clades (11,
12, 18; P. Baumann, N. A. Moran, and L. Baumann,
http://link.springer.de/link /service/books/10125/). The most
extensively studied endosymbionts are those from aphids (3,
10; Baumann et al., website). The clade constituting these
endosymbionts has been given the designation Buchnera
aphidicola, and phylogenies based on several molecules indicate
that the closest free-living relatives are members of the
Enterobacteriaceae. Previously we have sequenced about 130 kb of DNA from B. aphidicola of the aphid Schizaphis
graminum (Baumann et al., website), and recently the complete
sequence of the 640-kb genome of the endosymbiont of the aphid
Acyrthosiphon pisum has been determined (24).
These genetic studies indicate that in B. aphidicola there
has been a major reduction in the gene content with a retention of the genes necessary for a variety of housekeeping functions as well as the
synthesis of essential amino acids and riboflavin (3, 24;
Baumann et al., website). The biosynthetic functions of B. aphidicola are also supported by numerous nutritional studies (10; Baumann et al., website).
Psyllids, or "jumping plant lice" (Hemiptera: Psyllidae), differ
from aphids in many aspects of life history and biogeography but are
similar in that they feed on phloem sap (5, 14) and possess maternally transmitted endosymbionts (6, 11, 26; Baumann et al., website). Phloem sap of most plants is deficient in
essential amino acids (23), suggesting that B. aphidicola and the endosymbionts of psyllids have the same
functions related to host nutrition. The primary endosymbionts of
psyllids, designated Carsonella ruddii, constitute a unique
lineage within the 
3 subdivision of the
Proteobacteria (11, 26, 28). They are located
within host cells called bacteriocytes, where they are enclosed by
host-derived membrane vesicles; the multicellular structure containing
the bacteriocytes is called a bacteriome (6, 7, 11, 31). In a recent study of 32 psyllid species, the phylogenetic tree derived
from the 16S-23S rDNA of C. ruddii agreed with the tree derived from a host gene, a result consistent with a single infection of a psyllid ancestor and subsequent vertical transmission
(cospeciation) of endosymbionts and hosts (28). Some
psyllid species also contain morphologically diverse secondary (S)
endosymbionts (6, 11, 26); molecular phylogenetic analyses
indicate that S-endosymbionts result from multiple infections of hosts
and possible horizontal transmission among them (29).
Since C. ruddii appears to be present in all psyllids and
the S-endosymbionts are absent in about one-third of the species
(28), C. ruddii alone must be able to fulfill
all of the necessary functions of the endosymbiotic association.
The 16S and 23S rDNAs of C. ruddii have G+C contents lower
than those of any other known bacteria (11, 26, 28). In
addition, the 3' end of C. ruddii 16S rDNA lacks a sequence
complementary to the ribosomal binding site (RBS) of mRNA
(Shine-Dalgarno sequence) (28). These observations
suggested that the genome of C. ruddii may also have unusual
properties and led to the present study, in which we describe the
results of a sequence analysis of 37 kb of C. ruddii DNA.
| |
MATERIALS AND METHODS |
General methods.
Standard molecular biology methods were
used in this study (2, 22). Additional methods have been
described in our past publications and include the isolation of total
psyllid DNA, restriction enzyme and Southern blot analyses, and cloning
into 
ZAP (Strategene, La Jolla, Calif.) (28; Baumann et
al., website). The nucleotide sequence of C. ruddii DNA was
determined at the University of Arizona (Tucson) LMSE sequencing
facility. Besides T3 and T7 primers, custom-made oligonucleotide
primers were designed for sequencing. For sequence determination of
some DNA fragments, a double-stranded DNA nested deletion kit
(Pharmacia, Picataway, N.J.) was used.
Most of the sequence data involved the psyllids Pachypsylla
venusta and P. celtidis; additional psyllids used were
Acizzia uncatoides, Bactericera cockerelli,
Ctenarytaina eucalypti, C. longicauda, C. spatulata, Calophya schini, Heteropsylla cubana, and Trioza
eugenia. P. venusta and P. celtidis
contained only C. ruddii and lacked the S-endosymbiont
(28).
General approach.
In our previous studies on Buchnera
aphidicola (endosymbiont of aphids), we developed oligonucleotide
primers based on conserved protein sequences, amplified the DNAs by
PCR, cloned the fragments into pBluescript (Stratagene), and
subsequently determined their nucleotide sequence. These DNA fragments
were used as probes for restriction enzyme and Southern blot analyses
of endosymbiont DNA. Based on these results, appropriate restriction
enzyme-digested DNA fragments were cloned into ZAP (Stratagene) and
sequenced (3). Initially this approach was suitable for
the cloning of a 4.9-kb DNA fragment from C. ruddii-P.
venusta which contained atp genes (Fig.
1B). Subsequent attempts to clone a
portion of the 16S rDNA and the region upstream, as well as a portion
of the 23S rDNA and the region downstream (Fig. 1C), using these methods failed due to instability of the recombinants or insertion of
Escherichia coli sequences. Consequently, we used two
approaches to generate sets of overlapping 1.5- to 3.8-kb fragments. In
the first approach, we selected proteins that are highly conserved and
tend to be clustered in single regulatory units (Fig. 1)
(21). Synthetic oligonucleotide primers were designed
based on conserved amino acid sequences of homologous proteins. These
primers also contained, at the 5' ends, sequences for restriction
enzymes. Following amplification by PCR, the DNA was digested with the appropriate restriction enzymes and cloned into pBluescript
(Stratagene) or pWSK130 (a low-copy vector) (33), and the
nucleotide sequence was determined. The sequences of the
oligonucleotide primers and their positions on the sequenced DNA
fragments will be made available on request. In some cases we were not
able to extend the sequence using this approach and recourse was made
to unidirectional PCR.
View larger version (22K):
[in this window]
[in a new window]
|
FIG. 1.
Genetic maps of DNA fragments from C. ruddii.
(A) The 19.2-kb DNA fragment of C. ruddii-P.
venusta; (B) the 10.0-kb DNA fragment of C. ruddii-P.
venusta; (C) the 7.8-kb DNA fragment of C. ruddii-P. celtidis. Thick lines, structural genes;
thick striped lines, positions of probes used for restriction enzyme
and Southern blot analyses to obtain fragments for Vectorette II
unidirectional PCR; single-headed arrows, direction of transcription;
double-headed arrows with V on top, fragments obtained with Vectorette
II unidirectional PCR; double-headed arrows, overlapping DNA fragments
obtained by PCR; double-headed dashed arrows, DNA fragments also
obtained from endosymbionts of other psyllid species.
|
|
Unidirectional PCR.
We used the Vectorette II system
(Sigma-Genosys, St. Louis, Mo.) and the instructions provided by the
manufacturer. From the nucleotide sequence of endosymbiont DNA
fragments, we obtained a useable restriction enzyme site (close to the
end which was to be extended) and the sequence for two oligonucleotide
primers from which a probe was amplified by PCR. Restriction enzyme and Southern blot analyses were performed using this probe, the restriction enzyme that digests the site in the known sequence, and additional restriction enzymes. In most cases 1.6- to 3.5-kb DNA fragments were
selected. The endosymbiont-psyllid DNA was digested with a combination
of two restriction enzymes, and the appropriate-sized fragment was
eluted from agarose gels. The ends were filled in with the Klenow
fragment (2), and the DNA fragments were blunt-end ligated
to phosphorylated BamHI linkers (New England Biolabs, Beverly, Mass.) and subsequently digested with BamHI.
Following removal of the linkers, the fragments were ligated to
BamHI-compatible Vectorette II DNA. Using an oligonucleotide
primer derived from the known sequence, which also contained a
restriction enzyme site(s), and a Vectorette II primer, the DNA was
amplified by PCR, digested with the appropriate restriction enzymes,
and cloned into either pBluescript or pWSK130. The primers used for
unidirectional PCR are available upon request.
PCR conditions.
PCR amplification was performed in a 50-µl
volume containing 50 ng of total psyllid DNA, 5 mM MgCl2,
0.2 mM deoxynucleoside triphosphate mix, 1 µM primers, and 2 U of
Bio-X-ACT DNA polymerase in Optibuffer (Bioline, London, United
Kingdom). PCR products larger than 1 kb were amplified by 30 cycles
consisting of a 30-s denaturation at 94°C, a 2-min annealing at 48 to
50°C, and a 5-min elongation at 70°C. For PCR products smaller than
1 kb, the annealing and elongation times were reduced to 30 s
and 1 min, respectively.
Analysis of the DNA.
We used GeneJockey II (Biosoft,
Ferguson, Mo.) to identify open reading frames (ORFs) and Blast
searches (National Center for Biotechnology Information, Bethesda, Md.)
for proteins with amino acid sequence similarity. Alignment of amino
acids was performed using Gap (Genetics Computer Group, Madison,
Wis.). In comparative studies, sequences of B. aphidicola, Richettsia prowazekii, and Escherichia
coli were also included (accession numbers AF000398, AJ235269, and U00096, respectively).
Nucleotide sequence accession numbers.
The following are the
GenBank accession numbers (in parentheses) for the sequences of the
fragments obtained in this study: C. ruddii-P. venusta 19.2 kb (Fig. 1A), AF274444; C. ruddii-C. eucalypti
3.8 kb (Fig. 1A), AF250389; C. ruddii-P. celtidis 3.8 kb
(Fig. 1A), AF250390; C. ruddii-P. venusta 10.0 kb
(Fig. 1B), AF291051; and C. ruddii-P. celtidis 7.8 kb
(Fig. 1C), AF211141. In addition, sequences of rRNA operons
(complete or partial) for the following endosymbionts were
deposited: C. ruddii-P. venusta, AF211143; C. ruddii-C. eucalypti, AF211133; C. ruddii-A. uncatoides,
AF211124; C. ruddii-C. longicauda, AF211134; C. ruddii-C. spatulata, AF211135; C. ruddii-B. cockerelli, AF211126; C. ruddii-C. schini, AF211132;
C. ruddii-H. cubana, AF211138; and C. ruddii-T. eugenia, AF211151.
| |
RESULTS |
General properties of the C. ruddii DNA.
The three
DNA fragments of C. ruddii DNA were 19,209, 10,049, and
7,806 bp in length (total, 37,057 bp) (Fig. 1A, B, and C, respectively). Searches in databases (September 2000) identified 38 ORFs as corresponding to known genes; 37 were represented in the
E. coli genome, and 1 (aroQ) was found in
Haemophilus influenzae. The total G+C content of the DNA
fragments was 19.9 mol%. When the genes coding for rRNA (which have a
higher G+C content) were excluded, the G+C content was 18.0 mol%. All
genes on the longest fragment (Fig. 1A) are transcribed in one
direction. The genes on the other fragments (Fig. 1B and C) are
transcribed in both directions.
General properties of the ORFs.
A list of the C. ruddii genes together with the product designations, G+C contents,
and percent amino acid identities to E. coli homologs is
presented in Table 1. The highest G+C
contents are in the 16S and 23S rDNA (35.6 and 33.1%, respectively).
The range of G+C contents of protein-coding genes is 9.9 (atpF) to 28.3 (tuf) mol%. A plot of the percent
amino acid identity between the C. ruddii and
E. coli proteins against the mol% G+C content of the
C. ruddii genes (Fig.
2) indicates a correlation between the
conservation of the amino acid sequence of a protein and
the G+C content of the C. ruddii gene encoding the
protein. Less highly conserved protein sequences are encoded by genes
with lower G+C contents.
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 2.
Comparisons of the percent amino acid (AA) sequence
identity (ID) of homologous proteins of C. ruddii and
E. coli with the percent G+C content of the C. ruddii genes.
|
|
Amino acid composition and codon usage.
In C. ruddii, the reduction in G+C content has had a major effect on
polypeptide sequences, causing an increased frequency of amino acids
that utilize high-A+T-containing codons. Although such a shift occurs
in the endosymbiont B. aphidicola and the intracellular
pathogen R. prowazekii, it is most drastic in C. ruddii, in which biased base composition shows a more extreme effect on polypeptide sequences than in any previously studied bacterium (Fig. 3). Within the 32 polypeptide sequences inferred for C. ruddii that have
homologs in B. aphidicola, R. prowazekii, and E. coli, 50% of the residues consist of five amino acids for which
the corresponding codons have maximum A and T content (phenylalanine, lysine, isoleucine, asparagine, and tyrosine). In comparison, these
amino acids comprise only 29.3% of the amino acids of B. aphidicola, 29.4% of the amino acids of R. prowazekii,
and 22.3% of the amino acids of E. coli, for the same
genes. This shift also affects the charge of the proteins. The
calculated isoelectric points of the combined proteins of C. ruddii, B. aphidicola, R. prowazekii, and E. coli were
10.1, 9.9, 9.6, and 9.2, respectively.
View larger version (27K):
[in this window]
[in a new window]
|
FIG. 3.
Amino acid compositions and the A+T and G+C content of
codons of homologous proteins of C. ruddii (Cru),
B. aphidicola (Bap), R. prowazekii (Rpr), and
E. coli (Eco). The G+C contents are for the total coding
regions compared. The genes compared are listed in Fig. 1A and include
gidA, atpB, atpE, atpF, atpA, atpG, and atpD
(Fig. 1B) and trpS (Fig. 1C).
|
|
Intergenic spaces.
An inspection of the putative coding
regions of C. ruddii DNA indicates a major reduction or
elimination of intergenic spaces. A summary of the nucleotide sequences
found between 34 adjacent genes, which are transcribed in the same
direction, is presented in Table 2. In
85% of the adjacent genes there are overlapping regions between the
stop codons for the upstream genes and the initiating codons for the
downstream genes. The most frequent overlap arrangement (44%) is ATGA,
in which the last 3 nucleotides (nt) are used as a stop codon for the
upstream protein and the first 3 nt are used as the initiating
methionine for the downstream protein. In 35% of the cases, the
overlapping regions are longer. Even in the 15% of the adjacent gene
pairs that contain an intergenic space, it is short (7 nt or less). The
elimination of intergenic spaces also results in operon fusion. In
E. coli and many other organisms, the genes in Fig. 1A are
part of five different transcription units (L11 operon, 
operon, Str
operon, S10 operon, and spc operon) (13, 16). In C. ruddii, these operons appear to be part of a single transcription
unit.
The 3.8-kb
tuf-rpS3 DNA fragment designated by dashed
double-headed arrows in Fig.
1A was also sequenced from
C. ruddii of
two additional psyllid species.
Comparisons indicate that there
is both conservation and variation in
some of the protein initiation
and termination arrangements. In
C. ruddii-P. venusta,
C. ruddii-P. celtidis,
and
C. ruddii-C. eucalypti, the junctions between
rpS10-rpL3,
rpL3-rpL4, and
rpL4-rpL2
were the same (
;
the initiating codon is
overlined, and the stop codon is underlined).
There were differences in
the remaining junctions. Between
tuf-rpS10 of
C. ruddii-P. venusta and
C. ruddii-P. celtidis
there was
AT
TAA,
while in
C. ruddii-C. eucalypti the sequence was
.
Between
rpL2-rpS19 of
C. ruddii-P. venusta and
C. ruddii-P. celtidis the sequence was
, while in
C. ruddii-C. eucalypti the sequence was
TC
TAG. Between
rpS19-rpL22 of
C. ruddii-P. venusta and
C. ruddii-P. celtidis the sequence was
,
while in
C. ruddii-C. eucalypti the sequence was
TTA
6TAA. Between
rpL22-rpS3 of
C. ruddii-P. venusta and
C. ruddii-C. eucalypti the sequence was
GGA
8T
TAA, while in
C. ruddii-P. celtidis the
sequence was
GG
TAA. Similarly, the
sequence of a 2.8-kb 23S-
valS C. ruddii DNA fragment
(Fig.
1C) was determined for
C. ruddii of three additional
psyllid species. In
C. ruddii-P. celtidis,
C. ruddii-C. eucalypti,
C. ruddii-A.
uncatoides, and
C. ruddii-C. longicauda there was an
overlapping region between the initiating
ATG of
aphC and
the stop codon of
valS, consisting of 23, 14,
8, and 14 nt, respectively. Previously it was found that the
rpoB-rpoC junction (Fig.
1A, 2.7-kb fragment) of 13 strains of
C. ruddii involved the sequence
while in the related
species
C. ruddii-H.
cubana and
C. ruddii-H. texana, the sequence
in
the junctions was
TAAAAA
(
30). Similarly, in a previous
study of the
atpAGD region (Fig.
1B, 1.8-kb fragment) from 31
strains of
C. ruddii, it was found that in all cases the junction
region was
(
30).
For 22 pairs of genes transcribed in the same direction, the
flanking genes are the same in
C. ruddii as in
B. aphidicola,
R. prowazekii, and
E. coli, allowing direct comparison of the
homologous
intergenic space. For every one of these 22 cases,
the space between
genes in
C. ruddii is absent or smaller than
in any of these
other three organisms (Fig.
4). This
indicates
that
C. ruddii differs from all of these organisms
in the elimination
or reduction of intergenic spaces throughout the
genome.
View larger version (30K):
[in this window]
[in a new window]
|
FIG. 4.
Comparison of the nucleotide length of the intergenic
spaces between the same adjacent gene pairs (transcribed in the same
direction) of C. ruddii (Cru), B. aphidicola
(Bap), R. prowazekii (Rpr), and E. coli (Eco).
ORFA and ORFB of Cru are found in the positions
corresponding to atpH and atpC, respectively of
B. aphidicola, R. prowazekii, and E. coli.
Numbers on top of three bars indicate the lengths (in nucleotides) of
the intergenic spaces.
|
|
The same tendency to minimize sequence length is evident in the two
cases in which adjacent genes are transcribed in opposite
directions,
with end points flanking each other. In
argI-ORFC (Fig.
1B)
there is an overlap of 26 nt involving the sequence
TCAN
18TAG, in which the last triplet is the stop codon
for
argI and the complement of the first triplet is the stop
codon of the
putative ORFC. In the
ahpC-tal region (Fig.
1C) of
C. ruddii-P. celtidis, C. ruddii-P. venusta, C. ruddii-C. eucalypti, C. ruddii-C. longicauda, and
C. ruddii-C. spatulata, translation termination
involves the
sequence TTATAA, in which the last triplet is the
stop
codon for
tal while the complement of the first
triplet is
the stop codon of
ahpC. In
C. ruddii-A. uncatoides, termination
involves the
shorter sequence TTAA, in which the last 3 nt are
the stop
codon for
tal while the complement of the first 3 nt
(two of which overlap with the stop codon for
tal)
are the stop
codon for
ahpC.
Protein size.
In Fig. 5 the
sizes of 32 C. ruddii proteins are compared with those of
homologous proteins of B. aphidicola, R. prowazekii, and
E. coli. In 29 of the 32 cases, the C. ruddii
proteins were shorter than any of the homologs. The numbers of amino
acids in proteins of B. aphidicola, R. prowazekii, and
E. coli averaged 9.2, 9.1, and 9.5%, respectively, greater
than those in the C. ruddii proteins. The major size
difference appears in ribosomal protein L3, which has a substantial
deletion at the N terminus. We have also cloned and sequenced the 3.8 kb tuf-rpS3 fragment from C. ruddii-P. celtidis
and C. ruddii- C. eucalypti (Fig. 1A). A comparison of
the lengths of the proteins in C. ruddii from these three
psyllid species indicated that they are identical or nearly identical
in size. The number of amino acids is listed in parentheses following
the protein designation in the order C. ruddii-P. venusta,
C. ruddii-P. celtidis, and C. ruddii-C. eucalypti
(where the size is constant only one value is given): Tuf, partial 312, 312, 310; RpS10, 98; RpL3, 136; RpL4, 172; RpL2, 235, 235, 234; RpS19,
88; RpL22, 104, 101, 105; RpS3, partial 148, 148, 150.
View larger version (23K):
[in this window]
[in a new window]
|
FIG. 5.
Comparison of the percent increase in the amino acid
content of homologous proteins that are present in B. aphidicola (Bap), R. prowazekii (Rpr), and
E. coli (Eco) over that in C. ruddii (Cru).
|
|
rRNA operon.
Sequence comparison of the DNA region upstream of
rRNA operons of B. aphidicola indicated that a sequence
resembling the 
35, 10 promoter region is conserved and is followed
by downstream conserved sequences resembling boxA and
boxC (3; Baumann et al., website). In addition,
inverted repeats resembling rho-independent terminators following the
rRNA operons were found. We have used a similar comparative approach to
look for sequences conserved upstream of 16S rDNA which might
correspond to putative promoter regions. The results are presented in
Fig. 6A and indicate conservation of a
sequence resembling boxA; no sequences resembling the 35, 10 promoter region were found upstream of the putative
boxA, in the last 200 nt of trpS. Similarly,
there is no sequence conservation following 5S rDNA (Fig. 6B) and no
significant inverted repeats resembling rho-independent terminators
(25). These results suggest that the 16S-23S-5S rRNA genes
are part of a larger transcription unit that includes upstream and
downstream genes (Fig. 1C).
View larger version (32K):
[in this window]
[in a new window]
|
FIG. 6.
Comparisons of the intergenic sequence upstream of
16S (A) and downstream of 5S (B) in C. ruddii from different
psyllids. Ba, B. aphidicola; Ec, E. coli. Nucleotides in bold letters indicate conserved sequences
that are putative ends or beginnings of genes.
|
|
| |
DISCUSSION |
General properties.
C. ruddii has a unique
combination of properties distinguishing it from other known bacteria.
These properties consist of (i) extremely low G+C content (18% for the
protein-coding regions); (ii) elimination or reduction of intergenic
spaces, sometimes resulting in fusion of operons; (iii) absence of the
complement of an RBS at the 3' end of 16S rDNA (28) and an
RBS preceding any structural genes; (iv) a reduction in protein size;
(v) absence of conserved sequences corresponding to the 35, 10
promoter region preceding 16S rDNA; and (vi) absence of inverted
repeats characteristic of rho-independent terminators following 5S
rDNA. The absence of intergenic spaces and the complement of the RBS at
the 3' end of 16S rDNA suggest that long polycistronic mRNAs are made
and translational coupling occurs during protein synthesis (25). The organization of the rRNA genes is highly
conserved in bacteria, and they are generally arranged as a single
transcription unit with recognizable, conserved 35, 10 promoter
regions and a rho-independent terminator(s) (19). The
absence of these sequences in C. ruddii suggests that these
genes are part of a larger transcription unit. Outside of the rRNA
genes, the only conserved sequence that was found preceded 16S rDNA
(Fig. 6A) and had some resemblance to boxA, which is
involved in antitermination (4, 27). The absence of a
sequence complementary to the RBS at the 3' end of 16S-like rRNA has
also been noted in animal mitochondria (35).
The elimination or reduction of intergenic spaces and the reduction in
the protein sizes indicate a history of mutational
pressure and/or
selection favoring reduced sequence length. One
interpretation of the
tendency toward elimination of intergenic
spacers and reduction in gene
length in
C. ruddii is that mutations
are biased in favor of
deletions relative to insertions and that
selection to oppose these
deletions is often weak or ineffective
(due to genetic drift). An
analysis of mutational patterns in
pseudogenes of
Rickettsia
species indicated a mutational bias
favoring deletions
(
1). It is possible that
C. ruddii experiences
similar mutational bias but relaxed selection for conservation
of
function (or less efficient selection due to increased genetic
drift)
results in even greater shrinkage. Alternatively, selection
might have
favored the reduction of sequence length for some unknown
reason.
However, the length reduction occurs both in spacers (Fig.
4) and in
protein-coding sequences (Fig.
5), which are generally
subject to
different kinds of selection. The bias toward A+T in
DNA sequences is
most readily explained as the result of mutational
pressure. The
pattern in Fig.
2 suggests that this mutational
pressure is opposed by
selection for conservation of function,
with the result that genes
encoding proteins which are poorly
conserved have a higher A+T content.
Conservative selection opposing
the mutational bias appears to be
weaker or less efficient than
in other bacteria, with the result that
C. ruddii has the most
extreme bias toward A+T known for
bacteria (
15). A similarly
low G+C content has been found
in the genome of the eukaryote
Plasmodium (
20).
The unique combination of properties found in
C. ruddii is
also illustrated by a comparison of the G+C contents of homologous
rRNA
subunits of bacteria, plastids, and mitochondria with the
G+C contents
of their genomes (or representative
C. ruddii genomic
fragments). Since the results for small (16S and 16S-like) and
large
(23S and 23S-like) subunits of rDNA are very similar, only
the data for
23S rDNA are presented (Fig.
7). Two
different patterns
are discerned, one for bacteria and plastids and one
for and mitochondria.
The distinctness of the two patterns suggests the
existence of
different sets of constraints on allowable evolutionary
change,
although the reasons for these constraints are not understood.
In Fig.
7, arrow (a) designates
B. aphidicola, which is at
the
low end of the bacterial G+C content (the range for bacteria is
27 to 73 mol%). The remaining arrows designate entities which
do not fit
within the bacterial or mitochondrial pattern. Arrow
b corresponds to
the mitochondrion of the protozoan
Reclinomonas americana,
which is unique in that it retains many bacterial attributes
such as
the presence of
35,
10 promoter sequences and a
mitochondrion-encoded
2'
RNA polymerase
(
17). Arrow c designates
C. ruddii, which
is
distinct from the bacterial pattern and tends toward mitochondria.
Arrows d and e designate highly unusual, extrachromosomal DNA
molecules
which are found within membrane-bounded structures in
cells of the
Apicomplexa (genera
Plasmodium and
Toxoplasma)
(
34).
These structures appear to be remnants of plastids
which have
lost their photosynthetic function and are involved in fatty
acid
biosynthesis (
32). Their position is distinct from
plastids
and tends toward mitochondria.
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 7.
Comparison of the G+C content of 23S and 23S-like rDNA
with the G+C content of the genome.  , bacteria;  , mitochondria;
+, plastids; arrow a, B. aphidicola; arrow b,
Reclinomonas americana; arrow c, Cr-Pve; arrow d,
Toxoplasma gondii; arrow e, Plasmodium
falciparum. Data compiled in January 2000 from GenBank and data in
reference 15.
|
|
Gene content, gene order, and physiology.
The list of detected
genes presented in Table 1 indicates that C. ruddii encodes
proteins and RNAs which have housekeeping functions. This includes
rRNAs, ribosomal proteins, two subunits of RNA polymerase, elongation
factors, and tRNA synthases. In addition, we detected many of the
components of the ATP synthase. In E. coli and other
organisms, the genes presented in Fig. 1A are part of five different
operons (13, 16). In C. ruddii, these operons
appear to be fused. The order of the genes is identical to that in
E. coli (16), and the sole difference is the
absence of three genes. C. ruddii lacks rpL23,
rpL29, and rpL24, which in E. coli and many
other organisms are found following rpL4, rpL16, and
rpL14, respectively (Fig. 1A) (13, 16). In
B. aphidicola and many other organisms, the gene order of
the ATP synthase is atpBEFHAGDC (9).
In C. ruddii, part of this order is maintained (Fig. 1B).
However, we have not been able to detect atpH or
atpC. ORFA and ORFB are found in the locations where these
genes are expected, but they show no significant similarity to
atpH or atpC. The presence of genes encoding ATP
synthase suggests that C. ruddii may be able to generate ATP
from the proton motive force. Genes for transaldolase (Fig. 1B) and
transketolase (Fig. 1C) were detected, suggesting a functional
nonoxidative pentose phosphate cycle. In addition, genes encoding
proteins involved in the synthesis of amino acids of the glutamate and
aspartate family were found. These results suggest that C. ruddii may be able to synthesize essential amino acids for the
psyllid host as in the case of B. aphidicola, the
endosymbiont of aphids (10, 24; Baumann et al., website).
Comparison with B. aphidicola.
Both C. ruddii and B. aphidicola are associated with insect
hosts that feed on plant phloem sap and thus have similar nutritional needs. These endosymbiotic associations result from infections by two
different bacterial ancestors, both from the division of the
proteobacteria (11, 18, 28; Baumann et al., website). It
is likely that C. ruddii retains genes for essential amino acid pathways, as in B. aphidicola. Some of the same
genomewide features appear in both species and can be interpreted as
convergences; these include increased A+T content (18),
accelerated sequence evolution (18, 26, 28), and shortened
proteins (8). Although a slight reduction in protein
length has been noted previously for B. aphidicola relative
to E. coli (8), the reduction is much more
severe in C. ruddii, as is evident in Fig. 5. B. aphidicola has intergenic spaces similar to those found in other
bacteria (Fig. 4), and the 3'-end of its 16S rDNA has the complement of the RBS (3, 24; Baumann et al., website). The G+C contents of both the B. aphidicola genome and the B. aphidicola rRNA are comparable to those of other bacteria at the
lower end of the known scale (Fig. 7). In C. ruddii, the
modifications are considerably more drastic, resulting in an even lower
G+C content of the DNA and almost complete elimination of intergenic
spaces. B. aphidicola and some other bacteria with low G+C
contents have very small genomes, having lost most of the genes found
in ancestors (1, 24); future studies determining the
C. ruddii genome size will be of interest.
| |
ACKNOWLEDGMENTS |
This material is based on work supported by National Science
Foundation awards MCB-9807145 (to P.B.) and DEB-9978518 (to N.A.M. and
P.B.) and the University of California Experiment Station (to P.B.).
| |
FOOTNOTES |
*
Corrosponding author. Mailing address: Microbiology
Section, University of California, One Shields Ave., Davis, CA
95616-8655. Phone: (530) 752-0272. Fax: (530) 752-9014. E-mail:
pabaumann{at}ucdavis.edu.
| |
REFERENCES |
| 1.
|
Andersson, J. O., and S. G. Andersson.
1999.
Genome degradation is an ongoing process in Rickettsia.
Mol. Biol. Evol.
16:1178-1191[Abstract].
|
| 2.
|
Ausubel, F. M.,
R. Brent,
R. E. Kingston,
D. D. Moore,
J. G. Seidman,
J. A. Smith, and K. Struhl (ed.).
2000.
Current protocols in molecular biology.
John Wiley & Sons, Inc., New York, N.Y.
|
| 3.
|
Baumann, P.,
L. Baumann,
C. Y. Lai,
D. Rouhbakhsh,
N. A. Moran, and M. A. Clark.
1995.
Genetics, physiology, and evolutionary relationships of the genus Buchnera: intracellular symbionts of aphids.
Annu. Rev. Microbiol.
49:55-94[CrossRef][Medline].
|
| 4.
|
Berg, K. L.,
C. Squires, and C. L. Squires.
1989.
Ribosomal RNA operon anti-termination. Function of leader and spacer region boxB-boxA sequences and their conservation in diverse micro-organisms.
J. Mol. Biol.
209:345-358[CrossRef][Medline].
|
| 5.
|
Borror, D. J.,
C. A. Triplehorn, and N. F. Johnson.
1989.
An introduction to the study of insects, p. 335-345.
The W. B. Saunders Co., Fort Worth, Tex.
|
| 6.
|
Buchner, P.
1965.
Endosymbiosis of animals with plant microorganisms, p. 210-332.
Interscience, New York, N.Y.
|
| 7.
|
Chang, K. P., and A. J. Musgrave.
1969.
Histochemistry and ultrastructure of the mycetome and its "symbiotes" in the pear psylla, Psylla pyricola Foerster (Homoptera).
Tissue Cell
1:597-606[Medline].
|
| 8.
|
Charles, H.,
D. Mouchiroud,
J. Lobry,
I. Goncalves, and Y. Rahbe.
1999.
Gene size reduction in the bacterial aphid endosymbiont, Buchnera.
Mol. Biol. Evol.
16:1820-1822[Medline].
|
| 9.
|
Clark, M A., and P. Baumann.
1997.
The (F1F0) ATP synthase of Buchnera aphidicola (endosymbiont of aphids): genetic analysis of the putative ATP operon.
Curr. Microbiol.
35:84-89[CrossRef][Medline].
|
| 10.
|
Douglas, A. E.
1998.
Nutritional interactions in insect-microbial symbioses: aphids and their symbiotic bacteria Buchnera.
Annu. Rev. Entomol.
43:17-37[CrossRef][Medline].
|
| 11.
|
Fukatsu, T., and N. Nikoh.
1998.
Two intracellular symbiotic bacteria from the mulberry psyllid Anomoneura mori (Insecta, Homoptera).
Appl. Environ. Microbiol.
64:3599-3606[Abstract/Free Full Text].
|
| 12.
|
Fukatsu, T., and N. Nikoh.
2000.
Endosymbiotic microbiota of the bamboo pseudococcid Antonina crawii (Insecta, Homoptera).
Appl. Environ. Microbiol.
66:643-650[Abstract/Free Full Text].
|
| 13.
|
Hansmann, S., and W. Martin.
2000.
Phylogeny of 33 ribosomal and six other proteins encoded in an ancient gene cluster that is conserved across prokaryotic genomes: influence of excluding poorly alignable sites from analysis.
Int. J. Syst. Evol. Microbiol.
50:1655-1663[Abstract].
|
| 14.
|
Hodkinson, I. D.
1974.
The biology of the Psylloidea (Homoptera): a review.
Bull. Entomol. Res.
64:325-339.
|
| 15.
|
Holt, J. G. (ed.).
1984-1989.
Bergey's manual of systematic bacteriology, vol. I to IV.
The Williams & Wilkins Co., Baltimore, Md.
|
| 16.
|
Keener, J., and M. Nomura.
1996.
Regulation of ribosome synthesis, p. 1417-1431.
In
F. C. Neidhardt, et al. (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. ASM Press, Washington, D.C.
|
| 17.
|
Lang, B. F.,
G. Burger,
C. J. O'Kelly,
R. Cedergren,
G. B. Golding,
C. Lemieux,
D. Sankoff,
M. Turmel, and M. W. Gray.
1997.
An ancestral mitochondrial DNA resembling a eubacterial genome in miniature.
Nature
387:493-497[CrossRef][Medline].
|
| 18.
|
Moran, N. A., and A. Telang.
1998.
Bacteriocyte-associated symbionts of insects: a variety of insect groups harbor ancient prokaryotic endosymbionts.
Bioscience
48:295-304[CrossRef].
|
| 19.
|
Munson, M. A.,
L. Baumann, and P. Baumann.
1993.
Buchnera aphidicola (a prokaryotic endosymbiont of aphids) contains a putative 16S rRNA operon unlinked to the 23S rRNA-encoding gene: sequence determination, and promoter and terminator analysis.
Gene
137:171-178[CrossRef][Medline].
|
| 20.
|
Musto, H.,
H. Romero,
A. Zavala,
K. Jabbari, and G. Bernardi.
1999.
Synonymous codon choices in the extremely GC-poor genome of Plasmodium falciparum: compositional constraints and translational selection.
J. Mol. Evol.
49:27-35[CrossRef][Medline].
|
| 21.
|
Neidhardt, F. C., et al. (ed.).
1996.
Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed.
ASM Press, Washington, D.C.
|
| 22.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 23.
|
Sandström, J., and N. Moran.
1999.
How nutritionally imbalanced is phloem sap for aphids?
Entomol. Exp. Appl.
91:203-210[CrossRef].
|
| 24.
|
Shigenobu, S.,
H. Watanabe,
M. Hattori,
Y. Sakaki, and H. Ishikawa.
2000.
Mutualism as revealed at the genomic level: the whole genome sequence of Buchnera sp. APS, an endocellula bacterial symbiont of aphids.
Nature
407:81-86[CrossRef][Medline].
|
| 25.
|
Snyder, L., and W. Champness.
1997.
Molecular genetics of bacteria.
ASM Press, Washington, D.C.
|
| 26.
|
Spaulding, A. W., and C. D. von Dohlen.
1998.
Phylogenetic characterization and molecular evolution of bacterial endosymbionts in psyllids (Hemiptera: Sternorrhyncha).
Mol. Biol. Evol.
15:1506-1513[Free Full Text].
|
| 27.
|
Squires, C. L.,
J. Greenblatt,
J. Li,
C. Condon, and C. L. Squires.
1993.
Ribosomal RNA antitermination in vitro: requirement for Nus factors and one or more unidentified cellular components.
Proc. Natl. Acad. Sci. USA
90:970-974[Abstract/Free Full Text].
|
| 28.
|
Thao, M. L.,
N. A. Moran,
P. Abbot,
E. B. Brennan,
D. H. Burckhardt, and P. Baumann.
2000.
Cospeciation of psyllids and their prokaryotic endosymbionts.
Appl. Environ. Microbiol.
66:2898-2905[Abstract/Free Full Text].
|
| 29.
|
Thao, M. L.,
M. A. Clark,
L. Baumann,
E. B. Brennan,
N. A. Moran, and P. Baumann.
2000.
Secondary endosymbionts of psyllids have been acquired multiple times.
Curr. Microbiol.
41:300-304[CrossRef][Medline].
|
| 30.
| Thao, M. L., M. A. Clark, D. H. Burckhardt, N. A. Moran, and P. Baumann. Phylogenetic
analysis of vertically transmitted psyllid endosymbionts
(Carsonella ruddii) based on atpAGD and
rpoBC; comparisons with 16S-23S rDNA-derived phylogeny.
Curr. Microbiol., in press.
|
| 31.
|
Waku, Y., and Y. Endo.
1987.
Ultrastructure and life cycle of the symbionts in a Homopteran insect, Anomoneura mori Schwartz (Psyllidae).
Appl. Entomol. Zool.
22:630-637.
|
| 32.
|
Waller, R. F.,
P. K. Keeling,
R. G. K. Donald,
B. Striepen,
E. Handman,
N. Lang-Unnasch,
A. F. Cowman,
G. S. Besra,
D. S. Roos, and G. I. McFadden.
1998.
Nuclear-encoded proteins target to the plastid in Toxoplasma gondii and Plasmodium falciparum.
Proc. Natl. Acad. Sci. USA
95:12352-12357[Abstract/Free Full Text].
|
| 33.
|
Wang, R. F., and S. R. Kushner.
1991.
Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli.
Gene
100:195-199[CrossRef][Medline].
|
| 34.
|
Wilson, R. J. M., and D. H. Williamson.
1997.
Extrachromosomal DNA in the Apicomplexa.
Microbiol. Mol. Biol. Rev.
61:1-16[Abstract].
|
| 35.
|
Wolstenholme, D. R.
1992.
Animal mitochondrial DNA: structure and evolution.
Int. Rev. Cytol.
141:173-214[Medline].
|
Journal of Bacteriology, March 2001, p. 1853-1861, Vol. 183, No. 6
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.6.1853-1861.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Stoll, S., Feldhaar, H., Gross, R.
(2009). Promoter Characterization in the AT-Rich Genome of the Obligate Endosymbiont "Candidatus Blochmannia floridanus". J. Bacteriol.
191: 3747-3751
[Abstract]
[Full Text]
-
Sakharkar, K. R., Sakharkar, M. K., Verma, C., Chow, V. T. K.
(2005). Comparative study of overlapping genes in bacteria, with special reference to Rickettsia prowazekii and Rickettsia conorii. Int. J. Syst. Evol. Microbiol.
55: 1205-1209
[Abstract]
[Full Text]
-
Gomez-Valero, L., Latorre, A., Silva, F. J.
(2004). The Evolutionary Fate of Nonfunctional DNA in the Bacterial Endosymbiont Buchnera aphidicola. Mol Biol Evol
21: 2172-2181
[Abstract]
[Full Text]
-
Herbeck, J. T., Funk, D. J., Degnan, P. H., Wernegreen, J. J.
(2003). A Conservative Test of Genetic Drift in the Endosymbiotic Bacterium Buchnera: Slightly Deleterious Mutations in the Chaperonin groEL. Genetics
165: 1651-1660
[Abstract]
[Full Text]
-
Herbeck, J. T., Wall, D. P., Wernegreen, J. J.
(2003). Gene expression level influences amino acid usage, but not codon usage, in the tsetse fly endosymbiont Wigglesworthia. Microbiology
149: 2585-2596
[Abstract]
[Full Text]
-
Wernegreen, J. J., Degnan, P. H., Lazarus, A. B., Palacios, C., Bordenstein, S. R.
(2003). Genome Evolution in an Insect Cell: Distinct Features of an Ant-Bacterial Partnership. Biol. Bull.
204: 221-231
[Abstract]
[Full Text]
-
Sauer, C., Dudaczek, D., Holldobler, B., Gross, R.
(2002). Tissue Localization of the Endosymbiotic Bacterium "Candidatus Blochmannia floridanus" in Adults and Larvae of the Carpenter Ant Camponotus floridanus. Appl. Environ. Microbiol.
68: 4187-4193
[Abstract]
[Full Text]
-
Kikuchi, Y., Fukatsu, T.
(2002). Endosymbiotic Bacteria in the Esophageal Organ of Glossiphoniid Leeches. Appl. Environ. Microbiol.
68: 4637-4641
[Abstract]
[Full Text]
-
Baumann, L., Thao, M. L., Hess, J. M., Johnson, M. W., Baumann, P.
(2002). The Genetic Properties of the Primary Endosymbionts of Mealybugs Differ from Those of Other Endosymbionts of Plant Sap-Sucking Insects. Appl. Environ. Microbiol.
68: 3198-3205
[Abstract]
[Full Text]
Top
Abstract
Introduction
